A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation
Abstract
Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation.
Editor's evaluation
Authors provide valuable evidence identifying a lncRNA transcribed specifically in the pDC subtype from the +32Kb promoter region which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. With convincing methodology, they provide in-depth analysis about the possible role of lncIrf8, and its promoter region and cross-talk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region that is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. The work will be of interest to immunologists working on immune cell development.
https://doi.org/10.7554/eLife.83342.sa0Introduction
Lineage-determining transcription factors (TF) are master regulators of gene programs that frequently initiate self-reinforcing loops by autoactivation. TF autoactivation is important for cells to pass restriction points during development (referred to as points of no return) and to enforce cellular identity. Molecular circuitries of autoactivation have been studied for several TF, such as GATA-binding factor 1 (GATA1), PU.1 (encoded by Spi1), CCAAT enhancer-binding protein α and ε (C/EBPα and ε; Graf and Enver, 2009; Loughran et al., 2020; Nishimura et al., 2000; Okuno et al., 2005; Theilgaard-Mönch et al., 2022). A further example is interferon regulatory factor 8 (IRF8), which shows autoactivation in cooperation with basic leucine zipper ATF-like transcription factor 3 (BATF3; Anderson et al., 2021; Grajales-Reyes et al., 2015). An important principle in nature is negative feedback control to avoid signal overshooting and toxicity. Negative feedback control applies also to lineage-determining TF; however, there is a paucity on our knowledge of the molecular mechanisms involved.
IRF8 is a hematopoietic TF positioned at the center of the regulatory gene network for dendritic cell (DC) development (Anderson et al., 2021; Belz and Nutt, 2012; Chauvistré and Seré, 2020; Kim et al., 2020; Lin et al., 2015; Nutt and Chopin, 2020; Tamura et al., 2015; Verlander et al., 2022). IRF8 is a member of the interferon regulatory factor (IRF) family of TF. Initially members of this TF family were found to mediate the induction of interferon induced genes, but are now known to serve diverse functions in regulating the immune system (Honda and Taniguchi, 2006; Tamura et al., 2008). Irf8 knockout mice show abnormal development of classical DC type 1 (cDC1) and plasmacytoid DC (pDC) (Durai et al., 2019; Schiavoni et al., 2002; Sichien et al., 2016; Tsujimura et al., 2003). Irf8 is prominently upregulated during DC development by autoactivation (Grajales-Reyes et al., 2015; Lin et al., 2015), yet how Irf8 autoactivation is controlled and eventually limited, and the epigenetic mechanisms involved is largely unknown.
Irf8 expression in hematopoietic cells is induced and maintained by enhancers located at –50 kb,+32 kb,+41 kb and +56 kb relative to Irf8 transcription start site (TSS) (Anderson et al., 2021; Bagadia et al., 2019; Durai et al., 2019; Grajales-Reyes et al., 2015; Murakami et al., 2021; Schönheit et al., 2013). Enhancers are cis-regulatory sequences with multiple TF binding sites that cooperatively bind TF and thereby activate transcription, as demonstrated by many studies including our work (Davidson et al., 1986; Long et al., 2016; Wildeman et al., 1986; Zenke et al., 1986). Enhancers regulate complex gene networks and can also produce non-coding RNA, referred to as enhancer RNA (eRNA). eRNA serve as an indicator for enhancer activity and some eRNA have an activity on their own and act in cis or trans to regulate cell fate decisions (Sartorelli and Lauberth, 2020; Statello et al., 2021). Enhancer-associated long non-coding RNA (lncRNA) represent a class of lncRNA transcribed from active enhancers. Thus, eRNA and enhancer-associated lncRNA provide opportunities to detect enhancer activity and to investigate enhancer function.
DC are highly specialized immune cells that play a critical role in regulating innate and adaptive immune responses (Cabeza-Cabrerizo et al., 2021). DC develop from hematopoietic stem cells (HSC) via successive steps of lineage commitment and differentiation. More specifically, HSC develop into multipotent progenitors (MPP) that are committed to DC restricted common DC progenitors (CDP) and differentiate into classic DC (cDC) type 1 and type 2 (cDC1 and cDC2, respectively) and pDC (Anderson et al., 2021; Cabeza-Cabrerizo et al., 2021; Ginhoux et al., 2022; Nutt and Chopin, 2020; Rodrigues and Tussiwand, 2020). pDC were recently also shown to develop from lymphoid progenitors (Dress et al., 2019; Rodrigues et al., 2018; Rodrigues and Tussiwand, 2020). Differential expression of Irf8 regulates DC and monocyte specification in a dose-dependent manner (Cytlak et al., 2020; Murakami et al., 2021). Irf8 expression starts at the CDP stage, and is high in pDC and cDC1, which is attributed to the autoactivation of Irf8 during DC subsets specification (Grajales-Reyes et al., 2015; Lin et al., 2015). Interestingly, IRF8 can act as a transcriptional activator or repressor in hematopoiesis by interacting with different partner TF and binding to specific DNA sequences (Tamura et al., 2015).
As an activator, IRF8 binds to its own promoter in DC differentiation, which is considered as the autoactivation capacity of Irf8 (Grajales-Reyes et al., 2015; Lin et al., 2015). For instance, IRF8 interacts with partner TF, such as PU.1, to initiate Irf8 autoactivation at the CDP stage (Grajales-Reyes et al., 2015). Inversely, IRF8 inhibits C/EBPα activity in neutrophil differentiation (Kurotaki et al., 2014). IRF8 also represses C/EBPβ to generate and maintain DC lineage-specific enhancer landscapes (Bornstein et al., 2014). In addition, IRF8 is important for the Myc-Mycl transition in DC differentiation (Anderson lll et al., 2021). IRF8 represses Myc expression in progenitors, while IRF8 at high levels interacts with PU.1 and drives Mycl expression (Anderson lll et al., 2021). All this emphasizes the central position of IRF8 in coordinating the gene network that regulates DC differentiation and function.
During DC differentiation, the Irf8 gene locus shows high epigenetic dynamics, including histone modifications and TF binding identified by ChIP-seq (Chauvistré and Seré, 2020; Durai et al., 2019; Grajales-Reyes et al., 2015; Lin et al., 2015), chromatin accessibility measured by ATAC-seq (Kurotaki et al., 2019; Li et al., 2019), and three-dimensional chromatin structure remodeling determined by chromosome conformation capture (3 C) (Kurotaki et al., 2022; Schönheit et al., 2013). All this emphasizes the impact of epigenetic regulators on Irf8 gene activity in DC differentiation. Notably, Irf8 is flanked by multiple enhancers at –50 kb,+32 kb,+41 kb, and +56 kb that regulate Irf8 expression in hematopoietic cells (Anderson et al., 2021; Murakami et al., 2021). These four enhancers were found to be driven by PU.1, BATF3, E proteins and Runt-related transcription factor (RUNX)-core binding factor beta (CBFβ) (RUNX-CBFβ), respectively (Bagadia et al., 2019; Durai et al., 2019; Grajales-Reyes et al., 2015; Murakami et al., 2021; Schönheit et al., 2013).
Chromatin conformation, particularly enhancer promoter interactions, provides a platform for TF-driven gene regulation and serves as a driving force for cell-fate determinations (Misteli and Finn, 2021; Oudelaar and Higgs, 2021; Stadhouders et al., 2019). Schönheit et al. demonstrated Irf8 promoter interactions with its upstream enhancers by quantitative 3 C (Schönheit et al., 2013). In this study PU.1 was found to regulate chromatin remodeling between the –50 kb enhancer and the Irf8 promoter in myeloid differentiation. In a recent study Kurotaki et al., 2022 determined the higher-order chromatin structure in DC progenitors, cDC1 and cDC2 on a genome-wide scale by Hi-C. In this study, reorganization of chromatin conformation at DC-specific gene loci was observed during cDC differentiation, and IRF8 was found to promote chromatin activation in DC progenitors leading to cDC lineage-specific gene expression. However, high resolution maps of the physical chromatin interactions of the Irf8 promoter with upstream and downstream enhancers in the full complement of DC subsets, including pDC, are required for understanding Irf8 regulation during DC differentiation.
Frequently, chromatin data, including ATAC-seq and/or ChIP-seq data, are used to identify regulatory elements of gene transcription. Here we embarked on a different approach and searched for lncRNA, which by themselves might have regulatory functions or are indicative of enhancer activity. We identified a novel lncRNA transcribed from the Irf8 +32 kb enhancer, which is specifically expressed in pDC, referred to as lncIrf8. We found that the lncIrf8 promoter element but not lncIrf8 itself impacts pDC and cDC1 development. Thus, lncIrf8 acts as an indicator for the Irf8 +32 kb enhancer activity. Importantly, our study revealed a previously unrecognized negative feedback loop of Irf8 in DC differentiation. Irf8 first activates its expression by autoactivation via the +32 kb enhancer and second limits its own expression through the lncIrf8 promoter element in the +32 kb enhancer.
Results
lncIrf8 marks a pDC-specific Irf8 enhancer element
Irf8 expression in DC development is subject to complex epigenetic regulation. Here, we used an integrated approach with RNA-seq, ATAC-seq, ChIP-seq and Capture-C to track the dynamics of gene expression, histone modification and chromatin conformation in the sequel MPP, CDP, pDC, cDC1, and cDC2 (Figure 1, Figure 1—figure supplements 1 and 2).

Irf8 epigenetic signatures and promoter-enhancer interaction maps during DC differentiation.
(A) Gene expression and epigenetic signatures of Irf8 downstream region in MPP, CDP, pDC, all cDC, cDC1, and cDC2 are visualized by IGV browser. Gene expression was measured by RNA-seq, chromatin accessibility by ATAC-seq, H3K27ac and IRF8 binding by ChIP-seq. Positions of Irf8 3’ end, Irf8 enhancers, pDC specific lncIrf8 and cDC1 specific Tcons_00190258 lncRNA are indicated. For RNA-seq - and +strands are shown. Scale bar: 5 kb. (B) Physical interactions of Irf8 promoter with flanking sequences in MPP, CDP, pDC, cDC1, and cDC2 by nuclear-titrated (NuTi) Capture-C (turquoise), and CTCF binding by ChIP-seq in DC (Garber et al., 2012). Mean numbers of unique interactions normalized to a 300 kb region around the Irf8 promoter viewpoint (green triangle) and scaled by a factor of 1,000,000 are shown (n=2). The orientations of CTCF binding are indicated with blue and red arrows. Tcons_00190258 refers to the cDC1-specific lncRNA shown in (A). Scale bar: 100 kb. (C) Comparations of the chromatin interactions with Irf8 promoter in pDC, cDC1 and cDC2. Differential tracks were created by subtraction of the mean normalized tracks of (B). Pairwise comparisons are shown and color coded. Turquoise and orange tracks represent specific interactions with the Irf8 promoter in the indicated cell types. Scale bar: 100 kb. Purple bars and lines indicate the position of flanking enhancers relative to Irf8 TSS. The purple bars from left to right represent –50 kb, –34 kb, –26 kb, –16 kb,+27 kb,+32 kb,+38 kb,+41 kb,+47 kb,+56 kb and +62 kb enhancer, respectively (panels B and C). Irf8 +32 kb enhancer is highlighted by purple box.
We performed de novo transcript assembly of the RNA-seq data and detected two previously unknown transcripts without coding potential downstream of Irf8: a pDC specific lncRNA (Tcons_00190250) in the following referred to as lncIrf8 and a cDC1 specific lncRNA (Tcons_00190258; Figure 1A and Figure 1—figure supplement 1). lncIrf8 and Tcons_00190258 show the same expression pattern in pDC and cDC1, respectively, in BM and spleen (Figure 1—figure supplement 3), as revealed by reanalyzing scRNA-seq and bulk RNA-seq data (Pang et al., 2022; Rodrigues et al., 2018). lncIrf8 is transcribed within an enhancer region located 32 kb downstream of the Irf8 TSS labeled by H3K27ac and H3K4me1 and occupied by DC differentiation-associated TF, such as IRF8 and PU.1 (Figure 1A and Figure 1—figure supplement 1). This region is largely devoid of H3K9me3, a chromatin modification frequently associated with heterochromatin, indicating an open chromatin configureuration in DC (Figure 1—figure supplement 1). In addition, sequences of this region have been implicated in DC development and referred to as +32 kb enhancer (Durai et al., 2019). Thus, we proceeded to study lncIrf8 in detail.
ATAC-seq analysis revealed further details of the lncIrf8 region in CDP, pDC, cDC1 and cDC2 (Figure 1A, Figure 2A and Figure 1—figure supplement 1). In cDC1 the prominent ATAC-seq and IRF8 peaks mark the cDC1 specific +32 kb enhancer (Durai et al., 2019). In pDC the ATAC-seq peak is smaller and shifted further towards downstream but aligns well with the valley in the prominent H3K27ac peak. This ATAC-seq peak marks the lncIrf8 promoter and aligns with p300 (Durai et al., 2019) and H3K4me3 (Figure 2A and Figure 1—figure supplement 1). All this indicates that this chromatin region is open and transcriptionally active in pDC, enabling lncIrf8 transcription.

IncIrf8 promoter KO compromises pDC and cDC1 development in vitro.
(A) Genomic anatomy of lncIrf8 locus determined by 3’ and 5’ RACE PCR. Blue box, exon 2 and 3 (48 bp and 468 bp, respectively). The 1010 bp intron and polyA tail are shown. Data of RNA-seq, ATAC-seq, ChIP-seq of H3K27ac (enhancer mark) and H3K4me3 (active promoter mark, near TSS) are visualized by IGV browser for the indicated cell populations (pDC, all cDC, cDC1 and cDC2). Grey box, lncIrf8 promoter KO region; open box, cDC1 specific +32 kb enhancer by Durai et al., 2019. Irf8 +32 kb enhancer based on the H3K27ac enhancer mark is indicated with a purple line. Scale bar: 1 kb. (B) Gene expression of lncIrf8 and Irf8 in lncIrf8 promoter KO and control at day 0, 5, and 7 of Flt3L directed DC differentiation. Gene expression was determined by RT-qPCR and normalized to GAPDH. n=4. (C) Representative flow cytometry analysis of Flt3L directed DC differentiation of lncIrf8 promoter KO HoxB8 MPP and control (Lutz et al., 2022; Xu et al., 2022). pDC, all cDC, cDC1, and cDC2 were gated as in Figure 2—figure supplement 1E and are shown. Bar diagrams depict quantification of pDC, cDC1 and cDC2 normalized to living single cells on DC differentiation day 0, 3, 5, 7, and 9. n=6–7. (D) Representative phase-contrast microscopy images of lncIrf8 promoter KO HoxB8 MPP and control on day 7 of Flt3L directed DC differentiation. Scale bar: 200 μm. (E) Representative flow cytometry analysis of spontaneous DC differentiation of lncIrf8 promoter KO HoxB8 MPP and control with growth factors but without E2 (Lutz et al., 2022; Xu et al., 2022) at day 8. Gr1+ monocytes and CD11c+ DC are shown. Quantification of Gr1+ monocytes of living single cells on day 3, 6, 8, and 10 of spontaneous DC differentiation. n=6, lncIrf8 promoter KO; n=4, control. Empty gRNA vector or non-targeting gRNA vector HoxB8 MPP were used as controls. Data represent mean ± SD of at least three independent experiments with different HoxB8 MPP clones of lncIrf8 promoter KO and control without deletion. *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled.
Next, we determined the chromatin conformation of the Irf8 locus and the lncIrf8 region. We generated interaction profiles by nuclear-titrated (NuTi) Capture-C in MPP, CDP, pDC, cDC1, and cDC2 (Figure 1—figure supplement 2A and B) using Irf8 promoter as viewpoint. The Irf8 promoter shows multiple interactions with regions spanning up to ~100 kb upstream and downstream of Irf8 (Figure 1B and Figure 1—figure supplement 1). In pDC, the Irf8 promoter interactions are stronger with the upstream sequences than with downstream sequences (Figure 1C and Figure 1—figure supplement 2C). In cDC1 Irf8 promoter interactions are more confined to the regions downstream of Irf8 compared to MPP, CDP and pDC (Figure 1C and Figure 1—figure supplement 2C). This suggests that upstream and downstream sequences of Irf8 gene are involved in differentially regulating Irf8 expression and controlling the development of pDC and cDC1, respectively.
The CCCTC-binding factor (CTCF) is important for regulation of chromatin conformation through loop extrusion (Sanborn et al., 2015) and we therefore visualized CTCF binding sites in the Irf8 locus in DC (Garber et al., 2012). Interestingly, most of the Irf8 flanking enhancers (Durai et al., 2019; Grajales-Reyes et al., 2015; Murakami et al., 2021; Schönheit et al., 2013) are located within convergent CTCF binding sites upstream and downstream of the Irf8 gene (Figure 1B and Figure 1—figure supplement 1). There are also multiple interactions within this region without convergent CTCF binding sites, suggesting interactions with Irf8 promoter in a CTCF independent manner, such as by TF binding, active histone modifications and gene transcription (Figure 1B and Figure 1—figure supplement 1; Owens et al., 2022).
Surprisingly, in pDC H3K27ac at the lncIrf8 promoter is high, but this locus shows less interactions with the Irf8 promoter in pDC compared to CDP, cDC1 and cDC2 (Figure 1C and Figure 1—figure supplement 2C). In addition, in pDC the IRF8 protein occupancy at the lncIrf8 promoter is low and much higher in cDC (Figure 1A and Figure 1—figure supplement 1; Durai et al., 2019; Grajales-Reyes et al., 2015).
These observations warrant further studies and we thus proceeded to investigate the lncIrf8 locus in detail.
lncIrf8 promoter KO compromises pDC and cDC1 development
First, we annotated lncIrf8. Our de-novo transcript assembly of RNA-seq data revealed different isoforms of lncIrf8, with the most prominent isoform comprising exon 2 and 3 (Figure 1A, Figure 2A and Figure 1—figure supplement 1). Additionally, 3' end and 5' end RACE PCR confirmed the anatomy of this lncIrf8 isoform: two exons, one intron, and a polyA tail (Figure 2A). As expected lncIrf8 is not conserved across species (data not shown), which is in line with the general characteristics of lncRNA. Then second, we deleted 300 bp in the lncIrf8 promoter by CRISPR/Cas9 editing in conditionally immortalized HoxB8 MPP (Figure 2A and Figure 2—figure supplement 1A–D). The lncIrf8 promoter is located in the Irf8 +32 kb enhancer region and is in close proximity to the cDC1 specific +32 kb enhancer (Durai et al., 2019; Figure 2A and Figure 2—figure supplement 2). The 300 bp deletion comprises the H3K4me3 promoter mark and is confined to open chromatin identified by ATAC-seq and positioned in the valley of the H3K27ac mark (Figure 2A). Additionally, it contains binding sites for IRF8, PU.1, and BATF3 TF, which are important for DC development (Figure 2A and Figure 2—figure supplement 2B).
Generation of a precise deletion requires clonal cell populations, which is hardly achieved in somatic cells due to their limited lifespan. Therefore, we developed a Mx-Cas9-GFP system of conditionally immortalized HoxB8 MPP, which upon differentiation faithfully recapitulate DC development (Figure 2—figure supplement 1A, B; Xu et al., 2022). HoxB8 MPP were obtained from bone marrow of Mx-Cas9-GFP mice by infection with the estrogen (E2) inducible HoxB8-ER. These HoxB9 MPP exhibited an extended lifespan and robust clonogenic potential and differentiated into all DC subsets in vitro and in vivo (Xu et al., 2022). Infection of gRNA targeting lncIrf8 promoter in Mx-Cas9-GFP HoxB8 MPP and induction of Cas9 by interferon generated single-cell lncIrf8 promoter KO clones. Five out of 71 single-cell colonies with homozygous deletions were further studied and subjected to DC differentiation (Figure 2C–E, Figure 2—figure supplement 1C–G and Figure 2—figure supplement 3).
lncIrf8 promoter KO abolished lncIrf8 expression during DC differentiation compared to control without deletion (Figure 2B). Surprisingly, Irf8 expression was also severely compromised, which points to a cross-talk of the lncIrf8 promoter element with the Irf8 promoter. To determine whether lncIrf8 promoter KO also impacts DC subsets, CD11c+ DC, pDC and cDC subsets cDC1 and cDC2 were analyzed (Figure 2C and Figure 2—figure supplement 1E–G). Frequencies of pDC and cDC1 were severely reduced, while cDC2 were unaffected (Figure 2C). Accordingly, lncIrf8 promoter KO cultures contained mainly cDC2 and some undifferentiated cells and were more homogenous compared to control without deletion, which contain multiple DC subsets (Figure 2C and D, Figure 2—figure supplement 1F, G).
lncIrf8 promoter KO affected also the differentiation propensity of progenitors upon E2 withdrawal from MPP/CDP culture (Figure 1—figure supplement 2A and Figure 2—figure supplement 3). lncIrf8 promoter KO showed a marked increase in strongly adhesive cells compared to control (Figure 2—figure supplement 3B). lncIrf8 promoter KO cultures had higher frequencies of Gr1+ monocytes (Figure 2E, Figure 2—figure supplement 3I, J) and lower frequencies of all DC subsets CD11c+ DC, pDC, cDC1, and cDC2 (Figure 2—figure supplement 3C–G) compared to control without deletion.
The lncIrf8 promoter element is in close proximity to the cDC1 specific +32 kb enhancer previously described in mice by Durai et al., 2019 and thus we generated the cDC1 specific +32 kb enhancer KO following the same procedure as for the lncIrf8 promoter KO. Five out of 165 single-cell clones with homozygous deletions of cDC1 +32 kb enhancer were subjected to DC differentiation and further analyzed (Figure 2—figure supplement 4). Similar to lncIrf8 promoter KO, cDC1 specific +32 kb enhancer KO abolished lncIrf8 expression and also decreased Irf8 expression during DC differentiation (Figure 2—figure supplement 4D). The cDC1 +32 kb enhancer KO also compromised the frequency of cDC1 in Flt3L directed DC differentiation, while cDC2 were unaffected (Figure 2—figure supplement 4E). Frequencies of pDC were also compromised at day 7; however, this was not statistically significant. These observations are in line with previous studies in mice that cDC1 +32 kb enhancer KO compromised cDC1 differentiation and left pDC and cDC2 largely unaffected (Durai et al., 2019; Murakami et al., 2021). In addition, cDC1 +32 kb enhancer KO cultures had higher frequencies of Gr1+ monocytes upon spontaneous DC differentiation by withdrawal of E2 (Figure 2—figure supplement 4F) and thus showed a similar phenotype as the lncIrf8 promoter KO upon spontaneous DC differentiation (Figure 2E).
Given the novel phenotype of the lncIrf8 promoter KO, we proceeded to investigate the impact of the lncIrf8 promoter KO on DC differentiation in vivo in mice. We transplanted CD45.2 lncIrf8 promoter KO and CD45.2 control HoxB8 MPP into irradiated CD45.1 recipient mice (Figure 3—figure supplement 1A). DC in bone marrow and spleen were analyzed by flow cytometry on day 7 and 14 after cell transplantation (Figure 3, Figure 3—figure supplement 1A, B). In bone marrow, lncIrf8 promoter KO cells mostly differentiated into Gr1+ monocytes, and lower frequencies of all DC subsets were observed on day 7 for lncIrf8 promoter KO cells compared to control (Figure 3A–F). In spleen, frequencies of cell populations from lncIrf8 promoter KO and control were similar to bone marrow, including lower frequencies of all DC subsets for lncIrf8 promoter KO (Figure 3G–L). CD45.2 donor HoxB8 cells were largely lost at day 14 after cell transplantation (Figure 3B–F and Figure 3H–L).

lncIrf8 promoter KO comprises pDC and cDC1 development in vivo upon cell transplantation.
(A) Representative flow cytometry analysis of CD45.2 lncIrf8 promoter KO and control HoxB8 MPP in BM at day 7 after cell transplantation (for details see Figure 3—figure supplement 1A, B). Donor cell populations were gated from 7-AAD- CD45.2+ Lin- cells and Gr1+ monocytes, pDC, cDC1 and cDC2 are shown. (B–F) Quantification of Gr1+ monocytes, MHCII+ CD11c+ DC, pDC, cDC1, and cDC2 of living single cells in BM on day 7 and 14 after cell transplantation (n=3–4). (G) Representative flow cytometry analysis of lncIrf8 promoter KO and control HoxB8 MPP in spleen at day 7 after cell transplantation. Gating was as in panel (A). (H–L) Quantification of Gr1+ monocytes, MHCII+ CD11c+ DC, pDC, cDC1 and cDC2 on day 7 and 14 after cell transplantation (n=3–4). Data represent mean ± SD from 3 to 4 mice. *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled.
Thus, lncIrf8 promoter KO compromised pDC and cDC1 development both in vitro and in vivo.
lncIrf8 acts as an indicator of Irf8 +32 kb enhancer activity in pDC
Knockout of lncIrf8 promoter and thus abolishment of lncIrf8 expression severely diminished pDC and cDC1 development in vitro and in vivo. The lncIrf8 promoter is located within Irf8 +32 kb enhancer (Durai et al., 2019) and thus it was important to determine whether lncIrf8 itself plays a role in regulating pDC and cDC1 development. To address this question, we (i) overexpressed lncIrf8 in wild-type MPP and (ii) re-expressed lncIrf8 in lncIrf8 promoter KO MPP and monitored its impact on DC development (Figure 4 and Figure 4—figure supplement 1).

lncIrf8 overexpression leaves pDC and cDC1 development unaffected.
(A and B) Schematic representation of lncIrf8 overexpression in WT HoxB8 MPP and of plncIrf8-pA (lncIrf8 overexpression) and pGFP-pA (control) plasmids. A polyA signal AATAAA for transcription termination was inserted at the 3’ end of lncIrf8 and GFP. (C) Gene expression of lncIrf8 and Irf8 in plncIrf8-pA and pGFP-pA HoxB8 MPP on day 0, 3, 5, and 7 of Flt3L directed DC differentiation (n=4–5). Gene expression was by RT-qPCR and normalized to GAPDH. (D) Representative flow cytometry of DC subsets at day 5 and 9 of Flt3L directed DC differentiation of plncIrf8-pA and pGFP-pA HoxB8 MPP. Quantification of pDC and cDC1 of living single cells on Flt3L directed DC differentiation day 0, 3, 5, 7, and 9 (n=5) is shown. Gating for pDC and cDC1 was as in Figure 2—figure supplement 1E. (E) Heatmap representation of DC subsets of panel (D) at day 0, 3, 5, 7, and 9 of DC differentiation. Red, high frequency; white, intermediate frequency and blue, low frequency. Data represent mean ± SD of at least three independent experiments with different HoxB8 MPP clones of plncIrf8-pA and pGFP-pA. *p<0.05, **p<0.01, ***p<0.001, multiple t-tests. Data that have no difference (p>0.05) are not labeled.
lncIrf8 cDNA was cloned into a polyA lentivirus vector. An ‘AATAAA’ stop signal (Alvarez-Dominguez et al., 2015) was inserted at the 3’ end of lncIrf8 to avoid longer transcripts than lncIrf8 (plncIrf8-pA, Figure 4B). The respective pGFP-pA vector was used as control. lncIrf8 overexpressing single-cell clones were generated by limiting dilution (Figure 4—figure supplement 1A), expanded and subjected to DC differentiation. As expected lncIrf8 expression was markedly increased in plncIrf8-pA infected cells compared to control, while there were no significant differences in Irf8 expression between the two groups during DC differentiation (Figure 4C). Further, there were no differences in the frequencies of pDC, cDC1, and cDC2 between plncIrf8-pA infected cells and controls (Figure 4D and E, Figure 4—figure supplement 1B–D), indicating that lncIrf8 overexpression has no effect on Irf8 expression and DC differentiation.
To further extend this observation we performed a lncIrf8 rescue in lncIrf8 promoter KO MPP. lncIrf8 was re-expressed in lncIrf8 promoter KO MPP by lentiviral vector and cells were subjected to DC differentiation (Figure 4—figure supplement 1E). lncIrf8 RNA was effectively expressed and cells differentiated in response to Flt3L (Figure 4—figure supplement 1F). Yet frequencies of pDC and cDC1 were very low to absent and not rescued by lncIrf8 expression (Figure 4—figure supplement 1G, H).
In a nutshell, lncIrf8 overexpression and rescue had no effects on pDC and cDC1 development. This strongly suggests that lncIrf8 has no activity on its own in DC differentiation but rather acts as an indicator for the activation state of sequences within the Irf8 +32 kb enhancer. In addition, the lncIrf8 promoter comprises a sequence element with impact on pDC and cDC1 development.
Activation of lncIrf8 promoter promotes cDC1 development
Next, we proceeded to study the impact of the sequence element within lncIrf8 promoter on Irf8 expression and DC differentiation using CRISPR activation by dCas9-VP64 (Figure 5A and Figure 5—figure supplement 1). dCas9-VP64 is a mutated Cas9 deficient in nuclease activity, which is fused to the VP64 effector domain and confers gene activation. Targeting of dCas9-VP64 to the lncIrf8 promoter was achieved with specific gRNAs (Figure 5—figure supplement 1I). We also included targeting dCas9-VP64 to the Irf8 promoter to study the interplay with the lncIrf8 promoter. FACS sorted HoxB8 MPP expressing dCas9-VP64 and gRNA were subjected to DC differentiation and analyzed for lncIrf8 and Irf8 expression and DC subset composition (Figure 5B–E and Figure 5—figure supplement 1A–E).

Activation of lncIrf8 promoter promotes cDC1 development.
(A) Schematic representation of lncIrf8 and Irf8 promoter activation (top and middle, respectively) by CRISPR activation with dCas9-VP64. gRNAs were positioned upstream of lncIrf8 and Irf8 TSS for gene activation. dCas9-VP64 cells with non-targeting gRNA were used as control (bottom). Green and purple wavy lines represent Irf8 and lncIrf8 RNA, respectively. The number of wavy lines indicates levels of gene transcription determined by RT-qPCR in (C). Different length of blue arrows represents the frequencies of pDC and cDC1 according to panel B, D and E. Nc, No change; Ctrl, Control. (B) Representative flow cytometry analysis of CRISPR activation targeting the lncIrf8 and Irf8 promoters at day 7 and 9 of Flt3L directed DC differentiation. Two non-targeting gRNAs were used as controls and one representative non-targeting gRNA is shown (Non-targeting-VP64). Top row, CD11b+ B220- cDC and CD11b- B220+ pDC at day 7 of Flt3L directed DC differentiation; bottom row, CD11blow/- XCR1+ cDC1 and CD11b+ XCR1- cDC2 at day 9 of DC differentiation. For gating strategy see Figure 5—figure supplement 1B. (C) Gene expression of lncIrf8 and Irf8 in lncIrf8-VP64, Irf8-VP64 and non-targeting-VP64 HoxB8 MPP on day 0, 5, 7, and 9 of Flt3L directed DC differentiation (n=3). Gene expression analysis was by RT-qPCR and data are normalized to GAPDH. Data represent mean ± SD of three independent experiments. *p<0.05, **p<0.01, ****p<0.0001, two-way ANOVA, Tukey’s multiple comparisons test. Data that have no difference (p>0.05) are not labeled. (D and E) Quantification of pDC and cDC1 in percent of living single cells as in panel (B) on various days of Flt3L directed DC differentiation (n=3). Non-targeting-VP64 refers to both non-targeting-VP64 controls (n=6). Data represent mean ± SD of 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA, Tukey’s multiple comparisons test. Data that have no difference (p>0.05) are not labeled.
Activation of the lncIrf8 promoter by dCas9-VP64 caused a massive increase of lncIrf8 expression at DC differentiation day 0 (Figure 5C). The activation of the lncIrf8 promoter led also to Irf8 upregulations at DC differentiation day 5, 7, and 9 compared to non-targeting control (Figure 5A and C). This demonstrates the positive impact of the lncIrf8 promoter element on Irf8 expression during DC differentiation and is in accord with the physical interaction of lncIrf8 and Irf8 promoters by Capture-C (Figure 1B and Figure 1—figure supplement 1).
Intriguingly, in lncIrf8-VP64 cells lncIrf8 expression was downregulated at DC differentiation day 5, 7 and 9, while Irf8 expression was upregulated (Figure 5A and C). This indicates a repressive effect of IRF8 on lncIrf8 promoter and is in accord with IRF8 binding to the lncIrf8 region by ChIP-seq (Figure 1 and Figure 1—figure supplement 1). This observation suggests a negative feedback loop of IRF8 on the lncIrf8 promoter during DC differentiation (Figure 5A and C).
Activation of Irf8 promoter by dCas9-VP64 increased Irf8 expression at DC differentiation day 0, 5, 7, and 9, while expression of lncIrf8 was unaffected compared to non-targeting control (Figure 5A and C). As expected Irf8 promoter activation led to higher pDC frequencies (Figure 5B and D), and also increased cDC1 frequencies compared to the non-targeting controls (Figure 5B and E). Importantly, lncIrf8 promoter activation by dCas9-VP64 also increased cDC1 frequencies and this was particular prominent at day 9 of DC differentiation (Figure 5B and E). Frequencies of cDC2 were decreased at day 9 and other populations, including pDC, remained unchanged (Figure 5B and D, Figure 5—figure supplement 1C–E).
Taken together our CRISPR activation of the lncIrf8 and Irf8 promoters by dCas9-VP64 suggest a negative feedback loop of Irf8 for pDC and cDC1 development.
Negative feedback regulation of lncIrf8 and Irf8 promoters controls DC differentiation
To directly investigate the negative feedback loop of Irf8 regulation, we repressed lncIrf8 and Irf8 promoters by targeted repression with dCas9-KRAB and analyzed the DC subsets during DC differentiation (Figure 6A–F and Figure 5—figure supplement 1A, B, F-H). dCas9-KRAB is a nuclease deficient Cas9 fused to the KRAB effector domain, which confers gene repression when positioned with specific gRNA (Figure 5—figure supplement 1I).

Repression of lncIrf8 promoter compromises pDC and cDC1 development.
(A) Schematic representation of lncIrf8 and Irf8 promoter repression (top and middle, respectively) by CRISPR interference with dCas9-KRAB. gRNAs were positioned downstream of lncIrf8 and Irf8 TSS to block gene transcription. dCas9-KRAB cells with non-targeting gRNA were used as control (bottom). Green and purple wavy lines represent Irf8 and lncIrf8 RNA, respectively. The number of wavy lines indicates levels of gene transcription determined by RT-qPCR in (C). (B) Representative flow cytometry analysis of CRISPR interference targeting the lncIrf8 and Irf8 promoters at day 5 and 7 of Flt3L directed DC differentiation. Two non-targeting gRNAs were used as controls and one representative non-targeting gRNA is shown (Non-targeting-KRAB). cDC and pDC at day 5 and cDC1 and cDC2 at day 7 of Flt3L directed DC differentiation are shown similar to Figure 5B. (C) Gene expression of lncIrf8 and Irf8 in lncIrf8-KRAB, Irf8-KRAB and non-targeting-KRAB HoxB8 MPP on day 0, 3, 5, and 7 of Flt3L directed DC differentiation (n=3). Gene expression analysis was by RT-qPCR and data are normalized to GAPDH. Data represent mean ± SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA, Tukey’s multiple comparisons test. Data that have no difference (p>0.05) are not labeled. (D and E) Quantification of pDC and cDC1 in percent of living single cells as in panel (B) on various days of Flt3L directed DC differentiation (n=3). Non-targeting-KRAB refers to both non-targeting-KRAB controls (n=6). Data represent mean ± SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA, Tukey’s multiple comparisons test. Data that have no difference (p>0.05) are not labeled. (F) Representative phase-contrast microscopy image of lncIrf8-KRAB, Irf8-KRAB and non-targeting-KRAB on day 7 of Flt3L directed DC differentiation. Scale bar: 200 μm.
Targeted repression of the Irf8 promoter decreased Irf8 expression as expected but massively increased lncIrf8 expression compared to non-targeting control (Figure 6A and C). This is very much in line with Irf8 impacting lncIrf8 expression by a negative feedback loop. Positioning the dCas9-KRAB repressor in the IncIRF8 promoter led to downregulation of both Irf8 and lncIrf8 expression, which confirms the lncIrf8 promoter element acting on the Irf8 promoter (Figure 6A and C).
Interestingly, targeted repression of the Irf8 promoter severely compromised development of all DC subsets, including pDC, cDC1, and cDC2, compared to non-targeting controls and yielded CD11c+ cells with progenitor-like spherical morphology (Figure 6B and D–F, Figure 5—figure supplement 1F–H). This reemphasizes the pivotal role of IRF8 for DC development known from studies on Irf8 knockout mice (Murakami et al., 2021; Schiavoni et al., 2002; Sichien et al., 2016; Tsujimura et al., 2003). In addition, targeted repression of the lncIrf8 promoter (lncIrf8-KRAB) also compromised pDC and cDC1 development (Figure 6B, D and E). This result is very similar to the lncIrf8 promoter KO analyzed above (Figure 2A–C).
All these findings support a model of Irf8 regulating its own expression by a negative feedback loop acting on the Irf8 +32 kb enhancer to limit Irf8 autoactivation (Figure 7). This regulatory Irf8 +32 kb enhancer element is marked by lncIrf8. Irf8 expression starts in CDP and further increases in pDC and cDC1, with particularly high expression in pDC (Figure 1A, Figure 7A and B, Figure 1—figure supplement 1). The increase in Irf8 expression is proposed to be due to an increase in the interactions of the Irf8 promoter with upstream and downstream sequences. In pDC, the Irf8 promoter-enhancer interactions are more with upstream chromatin regions (Figure 1C and Figure 7A), which relates to high Irf8 expression. In cDC1, the Irf8 promoter interactions are stronger with the regions downstream of Irf8 (Figure 1C and Figure 7A) and the Irf8 +32 kb enhancer marked by lncIrf8 confers transcriptional repression.

Negative feedback loop of Irf8 through +32 kb enhancer governs DC differentiation.
(A) Schematic representation of Irf8 gene regulation during DC differentiation. Irf8 transcription is induced at the CDP stage by its flanking enhancers, including the +32 kb enhancer, and is further increased in pDC and cDC1 (green wavy lines; the number of wavy lines indicates levels of gene transcription as in Figure 5A and Figure 6A). The increase in Irf8 expression is due to an increase in the interactions of the Irf8 promoter with upstream and downstream sequences. Irf8 promoter interactions with upstream sequences are stronger in pDC and Irf8 promoter interactions with downstream sequences are stronger in cDC1. In pDC the +32 kb enhancer marked by lncIrf8 is less repressed by IRF8 repressor complex compared to cDC1, resulting in particularly high Irf8 expression and lncIrf8 transcription in pDC (purple wavy lines). This negative feedback inhibition of IRF8 on the +32 kb enhancer allows Irf8 to regulate its own expression and thus DC differentiation. (B) Negative feedback regulation of Irf8 through the +32 kb enhancer in cDC1 and pDC specification as described in panel (A). The RNA-seq and IRF8 ChIP-seq data are shown as in Figure 1A, Figure 1—figure supplement 1 and Figure 2—figure supplement 2A. (C) Recapitulating Irf8 and lncIrf8 transcription by repression and activation of the Irf8 +32 kb enhancer in HoxB8 MPP by targeted dCas9-KRAB and dCas9-VP64, respectively. Green and purple wavy lines represent gene expression as described in Figure 5A and Figure 6A.
In our model, we propose an IRF8 repressor complex that differentially acts on the Irf8 +32 kb enhancer element in a DC subset specific manner to limit Irf8 autoactivation. In cDC1 the +32 kb enhancer is repressed by the IRF8 repressor complex through negative feedback inhibition (prominent IRF8 binding in cDC by ChIP-seq; Figure 7A and B), which limits Irf8 autoactivation and expression. Conversely, in pDC there is less IRF8 repressor complex binding to the +32 kb enhancer, which results in high Irf8 and lncIrf8 transcription (Figure 7A and B). Recapitulating the IRF8 repressor complex with dCas9-KRAB and targeting lncIrf8 promoter in the +32 kb enhancer reduced Irf8 expression (Figure 7C). Conversely, activation of the +32 kb enhancer boosted lncIrf8 and Irf8 expression (Figure 7C). Thus, an intricate feedback loop of IRF8 on the +32 kb enhancer orchestrates Irf8 expression and thus DC differentiation.
Discussion
Hematopoiesis is a particularly well studied stem cell system and therefore provides an excellent model for studying TF in lineage commitment and cell differentiation, and the molecular principles involved (Belz and Nutt, 2012; Graf and Enver, 2009; Laurenti and Göttgens, 2018; Nutt and Chopin, 2020). This work revealed a previously unrecognized negative feedback loop of Irf8 in DC differentiation and shows how Irf8 autoactivation is controlled and ultimately limited. IRF8 is crucial for DC lineage specification both in humans and mice (Anderson et al., 2021; Belz and Nutt, 2012; Cabeza-Cabrerizo et al., 2021; Cytlak et al., 2020; Kurotaki et al., 2019; Nutt and Chopin, 2020). Irf8 is upregulated by autoactivation via the +32 kb enhancer (Grajales-Reyes et al., 2015; Lin et al., 2015). However, how Irf8 expression is controlled at late stages of DC differentiation and eventually limited is not known. Here we demonstrate that Irf8 expression is limited by a negative feedback loop via a sequence element marked by lncIrf8 in the Irf8 +32 kb enhancer.
Irf8 expression in hematopoiesis is regulated by its flanking enhancers, which determine lineage specification and DC subset development (Bagadia et al., 2019; Durai et al., 2019; Grajales-Reyes et al., 2015; Murakami et al., 2021; Schönheit et al., 2013). Frequently, enhancers are identified by ATAC-seq and ChIP-seq (Durai et al., 2019; Murakami et al., 2021), and here we embarked on a different approach and searched for eRNA and enhancer-associated lncRNA by RNA-seq. We identified a novel pDC-specific lncRNA (lncIrf8) transcribed from the Irf8 +32 kb enhancer. lncIrf8 itself lacks activities in DC differentiation but a lncIrf8 promoter element is crucial for pDC and cDC1 development. Upon deletion of this lncIrf8 promoter element pDC and cDC1 development was severely compromised, demonstrating that this sequence is important for both pDC and cDC1 development.
We propose a model where Irf8 expression during DC differentiation is in a first step initiated and activated through flanking enhancers, including the +32 kb enhancer by autoactivation (Grajales-Reyes et al., 2015; Lin et al., 2015; Figure 7). In a second step, the lncIrf8 promoter element confers feedback inhibition, which limits Irf8 expression. This feedback inhibition is different for pDC and cDC1, both of which express high levels of Irf8 (Bornstein et al., 2014; Grajales-Reyes et al., 2015; Lin et al., 2015). In cDC1, Irf8 expression is attributed to Irf8 autoactivation through the +32 kb enhancer driven by the BATF3-IRF8 complex (Durai et al., 2019; Grajales-Reyes et al., 2015). In pDC, Irf8 expression is even higher than in cDC1 (Bornstein et al., 2014; Lin et al., 2015), which we propose is due to less feedback inhibition at late stages of DC differentiation.
A candidate for mediating Irf8 feedback inhibition is IRF8 itself, since IRF8 works as transcriptional activator or repressor, depending on context. IRF8 activator or repressor function depends largely on the heterodimers (or even heterotrimers) with partner TF that bind to specific DNA sequences (Chang et al., 2018; Huang et al., 2016; Humblin et al., 2017; Tamura et al., 2015; Yoon et al., 2014). Modifications on IRF8 protein, such as phosphorylation and small molecule conjugation, also alter IRF8 activity (Chang et al., 2012; Konieczna et al., 2008). Potential IRF8 heterodimer partners, to form repressor complexes, are ETV6 or IRF2 (Huang et al., 2016; Humblin et al., 2017; Lau et al., 2018). The IRF8 repressor complex is proposed to bind to the +32 kb enhancer in cDC but not in pDC. This notion is in line with a prominent IRF8 signal at the +32 kb enhancer in cDC, which is absent in pDC (Durai et al., 2019; Grajales-Reyes et al., 2015; Figure 1A, Figure 7A and B, Figure 1—figure supplement 1 and Figure 2—figure supplement 2A).
Further support of our model stems from our CRIPSR activation/interference experiments (Figure 7C). CRIPSR activation of Irf8 promoter by dCas9-VP64 mimics Irf8 up-regulation during DC differentiation and causes an increase in pDC and cDC1 (Figure 5). CRIPSR interference of lncIrf8 promoter by dCas9-KRAB recapitulates transcriptional repressor binding to the +32 kb enhancer and causes Irf8 promoter inhibition (Figure 6).
We extended our study to delineate the chromatin conformation of the Irf8 promoter with flanking sequences by Capture-C. The Irf8 promoter was found to interact with its flanking enhancers already at the CDP stage and then interactions with specific upstream and downstream sequences are proposed to guide pDC and cDC specification, respectively. This is in accord with previous studies where some of these Irf8 flanking enhancers were required to maintain high levels of Irf8 expression (Anderson et al., 2021; Murakami et al., 2021).
We demonstrate that deletion of the lncIrf8 promoter element severely decreased Irf8 expression and abolished both pDC and cDC1 development in vitro and in vivo upon cell transplantation. These results are very similar to a previous study on cDC1 specific +32 kb enhancer knockout mice, which demonstrates the impact of +32 kb enhancer for cDC1 development in vivo (Durai et al., 2019). The lncIrf8 promoter is located in close proximity to the cDC1 specific +32 kb enhancer and thus can be expected to have overlapping functions. Indeed, deletion of the cDC1 +32 kb enhancer in HoxB8 MPP showed some similar activities on DC differentiation as the deletion of the lncIrf8 promoter element, including regulation of lncIrf8 and Irf8 expression, but mainly affected cDC1 differentiation. These observations indicate that the lncIrf8 promoter element has further functions compared to the cDC1 specific +32 kb enhancer, for example for pDC development, and the underlying mechanisms warrant further investigation.
eRNA and enhancer-associated lncRNA are indicative of enhancer activity, however whether the process of their transcription, the transcripts themselves, or both are functionally linked to enhancer activity, remains unclear (Sartorelli and Lauberth, 2020; Statello et al., 2021). Previous studies revealed that some eRNA and enhancer-associated lncRNA are indeed functionally connected with expression of the respective target genes (Sartorelli and Lauberth, 2020; Statello et al., 2021). Here, we found no apparent activity of lncIrf8 on its own in pDC and cDC1 development, as demonstrated by lncIrf8 overexpression and rescue experiments. lncIrf8 appears to rather serve as an indicator for Irf8 +32 kb enhancer activity. However, lncIrf8 might have additional functions in DC biology, which are not revealed in the current study and remain to be identified.
In conclusion, by analyzing the gene expression and epigenetic profiles of the Irf8 locus, we identified an enhancer element marked by lncIrf8 that regulates Irf8 and controls DC differentiation through a negative feedback loop. Our results suggest that Irf8 regulates itself by its flanking enhancers in DC fate determination: First, Irf8 induces its expression by autoactivation via flanking enhancers, including the Irf8 +32 kb enhancer, to initiate DC differentiation, and second limits its expression at late stages via the lncIrf8 promoter element within the same +32 kb enhancer by feedback inhibition. This molecular principle of feedback inhibition is expected to also apply to other TF and cell differentiation systems.
Materials and methods
Mice
Wild type C57BL/6, Mx-Cas9-GFP knock-in mice (Kühn et al., 1995; Platt et al., 2014; Xu et al., 2022), and CD45.1 recipient C57BL/6 mice were used in this study. Mice were kept under specific pathogen-free conditions in the central animal facility of RWTH Aachen University Hospital, Aachen, Germany. All the animal experiments were approved by the local authorities of the German State North Rhine-Westphalia, Germany according to the German animal protection law (reference number: 81–02.04.2018 .A228).
Cell culture
Request a detailed protocolMultipotent progenitors (MPP) were obtained from mouse bone marrow and expanded in vitro with a two-step protocol as described in Felker et al., 2010. Conditionally immortalized HoxB8 MPP were generated by retrovirus infection of bone marrow cells from wild-type or Mx-Cas9-GFP knock-in mice with an estrogen (E2) inducible HoxB8 estrogen receptor (ER) fusion gene (HoxB8-ER) (Redecke et al., 2013; Xu et al., 2022). MPP were grown in RPMI 1640 medium with 10% FCS (Gibco, 10270106), 100 U/ml penicillin/streptomycin, 2 mM L-glutamine and 50 μM β-ME with a four-cytokine cocktail of SCF, Flt3 ligand (Flt3L), IGF-1, and IL-6/soluble IL-6 receptor fusion protein (hyper-IL-6) as before (referred to as MPP growth medium) (Felker et al., 2010; Lutz et al., 2022; Xu et al., 2022) (Appendix 1-key resources table). E2 (1 µM) was added to activate HoxB8-ER and to maintain the conditionally immortalized state of HoxB8 MPP. Cell density was adjusted to 1.5 million cells/ml every day. HEK293T cells for retrovirus and lentivirus production were grown in DMEM supplemented with 10% FCS (PAA, A01125-499), 100 U/ml penicillin/streptomycin, 2 mM L-glutamine (Appendix 1-key resources table).
In vitro DC differentiation with HoxB8 MPP
Request a detailed protocolHoxB8 MPP were differentiated into DC using a two-step protocol modified from Felker et al., 2010 and described in Lutz et al., 2022; Xu et al., 2022. In brief, 0.75 million cells/ml were grown in MPP growth medium with 50 ng/ml Flt3L (Peprotech, 300–19) and reduced E2 (0.01 μM) for two days and cell density was kept to 0.75 million cells/ml. To induce DC differentiation, HoxB8 MPP were then washed with PBS to remove cytokines and E2, and cultured in RPMI 1640 medium supplemented with FCS, penicillin/streptomycin, L-glutamine, β-ME (same concentrations as above), and Flt3L (50 ng/ml, Peprotech) (referred to as DC differentiation day 0). Partial medium changes were performed on differentiation day 3 and 6. Spontaneous DC differentiation of HoxB8 MPP was achieved simply by removing E2 from growth medium (SCF, Flt3L, IGF1 and hyper-IL6), and culturing the cells at 1.5 million cells/ml (Lutz et al., 2022; Xu et al., 2022).
Nuclear-Titrated (NuTi) Capture-C
Request a detailed protocolWild-type bone marrow cell-derived MPP, CDP, cDC1, cDC2, and pDC were generated in vitro with the two-step protocol as described in Felker et al., 2010. Cell populations were sorted by BD FACSAria IIu or BD FACSMelody, MPP are Gr1- CD117hi CD135low/-, CDP are Gr1- CD117int CD135+ CD115+, cDC1 are CD11c+ CD11blow/- XCR1+, cDC2 are CD11c+ CD11b+ XCR1-, pDC are CD11c+ CD11b- B220+.
The chromatin conformation capture (3 C) library preparation protocol used in this study was modified from Downes et al., 2021; Downes et al., 2022 with the reagents listed in Appendix 1-key resources table. MPP, CDP, cDC1, cDC2 and pDC were fixed with formaldehyde (final concentration 2%) and subjected to nuclear isolation according to the protocol in Downes et al., 2022; Li et al., 2019. Nuclei (15–20 million per sample) were digested with DpnII and DNA fragments were ligated by T4 DNA HC ligase. DNA was extracted and purified with Phenol-Chloroform-Isoamyl alcohol (PCI, 25:24:1). DpnII digestion efficiency was determined by SYBR qPCR with the primers listed in Appendix 1-key resources table and the quality of 3 C libraries was investigated by agarose gel (1%) electrophoresis; 3 C samples were used only if the DpnII digestion efficiency was more than 70%.
For Irf8 promoter viewpoint 2 oligonucleotide probes targeting Irf8 promoter were designed with the design tool Oligo (https://oligo.readthedocs.io/en/latest/index.html). Oligonucleotide probes are positioned adjacent to the DpnII cut sites on a restriction fragment spanning the Irf8 promoter (chr8:123,259,948–123,260,530) and 70 bp ssDNA biotinylated oligonucleotides were synthetized by Sigma-Aldrich (listed in Appendix 1-key resources table).
To enrich for fragments containing ligation events with Irf8 promoter viewpoint, NuTi Capture-C was performed as previously described (Downes et al., 2021; Downes et al., 2022). Briefly, the 3 C libraries prepared from MPP, CDP, cDC1, cDC2 and pDC were sonicated using Covaris S220 to an average size of ~200 bp using standard settings recommended by the manufacturer (time: 180 s, duty cycle: 10%, peak incident power: 175 W, cycles per burst: 200, temperature: 5–9°C). End repair was performed with the NEBNext Ultra II kit (New England Biolabs, E7645S) using 2 µg of sonicated 3 C library in duplicate for each sample. Illumina NEBNext Indices (New England Biolabs, E7500S, and E7335S) were added with a total of 6 cycles of amplification to allow for multiplexing. After this step, duplicates were pooled to increase sample complexity. We enriched samples as per NuTi Capture-C protocol, with two capture rounds in a multiplexed reaction, using 2 µg of each indexed sample as an input for the first capture. The hybridization with biotinylated probes was performed with the KAPA Hyper Capture Reagent Kit (Roche, 9075828001). Each ssDNA biotinylated probe was used at a concentration of 2.9 nM, with a final pool concentration of 5.8 nM, and 4.5 µl of the pooled oligonucleotides were used per sample. Captured DNA was pulled-down with M-270 Streptavidin Dynabeads (Invitrogen, 65305), washed and amplified off the beads with a total of PCR 14 cycles. The DNA obtained after the first capture was used as an input in the second capture round. The experiments were performed for the first and the second biological replicate separately, and then sequenced with NextSeq 550 Illumina System with 300 paired-end or 150 paired-end, respectively.
NuTi Capture-C data analysis
Request a detailed protocolThe Capture-C data was analyzed with CapCruncher (v0.1.1a1) pipeline (https://github.com/sims-lab/CapCruncher; sims-lab, 2022) in capture mode (Downes et al., 2022). The reads were aligned to the mm9 genome assembly with Bowtie2 (Langmead and Salzberg, 2012), with specific options: -p6--very-sensitive. Viewpoint coordinates used were: chr8:123,259,948–123,260,530, 1000 bp around viewpoint was excluded. The data were normalized to ~300 kb region around the viewpoint (chr8:123,132,865–123,433,117).
The Capture-C profiles in Figure 1 and Figure 1—figure supplement 1 show the mean number of unique interactions in two biological replicates, normalized to ~300 kb region around the viewpoint. Differential tracks were created by subtraction of the mean normalized tracks.
ATAC-seq, ChIP-seq, RNA-seq, and scRNA-seq
Request a detailed protocolATAC-seq analysis of MPP, CDP, cDC1, cDC2, and pDC was performed by Omni-ATAC-seq (Corces et al., 2017) with minor modifications as described in Li et al., 2019. RNA-seq analysis and ChIP-seq analysis was done as previously described (Allhoff et al., 2016; Lin et al., 2015).
To determin lncRNA expression in mouse ex-vivo pDC, scRNA-seq data of BM and splenic pDC were downloaded from GSE114313 (Rodrigues et al., 2018) and BAM files were converted back into FASTQ files using bamtofastq (https://support.10xgenomics.com/docs/bamtofastq). For visualization of lncRNA expression, we created a custom reference genome of mm9 by following the tutorial: available here. Cellranger pipeline was then processed to generate an expression count matrix of pDC. We next used scanpy (Wolf et al., 2018) to analyze the scRNA-seq data. We filtered cells based on the number of detected genes (<200 or>3500), and the proportion of mitochondrial genes (>10%). After data quality control, we retained 7044 cells for splenic pDC and 8158 cells for BM pDC, respectively. We then log-normalized the count matrix using a scaling factor of 10,000 and selected the top 3000 highly variable genes, which were used for dimensionality reduction based on principle component analysis. The cells were visualized using uniform manifold approximation and projection (UMAP) (Becht et al., 2019).
Plasmids
psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) for lentiviral packaging and envelope expressing plasmids were kind gifts from Didier Trono, EPFL, Lausanne, Switzerland. The gRNA expressing vector pLKO5.sgRNA.EFS.tRFP (Addgene #57823) and pLKO5.sgRNA.EFS.tRFP657 (Addgene #57824) were kind gifts from Benjamin Ebert, Harvard Medical School, Boston, USA (Heckl et al., 2014).
For lncIrf8 overexpression and rescue, lncIrf8 cDNA was introduced into polyA containing lentivirus vector pGFP-pA to generate plncIrf8-pA. pGFP-pA was constructed from pCIG3 (Addgene #78264; Caviness et al., 2014) by Gibson Assembly (New England Biolabs, E5510S; Gibson et al., 2009). In brief, the WPRE element was removed and a polyA signal ‘AATAAA’ was inserted at the 3’ end of GFP to construct pGFP-pA. lncIrf8 cDNA was sub-cloned into pGFP-pA using XhoI and SalI with the primers shown in Appendix 1-key resources table to obtain plncIrf8-pA. CRISPR activation and repression of lncIrf8 and Irf8 promoters were achieved by dCAS9-VP64_GFP (Addgene #61422) (Konermann et al., 2015) and pTet-KRAB-dCas9-GFP (Xu et al., 2022), respectively.
Lentivirus infection
Request a detailed protocolgRNAs, lncIrf8, dCas9-VP64-GFP, and KRAB-dCas9-GFP were delivered into HoxB8 MPP by lentiviral infection. Briefly, lentiviral particles were produced by calcium phosphate transfection (Graham and van der Eb, 1973) of HEK293T cells with psPAX2, pMD2.G, and the lentiviral transfer vector. At 48 and 72 hours after HEK293T cell transfection, supernatant containing virus particles was collected and concentrated using chondroitin sulfate sodium salt (CSS) and polybrene precipitation (Landazuri et al., 2007; Appendix 1-key resources table). HoxB8 MPP were infected twice with concentrated virus and subjected to Ficoll (Pancoll) purification to remove precipitate and dead cells.
Genetically modified HoxB8 MPP cell lines
lncIrf8 promoter and cDC1 specific +32 kb enhancer KO:
Request a detailed protocolMx-Cas9-GFP HoxB8 MPP were used to generate lncIrf8 promoter and cDC1 specific +32 kb enhancer knockout (KO) HoxB8 MPP by CRISPR/Cas9. Briefly, pairs of gRNAs each for lncIrf8 promoter KO and cDC1 specific +32 kb enhancer KO (Appendix 1-key resources table) were designed with the IDT online gRNA design tool. For cDC1 specific +32 kb enhancer KO we additionally used the same gRNA sequences from Durai et al., 2019 listed in Appendix 1-key resources table. gRNAs were cloned into pLKO5.sgRNA.EFS.tRFP and pLKO5.sgRNA.EFS.tRFP657 vectors (Heckl et al., 2014) using BsmBI-v2 (Appendix 1-key resources table), respectively.
One 10 cm dish (Bio-One) with 1.8 million HEK293T cells (70%–80% confluency) in 10 ml DMEM plus supplements (see above) was used to produce gRNA expressing lentivirus particles. HEK293T cells were transfected with 10 μg gRNA vector, 7.5 μg psPAX2, and 2.5 μg pMD2.G per gRNA by calcium phosphate transfection and lentivirus particles were harvested 48 and 72 h after transfection.
The gRNA expressing lentiviral particles were used to infect 3 million Mx-Cas9-GFP HoxB8 MPP. Cas9 and GFP expression were induced by mIFNα (1000 IU/ml; Appendix 1-key resources table) for 4 h, followed by FACS sorting for cells expressing the two gRNAs and Cas9 (GFP+ RFP+ RFP657+ cells). Single-cell clones were obtained by single-cell FACS sorting or limiting dilution.
Single-cell clones were genotyped by genomic PCR with primers listed in Appendix 1-key resources table. PCR products were sequenced by Eurofins Genomics and sequences were analyzed by SnapGene. Potential off-targets were routinely predicted by online software tools such as CRISPR-Cas9 gRNA checker (https://eu.idtdna.com/site/order/designtool/in-dex/CRISPR _SEQUENCE). For lncIrf8 promoter KO, 5 out of 71 single-cell colonies with homozygous deletions were subjected to off-target analysis. The top 2–5 predicted coding or non-coding targets were selected and HoxB8 MPP clones without off-target effects, or where off-target effects occurred in genes that were not expressed in MPP, CDP and DC subsets, were used for further studies (Table 1). For cDC1 specific +32 kb enhancer KO, 5 out of 165 single-cell clones with homozygous deletions were subjected to DC differentiation and further analyzed. Potential off-target genes of self-designed gRNAs for cDC1 +32 kb enhancer KO were not expressed in MPP, CDP, and DC subsets, and thus these gRNAs were used in the study.
Off-target analysis.
Potential off-targets | KO bulk(100 cells) | KO single-cell clones | Gene expression | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
6 | 7 | 19 | 21 | 24 | MPP | CDP | cDC | pDC | ||||
gRNA1 | Chr 13: –48032179 | Gm36101 | No | No | No | No | No | No | No | No | No | No |
Chr 2: –131293076 | Non-coding | No | No | No | No | No | No | No | No | No | No | |
gRNA2 | Chr 1: –136050990 | AsCl5 | 16 bp deletion | 16 bp deletion | 16 bp deletion | No | 1 bp insertion | No | No | No | No | No |
Chr 6:+119352090 | CACNA2D4 | No | No | No | No | No | No | No | No | No | Yes | |
Chr 3:+33982811 | Non-coding | 3–29 bp deletion; 1–3 bp insertion | 3–29 bp deletion | 3 bp deletion | 1–2 bp insertion | No | No | No | No | No | No | |
Chr 2: –132060887 | Non-coding | No | No | No | No | No | No | No | No | No | No | |
Chr 2: –150624255 | Non-coding | No | No | No | No | No | No | No | No | No | No |
-
Top potential off-targets of gRNA1 and gRNA2 predicted by CRISPR-Cas9 gRNA checker (see Materials and methods) were analyzed in lncIrf8 promoter KO bulk culture and KO single-cell clones by PCR analysis of genomic DNA. Potential off-target genes (coding) and non-coding sequences are listed. The absence of off-targets (No) and off-target deletions/insertion are shown.
lncIrf8 overexpression and lncIrf8 knockout rescue
Request a detailed protocollncIrf8 overexpression was performed in wild-type HoxB8 MPP. Lentiviral particles expressing lncIrf8 were produced from ten 6 cm dishes (Bio-One), each consisting of 0.75 million HEK293T cells (70–80% confluency) in 5 ml DMEM with supplements (see above). HEK293T cells were transfected with 5 μg plncIrf8-pA or pGFP-pA, 2.5 μg psPAX2, and 2.5 μg pMD2.G. Lentivirus particles were concentrated as above and used to infect 0.5 million HoxB8 MPP; single-cell clones were generated by limiting dilution and genotyped using the primers listed in Appendix 1-key resources table. Two out of 47 HoxB8 MPP colonies with plncIrf8-pA and 3 out of 19 HoxB8 MPP colonies with pGFP-pA (control) were expanded and subjected to Flt3L-directed DC differentiation.
lncIrf8 knockout rescue was carried out in lncIrf8 promoter KO HoxB8 MPP. FACS sorted cells that genotyped as lncIrf8 promoter KO homozygous deletion cells, were infected with lentiviral particles expressing lncIrf8. Lentiviral infection conditions were the same as for lncIrf8 overexpression in wild-type HoxB8 MPP (see above).
CRISPR activation and CRISPR interference
Request a detailed protocolCRISPR activation and CRISPR interference were performed by infecting wild-type HoxB8 MPP with lentiviral particles expressing dCAS9-VP64_GFP and pTet-KRAB-dCas9-GFP, respectively. The virus particles were produced as in the lncIrf8 overexpression experiments. In brief, virus particles from ten 6 cm dishes were used to infect 0.5 million wild-type HoxB8 MPP, followed by FACS sorting for GFP+ cells expressing dCas9-VP64 or dCas9-KRAB. Doxycycline (1 μg/ml) was used to induce dCas9-KRAB expression 2 days before cell sorting.
gRNAs targeting lncIrf8 and Irf8 promoters were cloned into pLKO5.sgRNA.EFS.tRFP as above. The dCas9-VP64-GFP and dCas9-KRAB-GFP HoxB8 MPP were then infected with specific gRNAs for gene activation and repression. The conditions for gRNA infection were the same as in lncIrf8 promoter KO experiments. Doxycycline (1 μg/ml) was given to the sorted dCas9-KRAB-GFP cells every 3 days to ensure sustained dCas9-KRAB expression.
Flow cytometry analysis and cell sorting
Request a detailed protocolDC subsets were analyzed by flow cytometry using FACS Canto II or LSR Fortessa (both from BD Biosciences). The information for flow cytometry antibodies is shown in Appendix 1-key resources table. For live/dead staining, cells were incubated with 3 μl 7-AAD per test for 5–10 min before flow cytometry measurement. Cells were sorted by FACS Aria IIu or FACS Melody, and flow cytometry data were analyzed by FlowJo V10 (all from BD Biosciences). Data on DC frequencies were subjected to the hierarchical clustering and represented in heatmap format using the online tool MORPHEUS (https://software.broadinstitute.org/morpheus/).
Cell transplantation
Request a detailed protocolCD45.1 recipient mice were sublethal irradiated (6.0 Gy, CP-160 Faxitron) 1 day before transplantation. lncIrf8 promoter KO and control HoxB8 MPP (single-cell clones) were expanded in vitro as described above. Cells were injected into recipient mice via the tail vein (5 million cells in 100 μl PBS per mouse). Donor cells from bone marrow and spleen were subjected to flow cytometry analysis at 7 and 14 days after cell transplantation.
RT-qPCR
Request a detailed protocolRNA was isolated by using the NucleoSpin RNA kit (Macherey-Nagel, 740955.250) according to the manufacturer’s instructions. RNA was subjected to cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) with Murine RNase Inhibitor (New England Biolabs, M0314S) (Appendix 1-key resources table). RT-qPCR was performed by a StepOnePlus Real-Time PCR system (Applied Biosystems) with SYBR-green fluorescence (Applied Biosystems, 4385610). The primers for qPCR were listed in Appendix 1-key resources table. Mouse GAPDH was used for normalization of lncIrf8 and Irf8 gene expression.
Identification of lncIrf8
Request a detailed protocolDe-novo transcript assembly of RNA-seq data was used to search for unknown transcripts with no coding potential and this identified the pDC specific lncRNA Tcons_00190250 (referred to as lncIrf8) and the cDC1-specific lncRNA Tcons_00190258. In brief, paired end 2×100 bps reads from RNA-seq of MPP, CDP, cDC1, cDC2, and pDC were aligned to mm9 genome using Star aligner (version 2.4) (Dobin et al., 2013) and run for Cufflinks (version 2.0; Trapnell et al., 2012). Data were subjected to lenient filtering with the parameters: min isoform fraction 0.1% and pre-RNA-fraction of 0.1%, and ribosomal genes were also filtered. Next, all the predicted transcripts were merged with cuffmerge and all transcripts with no overlap with known transcripts in mouse GENCODE were selected for further analysis (Frankish et al., 2019). The coding potential and conservation of coding elements of the lncRNAs were checked with CPAT (Wang et al., 2013) and PhyloCSF (Lin et al., 2011), respectively. lncIrf8 (Tcons_00190250) and Tcons_00190258 show low scores in both analyses, which faithfully supports their role as non-coding genes and exhibit low cross-species conservation.
To further characterize the major transcripts of lncIrf8, 3' and 5' end Rapid Amplification of cDNA Ends (RACE) PCR was performed using template-switching RT enzyme mix (New England Biolabs, M0466) and TA cloning kit (Thermo Fisher Scientific, K202020) (Appendix 1-key resources table). The primers (listed in Appendix 1-key resources table) used for RACE PCR were synthesized by Eurofins Genomics except for 5' RACE-TSO, which was from IDT.
3' RACE PCR
Request a detailed protocolReverse transcript (RT) and template-switching: 4 μl (10 ng to 1 μg) total RNA (from DC differentiation day 5) were incubated at 80°C for 3 min and cooled rapidly on ice. RNA was then incubated with template-switching RT buffer, 1 mM dNTP, 5 mM DTT, 10 μM QT primer (Scotto-Lavino et al., 2006) and 1 μl RT enzyme in 10 μl at room temperature for 5 min, followed by 1 hr at 42°C, 10 min at 50°C, and 85°C for 5 min to inactive the RT enzyme mix and sample was then kept at 4°C and diluted with 20 μl MilliQ water.
First-round PCR: 1 μl of diluted sample was subjected to the first round PCR with Q0 primer and 3' RACE-GSP-lncIrf8-F1 (10 μM) using Q5 high fidelity DNA polymerase. Second-round PCR: the PCR products from the first round PCR (1:20 dilution) was then used as template for the second round of PCR by using Q1 primer (10 μM), 3' RACE-GSP-lncIrf8-F2 (10 μM). Q0 and Q1 primers and the PCR programs are described in Scotto-Lavino et al., 2006. Products from the second-round PCR were purified using the PCR clean-up kit (Macherey-Nagel, 740609.50).
A-tailing and TA cloning: a reaction containing 5 μl of PCR clean-up product in Taq buffer, 1 mM MgCl2, 0.4 mM dATP and 1 μl Taq DNA polymerase in 25 μl was prepared and incubated at 70°C for 20 min for A-tailing of PCR products. Five μl of the A-tailed products were subjected to TA cloning into pCR2.1 vector according to the manufacturer’s instruction followed by Sanger sequencing.
5' RACE PCR
Request a detailed protocolIn order to identify the TSS of lncIrf8, template RNA from bone marrow cell-derived pDC was used to perform 5' RACE PCR using template-switching RT enzyme mix (New England Biolabs, M0466) according to the manufacturer’s instructions. Template switching was by 5' RACE-GSP-lncIrf8-R2 primer (10 μM) and template switching oligo (TSO) (75 μM). Similar to the 3' RACE PCR, two rounds of PCR were used to improve PCR specificity. In brief, 5' RACE-GSP-lncIrf8-R2 (10 μM) and TSO-specific primer (10 μM) were used to perform the first-round PCR, 5' RACE-GSP-lncIrf8-R1 primer (10 μM) and the TSO-specific primer (10 μM) were used to perform the second round PCR, followed by fragments A-tailing, TA cloning, and Sanger sequencing as described above for 3' RACE PCR.
Identification of transcription factor binding sites
Request a detailed protocolTranscription factor binding sites (TFBS) in the Irf8 +32 kb enhancer were predicted using a motif matching tool based on the MOODS (Korhonen et al., 2009) and position weight matrixes (PWMs) were obtained from the JASPAR database (Fornes et al., 2020). The bit-score cut-off thresholds were determined by applying the dynamic programming approach as described in Wilczynski et al., 2009 with an FPR of 0.0001. DC TF were considered and are depicted.
Statistical analysis
Request a detailed protocolStatistical analyses were performed using Prism (GraphPad). Unpaired t test and Multiple t-tests were used to compare data from two groups, two-way ANOVA with Tukey’s multiple comparisons test was used to analyze data from more than two groups.
Appendix 1
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (M. musculus) | C57BL/6 mice (CD45.1 and CD45.2) | Jackson Laboratory | ||
Genetic reagent (M. musculus) | Mx-Cas9-GFP mice | Medical Faculty, RWTH Aachen University Xu et al., 2022 | ||
Cell line (H. sapiens) | HEK293T | ATCC | https://www.atcc.org | Lentivirus and retrovirus production |
Cell line (M. musculus) | lncIrf8 promoter KO HoxB8 MPP; Control HoxB8 MPP | This paper | lncIrf8 promoter KO | |
Cell line (M. musculus) | plncIrf8-pA HoxB8 MPP; pGFP-pA HoxB8 MPP | This paper | lncIrf8 overexpression and rescue | |
Cell line (M. musculus) | Irf8-VP64 HoxB8 MPP; lncIrf8-VP64 HoxB8 MPP; Non-targeting-VP64-1 HoxB8 MPP; Non-targeting-VP64-2 HoxB8 MPP | This paper | Irf8 and lncIrf8 promoters activation | |
Cell line (M. musculus) | Irf8-KRAB HoxB8 MPP; lncIrf8-KRAB HoxB8 MPP; Non-targeting-KRAB-1 HoxB8 MPP; Non-targeting-KRAB-2 HoxB8 MPP | This paper | Irf8 and lncIrf8 promoters repression | |
Antibody | APC/Cyanine 7 anti-mouse/human B220 (rat monoclonal) | Biolegend | Cat# 103223; RRID: AB_313006 | FACS (1:400) |
Antibody | Brilliant Violet 510 anti-mouse/human CD11b (rat monoclonal) | Biolegend | Cat# 101245; RRID: AB_2561390 | FACS (1:400) |
Antibody | PE/Cyanine 7 anti-mouse CD11c (Armenian hamster monoclonal) | Biolegend | Cat# 117317; RRID: AB_493569 | FACS (1:400) |
Antibody | APC anti-mouse CD115 (rat monoclonal) | eBioscience | Cat# 17-1152-80; RRID: AB_1210789 | FACS (1:400) |
Antibody | PE/Cyanine 7 anti-mouse CD117 (rat monoclonal) | eBioscience | Cat# 25-1172-82; RRID: AB_469646 | FACS (1:400) |
Antibody | PE anti-mouse CD135 (rat monoclonal) | eBioscience | Cat# 12-1351-82; RRID: AB_465859 | FACS (1:400) |
Antibody | Biotin anti-mouse CD19 (rat monoclonal) | Biolegend | Cat# 115503; RRID: AB_313638 | FACS (1:800) |
Antibody | Biotin anti-mouse CD3e (Armenian hamster monoclonal) | eBioscience | Cat# 13-0031-82; RRID: AB_466319 | FACS (1:800) |
Antibody | APC/Cyanine 7 anti-mouse CD45.2 (mouse monoclonal) | Biolegend | Cat# 109823; RRID: AB_830788 | FACS (1:400) |
Antibody | Biotin anti-mouse F4/80 (rat monoclonal) | Biolegend | Cat# 123105; RRID: AB_893499 | FACS (1:800) |
Antibody | Alexa Fluor 700 anti-mouse Gr1 (rat monoclonal) | Biolegend | Cat# 108421; RRID: AB_493728 | FACS (1:400) |
Antibody | PerCP/Cyanine 5.5 anti-mouse Gr1 (rat monoclonal) | eBioscience | Cat# 45-5931-80; RRID: AB_906247 | FACS (1:400) |
Antibody | Alexa Fluor 700 anti-mouse Ly6C (rat monoclonal) | Biolegend | Cat# 128023; RRID: AB_10640119 | FACS (1:400) |
Antibody | Biotin anti-mouse Ly6G (rat monoclonal) | Biolegend | Cat# 127603; RRID: AB_1186105 | FACS (1:800) |
Antibody | Brilliant Violet 785 anti-mouse MHCII (rat monoclonal) | Biolegend | Cat# 107645; RRID: AB_2565977 | FACS (1:2000) |
Antibody | Biotin anti-mouse NK1.1 (mouse monoclonal) | eBioscience | Cat# 14-5941-82; RRID: AB_467736 | FACS (1:800) |
Antibody | Super Bright anti-mouse Siglec-H (rat monoclonal) | Invitrogen | Cat# 63-0333-82 RRID: AB_2784853 | FACS (1:400) |
Antibody | PE/Dazzle 594 Streptavidin | Biolegend | Cat# 405247 | FACS (1:1000) |
Antibody | Biotin anti-mouse Ter119 (rat monoclonal) | eBioscience | Cat# 14-5921-82; RRID: AB_467727 | FACS (1:800) |
Antibody | Brilliant Violet 421 anti-mouse/rat XCR1 (mouse monoclonal) | Biolegend | Cat# 148216; RRID: AB_2565230 | FACS (1:400) |
Antibody | 7-Aminoactinomycin D (7-AAD) | Thermo Fisher Scientific | Cat# A1310 | FACS (3 μl per test) |
Chemical compound, drug | β-estradiol (E2) | Sigma-Aldrich | Cat# E2758 | |
Chemical compound, drug | β-mercaptoethanol (β-ME) | Gibco | Cat# 31350010 | |
Chemical compound, drug | BsmBI-v2 | New England Biolabs | Cat# R0739S | |
Chemical compound, drug | Chondroitin sulfate sodium salt from shark cartilage (CSS) | Sigma-Aldrich | Cat# C4384 | |
Chemical compound, drug | cOmplete Mini | Roche | Cat# 11836153001 | |
Chemical compound, drug | dATP | New England Biolabs | Cat# N0440S | |
Chemical compound, drug | Dimethysulfoxide (DMSO) | Sigma-Aldrich | Cat# D8418 | |
Chemical compound, drug | Doxycycline hyclate | Sigma-Aldrich | Cat# D9891 | |
Chemical compound, drug | DpnII | A kind gift from A. Marieke Oudelaar, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany. DpnII enzyme with a similar activity is also available from New England Biolabs. | Cat# R0543M | |
Chemical compound, drug | DMEM | Gibco | Cat# 41965039 | |
Chemical compound, drug | DTT | Thermo Fisher Scientific | Cat# 20290 | |
Chemical compound, drug | EDTA | Gibco | Cat# 15575–038 | |
Chemical compound, drug | Fetal calf serum (FCS) | PAA | Cat# A01125-499 | |
Chemical compound, drug | Fetal calf serum (FCS) | Gibco | Cat# 10270106 | |
Chemical compound, drug | Formaldehyde (37%) | AppliChem | Cat# A0877 | |
Chemical compound, drug | Recombinant human Flt3-Ligand (Flt3L) | Peprotech | Cat# 300–19 | |
Chemical compound, drug | Recombinant murine stem cell factor (SCF) | Peprotech | Cat# 250–03 | |
Chemical compound, drug | Human IGF-1 long range | Sigma-Aldrich | Cat# 85,580 C | |
Chemical compound, drug | Recombinant IL-6/soluble IL-6 receptor fusion protein | A kind gift from S. Rose-John, Kiel, Germany Fischer et al., 1997. R&D Systems provides a similar product with the same activity. | Cat# 9038 SR | |
Chemical compound, drug | HEPES | Sigma-Aldrich | Cat# H4034 | |
Chemical compound, drug | L-glutamine | Gibco | Cat# 25030081 | |
Chemical compound, drug | M-270 Streptavidin Dynabeads | Invitrogen | Cat# 65305 | |
Chemical compound, drug | Mouse interferon α (mIFNα) | Miltenyi Biotec | Cat# 130-093-131 | |
Chemical compound, drug | Murine RNase Inhibitor | New England Biolabs | Cat# M0314S | |
Chemical compound, drug | Pancoll human, density 1.077 g/ml (Ficoll) | PAN-Biotech | Cat# P04-601000 | |
Chemical compound, drug | Penicillin/streptomycin | Gibco | Cat# 15140122 | |
Chemical compound, drug | Phenol-Chloroform-Isoamyl alcohol (PCI) | Sigma-Aldrich | Cat# 77617 | |
Chemical compound, drug | Phosphate buffered saline (PBS) | Gibco | Cat# 10010023 | |
Chemical compound, drug | Polybrene (PB, Hexadimethrine bromide) | Sigma-Aldrich | Cat# H9268 | |
Chemical compound, drug | Q5 high fidelity DNA polymerase | New England Biolabs | Cat# M0491L | |
Chemical compound, drug | RPMI 1640 | Gibco | Cat# 31870025 | |
Chemical compound, drug | SalI | New England Biolabs | Cat# R0138S | |
Chemical compound, drug | Supernatant from Flt3L-producing B16F1 cells (1%) | Homemade. Flt3L from Peprotech has the same activity (1:1000) | Cat# 300–19 | |
Chemical compound, drug | Supernatant from CHO KLS C6 cells expressing soluble murine SCF (1%) | Homemade. Peprotech provides a similar product with the same activity (1:1000). | Cat# 250–03 | |
Chemical compound, drug | SYBR-green fluorescence | Applied Biosystems | Cat# 4385610 | |
Chemical compound, drug | T4 DNA HC ligase | Life Tech | Cat# EL0013 | |
Chemical compound, drug | Taq DNA polymerase | Homemade | ||
Chemical compound, drug | Taq buffer (10 x) | Thermo Fisher Scientific | Cat# B33 | |
Chemical compound, drug | Template-switching RT enzyme mix | New England Biolabs | Cat# M0466 | |
Chemical compound, drug | XhoI | New England Biolabs | Cat# R0146S | |
Commercial assay or kit | Gibson Assembly kit | New England Biolabs | Cat# E5510S | |
Commercial assay or kit | High-Capacity cDNA Reverse Transcription Kit | Applied Biosystems | Cat# 4368814 | |
Commercial assay or kit | KAPA Hyper Capture Reagent Kit | Roche | Cat# 9075828001 | |
Commercial assay or kit | NEBNext Ultra II DNA Library Prep Kit for Illumina | New England Biolabs | Cat# E7645S | |
Commercial assay or kit | NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) | New England Biolabs | Cat# E7335S | |
Commercial assay or kit | NEBNext Multiplex Oligos for Illumina (Index Primers Set 2) | New England Biolabs | Cat# E7500S | |
Commercial assay or kit | NucleoSpin RNA kit | Macherey-Nagel | Cat# 740955.250 | |
Commercial assay or kit | PCR clean-up kit | Macherey-Nagel | Cat# 740609.50 | |
Commercial assay or kit | TA cloning kit | Thermo Fisher Scientific | Cat# K202020 | |
Sequence-based reagent | 5'RACE-TSO | New England Biolabs | 5’ RACE PCR primers | GCTAATCATTGCAAGCAGTGGTATC AACGCAGAGTACATrGrGrG |
Sequence-based reagent | 5'RACE-TSO-Specific | New England Biolabs | 5’ RACE PCR primers | CATTGCAAGCAGTGGTATCAAC |
Sequence-based reagent | 5'RACE-GSP-lncIrf8-R1 | New England Biolabs | 5’ RACE PCR primers | TGTCAGTGATGGGGGCTGGAGAAAT |
Sequence-based reagent | 5'RACE-GSP-lncIrf8-R2 | New England Biolabs | 5’ RACE PCR primers | GCTCAGGATGCCAGGTCCCTTCTT |
Sequence-based reagent | 3'RACE-QT | Scotto-Lavino et al., 2006 | 3’ RACE PCR primers | CCAGTGAGCAGAGTGACGAGGACTC GAGCTCAAGCTTTTTTTTTTTTTTTTT |
Sequence-based reagent | 3'RACE-Q0 | Scotto-Lavino et al., 2006 | 3’ RACE PCR primers | CCAGTGAGCAGAGTGACG |
Sequence-based reagent | 3'RACE-QI | Scotto-Lavino et al., 2006 | 3’ RACE PCR primers | GAGGACTCGAGCTCAAGC |
Sequence-based reagent | 3'RACE-GSP-lncIrf8-F1 | This paper | 3’ RACE PCR primers | ATTTCTCCAGCCCCCATCACTGACA |
Sequence-based reagent | 3'RACE-GSP-lncIrf8-F2 | This paper | 3’ RACE PCR primers | AAGAAGGGACCTGGCATCCTGAGC |
Sequence-based reagent | lncIrf8-F | This paper | Genotyping primers | TCCTGAAGGGACAGGCAAG |
Sequence-based reagent | lncIrf8-R | This paper | Genotyping primers | CTTGGACATTGAGGACGCC |
Sequence-based reagent | cDC1 +32 kb-F1 | This paper | Genotyping primers | GTGACTGCAAGTAAGTTCTTCGG |
Sequence-based reagent | cDC1 +32 kb-F2 | This paper | Genotyping primers | AAGTAGAGATTCCCTTTCTAAGCC |
Sequence-based reagent | cDC1 +32 kb-R | This paper | Genotyping primers | ATCAGGCTGGGTGGTGGTT |
Sequence-based reagent | Sc-lncIrf8-F | This paper | Cloning primers | ACACTCGAGACTGTCAGATGCAGGGG; the underline sequences represent cloning sites |
Sequence-based reagent | Sc-lncIrf8-R | This paper | Cloning primers | AAAAAAGTCGACGCATCAGATTTAATATA GAACTAGGACA; the underline sequences represent cloning sites |
Sequence-based reagent | CMV-lncIrf8-F | This paper | Genotyping primers | TGGGCGTGGATAGCGGTTT |
Sequence-based reagent | CMV-lncIrf8-R | This paper | Genotyping primers | CACTGAGACTTAGCAAGGGGGA |
Sequence-based reagent | CMV-GFP-F | This paper | Genotyping primers | TGGGCGTGGATAGCGGTTT |
Sequence-based reagent | CMV-GFP-R | This paper | Genotyping primers | TGGGGGTGTTCTGCTGGTAG |
Sequence-based reagent | mlncIrf8-tQ-F | This paper | RT-qPCR primers | ACTGTCAGATGCAGGGG |
Sequence-based reagent | mlncIrf8-tQ-R | This paper | RT-qPCR primers | TCACAATCGTCTGTAACTCCG |
Sequence-based reagent | mIrf8-tQ-F | This paper | RT-qPCR primers | GAGCGAAGTTCCTGAGATGG |
Sequence-based reagent | mIrf8-tQ-R | This paper | RT-qPCR primers | TGGGCTCCTCTTGGTCATAC |
Sequence-based reagent | mGAPDH-tQ-F | This paper | RT-qPCR primers | ACCTGCCAAGTATGATGACATCA |
Sequence-based reagent | mGAPDH-tQ-R | This paper | RT-qPCR primers | GGTCCTCAGTGTAGCCCAAGAT |
Sequence-based reagent | m3C-F | Downes et al., 2021; Downes et al., 2022 | Capture-C qPCR primers | GGAGAAAGAAGGCTGGTGTTAT |
Sequence-based reagent | m3C-R | Downes et al., 2021; Downes et al., 2022 | Capture-C qPCR primers | TATCTGAGTTGGACAGCATTGG |
Sequence-based reagent | m3C-control-F | Downes et al., 2021; Downes et al., 2022 | Capture-C qPCR primers | TTATCTTGCATTTGCCAACTCG |
Sequence-based reagent | m3C-control-R | Downes et al., 2021; Downes et al., 2022 | Capture-C qPCR primers | TGGGTTTCCCTGATTCTGAAA |
Sequence-based reagent | Irf8_P_L | This paper | Capture probes | GATCCGTGCATCACCAGCCTCC TTGACCTTAGGCAGACGCCCCA GCCCCCCGGCCATTTTTGGGGCAGCC |
Sequence-based reagent | Irf8_P_R | This paper | Capture probes | CCAAATGAACAAACACCTCTCCC TTTAAAATCTGCCTGATGGCCAA CTTCATAATGAAGAGAAATAGATC |
Sequence-based reagent | gRNA-1-F | This paper | gRNAs | CACCGTCCATTATACTAAGATACCC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-1-R | This paper | gRNAs | AAACGGGTATCTTAGTATAATGGAC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-2-F | This paper | gRNAs | CACCGGTGCCGAGAAAGGACACGT; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-2-R | This paper | gRNAs | AAACACGTGTCCTTTCTCGGCACC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO1-5‘F | Durai et al., 2019 | gRNAs | CACCGTTGTGATCTTTGAGGTAGA; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO1-5’R | Durai et al., 2019 | gRNAs | AAACTCTACCTCAAAGATCACAAC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO1-3‘F | Durai et al., 2019 | gRNAs | CACCGAACTGGCCTGGGGCAGGTC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO1-3’R | Durai et al., 2019 | gRNAs | AAACGACCTGCCCCAGGCCAGTTC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO2-5’F | This paper | gRNAs | CACCGACATTCTGCACCCCAGTCA; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO2-5’R | This paper | gRNAs | AAACTGACTGGGGTGCAGAATGTC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO2-3’F | This paper | gRNAs | CACCGAGGATCGCACCTCACCTACT; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-KO2-3’R | This paper | gRNAs | AAACAGTAGGTGAGGTGCGATCCTC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-Irf8-VP64-F | This paper | gRNAs | CACCGACGGTCGCGCGAGCTAATTG; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-Irf8-VP64-R | This paper | gRNAs | AAACCAATTAGCTCGCGCGACCGTC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-Irf8-KRAB-F | This paper | gRNAs | CACCGCGGCAGGTAGGACGCGATG; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-Irf8-KRAB-R | This paper | gRNAs | AAACCATCGCGTCCTACCTGCCGC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-lncIrf8-VP64-F | This paper | gRNAs | CACCGGTGCCGAGAAAGGACACGT; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-lncIrf8-VP64-R | This paper | gRNAs | AAACACGTGTCCTTTCTCGGCACC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-lncIrf8-KRAB-F | This paper | gRNAs | CACCGAGTCACTCGTCCTTTGGGG; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-lncIrf8-KRAB-R | This paper | gRNAs | AAACCCCCAAAGGACGAGTGACTC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-non-targeting-1-F | Manguso et al., 2017 | gRNAs | CACCGCGAGGTATTCGGCTCCGCG; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-non-targeting-1-R | Manguso et al., 2017 | gRNAs | AAACCGCGGAGCCGAATACCTCGC; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-non-targeting-2-F | Manguso et al., 2017 | gRNAs | CACCGATGTTGCAGTTCGGCTCGAT; the underline sequences represent cloning sites |
Sequence-based reagent | gRNA-non-targeting-2-R | Manguso et al., 2017 | gRNAs | AAACATCGAGCCGAACTGCAACATC; the underline sequences represent cloning sites |
Software, algorithm | Bamtofastq | 10 x Genomics | https://support.10xgenomics.com/docs/bamtofastq | |
Software, algorithm | Bowtie2 | Langmead and Salzberg, 2012 | http://bowtie-bio.sourceforge.net | |
Software, algorithm | CapCruncher (v0.1.1a1) | Downes et al., 2022 | https://github.com/sims-lab/CapCruncher | |
Software, algorithm | CPAT | Wang et al., 2013 | http://code.google.com/p/cpat/ | |
Software, algorithm | Cufflinks (version 2.0) | Trapnell et al., 2012 | http://cufflinks.cbcb.umd.edu/ | |
Software, algorithm | FlowJo V10 | BD Biosciences | ||
Software, algorithm | IGV browser | Broad Institute | ||
Software, algorithm | MOODS | Korhonen et al., 2009 | https://www.cs.helsinki.fi/group/pssmfind/ | |
Software, algorithm | Oligo | Oudelaar et al., 2020 | https://oligo.readthedocs.io/en/latest/index.html | |
Software, algorithm | PhyloCSF | Lin et al., 2011 | http://compbio.mit.edu/PhyloCSF | |
Software, algorithm | Prism | GraphPad | ||
Software, algorithm | Scanpy | Wolf et al., 2018 | ||
Software, algorithm | Star aligner (version 2.4) | Dobin et al., 2013 | http://code.google.com/p/rna-star/ | |
Software, algorithm | Snapgene | GSL Biotech | ||
Software, algorithm | UMAP | Becht et al., 2019 |
Data availability
ATAC-seq (MPP, CDP and cDC2), Capture-C targeting Irf8 promoter (MPP, CDP, cDC1, cDC2 and pDC), IRF8 ChIP-seq (cDC and pDC), and RNA-seq (MPP, CDP, cDC1, cDC2 and pDC) data generated in this study have been deposited in Gene Expression Omnibus and are accessible through GEO Series accession numbers GSE198651.ATAC-seq data of cDC1 and pDC (GSE118221) are published (Lin et al., 2015), and the ATAC-seq data of MPP, CDP and cDC2 of the same study are described here (GSE198651). ChIP-seq of CTCF in DC (GSE36099) (Garber et al., 2012), H3K27ac (GSE73143) (Allhoff et al., 2016), H3K4me1 and PU.1 (GSE57563) (Allhoff et al., 2014), H3K4me3 and H3K9me3 (GSE64767) (Lin et al., 2015) in MPP, CDP, cDC and pDC were re-analyzed in this study. scRNA-seq data were reanalyzed from GSE114313 (Rodrigues et al., 2018). Bulk RNA-seq data of splenic pDC, cDC1 and cDC2 were reanalyzed from GSE188992 (Pang et al., 2022).The sequence of the pDC specific lncRNA (lncIrf8 identified by RACE PCR) and the cDC1 specific lncRNA (Tcons_00190258 identified by RNA-seq) has been submitted to GenBank. The GenBank accession numbers for lncIrf8 and Tcons_00190258 are ON134061 and ON134062, respectively.
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NCBI Gene Expression OmnibusID GSE198651. A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation.
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NCBI NucleotideID ON134061. Mus musculus lncIRF8 lncRNA, partial sequence.
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NCBI NucleotideID ON134062. Mus musculus TCONS_00190258 lncRNA, partial sequence.
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NCBI Gene Expression OmnibusID GSE118221. Identification of Transcription Factor Binding Sites using ATAC-seq.
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NCBI Gene Expression OmnibusID GSE36099. A high throughput in vivo protein-DNA mapping approach reveals principles of dynamic gene regulation in mammals (ChIP-Seq).
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NCBI Gene Expression OmnibusID GSE73143. Differential Peak Calling of ChIP-Seq Signals with Replicates with THOR.
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NCBI Gene Expression OmnibusID GSE57563. Detecting differential peaks in ChIP-seq signals with ODIN.
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NCBI Gene Expression OmnibusID GSE64767. Epigenetic Program and Transcription Factor Circuitry of Dendritic Cell Development.
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NCBI Gene Expression OmnibusID GSE114313. A distinct lineage of origin reveals heterogeneity of plasmacytoid dendritic cells II (scRNAseq).
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NCBI Gene Expression OmnibusID GSE188992. Bulk RNAseq of Mouse Splenic Dendritic Cell Subsets.
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Decision letter
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Florent GinhouxReviewing Editor; Agency for Science Technology and Research, Singapore
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Satyajit RathSenior Editor; Indian Institute of Science Education and Research (IISER), India
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Stephen L NuttReviewer; Walter and Eliza Hall Institute of Medical Research, Australia
Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.
Decision letter after peer review:
Thank you for submitting your article "A lncRNA identifies IRF8 enhancer element in negative feedback control of dendritic cell differentiation" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Satyajit Rath as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Stephen L Nutt (Reviewer #2).
The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.
Essential revisions:
1) Demonstrate the presence of lncRNA selectively in ex-vivo splenic pDC sub-type and confirm its expression in human pDCs.
2) To clarify the role of irf8 in pdc development in regards to published data comparing the lncIrf8 and +32kb activity in their system: The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs (Figure 2C, Figure 2 Suppl 3D). However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO? In addition, it is essential that authors delete the +32 Kb enhancer region (as defined in Durai 2019) in their culture system and analyze the same with respect to +32Kb lncIRF8 promoter deletion in terms of expression of IRF8, lncIRF8 and its effect on the development of pDC vs cDC1.
3)( Provide evidence that IRF8 binds in the lncIRF8 promoter. Authors showed show DNA binding motifs for Irf8 in the lncIrf8, but no evidence of actual binding. Perhaps the +32kb enhancer is also physically involved, but either way more evidence is needed to fully support the authors' models. Instead, IRF8 binds in the more 5' part of the +32kb enhancer identified by Durai 2019. How then is a putative IRF8 repressor complex linked to the lncIRF8 promoter as the authors propose? Moreover, the model does not appear to address why the IRF8 repressor complex binding to the lncIRF8 would be active in cDC1 and not pDC.
4) Authors should acknowledge/discuss clearly that one limitation of their study is that lncIRF8 might have other roles in DC/pDC biology which is yet unidentified in the current study.
Reviewer #1 (Recommendations for the authors):
1. While refining the cross-talk between promoter and enhancers authors have completely overlooked the functional significance of lncIRF8 in pDCs. Authors have demonstrated the expression of lncIRF8 is specific to pDCs, it is not evident whether all pDCs express it or a fraction of the same.
a. Further authors should confirm the expression of lncRNA in ex vivo pDCs from various murine tissues. Is the expression of lncRNA also conserved in human pDCs?
b. How is the expression of lncIRF8 modulated upon CpG stimulation/viral infection of pDCs?
c. Is there any significance of lncIRF8 in pDC functions (TLR stimulations, cytokine production, etc.) these aspects could be studied without much effort as authors already have systems that can knockout lncIRF8 promoter or overexpress lncIRF8.
2. Though authors have identified that lncIRF8 is specifically expressed in pDC subtype but while understanding its significance and regulation, it was preferred to study bulk DC cultures overlooking the finding regarding specificity. Experiments performed by employing dCas9 VP64/KRAB-GFP constructs, do provide some direction in terms of mechanistic understanding of the cross-talk between the promoter of IRF8 and lncIRF8. This data needs to be further strengthened by sorting pDC, cDC1 and cDC2 populations and studying the effect of the genetic manipulations on IRF8 and lncRNA expression in each cell type.
3. One of the most important findings of the submitted study is to demonstrate that knocking out the lncIRF8 promoter also diminishes the expression of IRF8, pDC as well as cDC1 development (Figure 2). Once the IRF8 expression is suppressed the analysis of pDC and cDC1 differentiation is redundant. It is previously reported that the deletion of +32 Kb enhancer (Durai et al. 2019) in a mouse model did not affect pDC but specifically depleted cDC1 development. Authors should examine the deletion of only the +32 kb enhancer region in their culture system to sort out the hierarchy between the +32 Kb enhancer and +32 Kb lncRNA promoter in terms of DC subtype development (cDC1 Vs pDC). Does the deletion of the lncIRF8 promoter lead to epigenetic changes at the overall +32 region? It is expected that due to interactions and chromatin loop formation between IRF8 promoter with various enhancers elements; deletion of lncIRF8 promoter might play an important role in defining these interactions in a cell type-specific manner. These interactions become more valuable by the fact that activation of the lncIRF8 promoter leads to enhanced cDC1 differentiation. Does this simply mean more open chromatin conformation around the cDC1-specific +32 Kb region rather than correlating with the lncIRF8?
Can authors analyze the epigenetic changes by performing similar experiments in some of the available pDC and cDC1 cell lines? As these cell lines are likely to have active IRF8 transcription and it would help to measure the effect of lncIRF8 promoter deletion/mutation/activation/repression on IRF8 expression and specific DC subtype maintenance.
4. Based on the various earlier published reports and results from the current study authors have shown a schematic representation of the pDC and cDC1-specific interactions. Can these interactions be validated in primary/ex-vivo DCs and be correlated with lncIRF8 as well as IRF8 expression? Will it be possible for authors to validate these interactions in light of the lncIRF8 promoter deletion/mutation/activation/repression? Authors may be careful to perform these experiments in purified pDC and cDC1 fractions to get meaningful data.
In case it is not possible to perform experiments with primary DCs/genetically modified HOXB8 cultures; if required, some of these experiments may be performed in the cell lines wherein overall interaction between various enhancer elements and promoters can be validated.
5. Authors have also identified a TCONS_00190258 which is specifically expressed in the cDC1 subtype. Why not look at the expression levels of this transcript in the various experiments in the current study as enhancer element crosstalk will help in cell type-specific transcription of IRF8, lncIRF8, and TCONS_00190258?
Reviewer #2 (Recommendations for the authors):
I find the quality and interpretation of the genomics data to be excellent and I have no major concerns with that data. I do have some specific comments around the interpretation of the cellular data and the model presented.
Specific comments
1. The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs (Figure 2C, Figure 2 Suppl 3D). However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO?
2. There does not appear to be compelling evidence that IRF8 binds in the lncIRF8 promoter in cDC1. Instead, IRF8 binds in the more 5' part of the +32kb enhancer identified by Durai 2019. How then is a putative IRF8 repressor complex linked to the lncIRF8 promoter as the authors propose? Moreover, the model does not appear to address why the IRF8 repressor complex binding to the IRF8 repressor complex would be active in cDC1 and not pDC.
3. The authors use 2 versions of the HoxB8 MPP differentiation system (lines 207-234). Firstly, it is unclear, why two similar approaches are used, but more importantly, the second system outlined in figure 2 Suppl 3 shows that the lncIRF8 promoter is also required for cDC2 differentiation. This is a concern, as IRF8 is not expressed in cDC2, and Irf8-/- mice produce cDC2.
https://doi.org/10.7554/eLife.83342.sa1Author response
Essential revisions:
1) Demonstrate the presence of lncRNA selectively in ex-vivo splenic pDC sub-type and confirm its expression in human pDCs.
Point 1: Presence of lncRNA selectively in ex-vivo splenic pDC sub-type.
This is interesting and we addressed this point by re-analyzing public scRNA-seq and bulk RNA-seq datasets for spleen. We also included the analysis of lncRNA expression in bone marrow. The scRNA-seq data of murine ex-vivo spleen pDC and bone marrow pDC were obtained from GSE114313 (Rodrigues et al., Nat. Immunol., 2018). The bulk RNA-seq dataset of splenic DC subsets pDC, cDC1 and cDC2 are from GSE188992 (Pang et al., Front. Immunol., 2022).
These new results are now shown in new Figure 1—figure supplement 3 (page 32) and described in the Results section, page 6, lines 151-154 as follows:
“lncIrf8 and TCONS_00190258 show the same expression pattern in pDC and cDC1, respectively, in BM and spleen (Figure 1—figure supplement 3), as revealed by reanalyzing scRNA-seq and bulk RNA-seq data (Rodrigues et al., 2018; Pang et al., 2022).”
The Materials and methods section was updated accordingly (page 17, lines 536-548).
More specifically, the scRNA-seq data from GSE114313 (Rodrigues et al., Nat. Immunol., 2018) were downloaded, BAM files were converted back into FASTQ files and a custom reference genome of mm9 was created now including the novel lncIrf8 (GenBank: ON134061) and Tcons_00190258 lncRNA (GenBank: ON134062). Data were subjected to bioinformatic processing as described in the Materials and methods section on page 17, lines 536-548 and visualized in UMAPs.
In Dress et al., Nat. Immunol., 2019 authors are critical on the pDC-like cells in GSE114313. Therefore to better define the pDC population in GSE114313, we included the pDC marker genes Tcf4, Bst2 and Siglec-H in our analysis and used the cDC marker genes Zbtb46, Cx3cr1 and Xcr1 as negative controls. We observed that lncIrf8 is expressed in bone marrow (BM) and spleen pDC (new Figure 1—figure supplement 3, page 32). Expression of the cDC1 specific Tcons_00190258 lncRNA in BM and spleen pDC is very low to absent.
To further extend this analysis, we analyzed the bulk RNA-seq dataset of splenic DC subsets pDC, cDC1 and cDC2 from GSE188992 (Pang et al., Front. Immunol., 2022). lncIrf8 and Tcons_00190258 lncRNA were visualized by IGV browser. As expected, lncIrf8 and Tcons_00190258 lncRNA were expressed in pDC and cDC1 of spleen, respectively, and the expression pattern was the same as in our in vitro bone marrow DC (new Figure 1—figure supplement 3).
Point 2: Confirm its expression in human pDCs.
To address this point, we checked the transcripts around the Irf8 gene in different species using data from NCBI, including mouse, human and zebrafish. We found a lncRNA downstream of human Irf8 gene, referred to as LINC02132, but no lncRNA was observed downstream of zebrafish Irf8 gene (see Author response image 1A).

Mouse lncIrf8 is not conserved across species.
(A) Visualization of lncRNA downstream of Irf8 gene in mouse, human and zebrafish using data from NCBI. Red box indicates lncRNA. (B) Sequence alignment of mouse lncIrf8 and human LINC02132 using MEGA 6.0. (C) Visualization of human LINC02132 by UCSC browser. Data of gene transcription, H3K27ac signal and DNase signal are from ENCODE. (D) Gene expression of human LINC02132 in different tissues. Data are from GTEx RNA-seq in 54 tissues of 17382 samples, 948 donors.
We found that the sequences of mouse lncIrf8 and human LINC02132 are not conserved (Author response image 1B). The human LINC02132 locus also shows potential enhancer activities, as revealed by H3K27ac and open chromatin (DNase) signals (Author response image 1C). The human LINC02132 is mainly expressed in lymphocytes, spleen and whole blood (Author response image 1D). Data of H3K27ac (7 cell lines) and DNase signal are from ENCODE; gene expression of human LINC02132 are from GTEx RNA-seq (54 tissues, 17382 samples, 948 donors).
Taken together, the pDC specific lncIrf8 is not conserved across species, which is in line with the general characteristics of lncRNA. We have now included this information in the Results section, page 7, lines 200-202.
2) To clarify the role of irf8 in pdc development in regards to published data comparing the lncIrf8 and +32kb activity in their system: The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs (Figure 2C, Figure 2 Suppl 3D). However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO? In addition, it is essential that authors delete the +32 Kb enhancer region (as defined in Durai 2019) in their culture system and analyze the same with respect to +32Kb lncIRF8 promoter deletion in terms of expression of IRF8, lncIRF8 and its effect on the development of pDC vs cDC1.
Point 1: Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO?
We appreciate the point raised and know the work by Sichien et al., 2016 on WT:Irf8-/- BM chimeras and the conclusions reached from their work that IRF8 is dispensable for pDC development. Previous studies, including the study of Sichien et al., 2016, showed that pDC are reduced in Irf8 knockout mice (Murakami et al., Nat. Immunol., 2021; Schiavoni et al., J. Exp. Med., 2002; Tsujimura et al., J. Immunol., 2003). Thus, there is the possibility that the conclusions reached by Sichien et al., 2016 on the impact of IRF8 on pDC function, is on the pDC population, which is left in the Irf8-/- mice. In addition the results in Sichien et al., 2016 are based on WT:Irf8-/- BM chimeras upon transplantation in lethally irradiated mice and thus the divergent results might be due to the experimental set-up WT:Irf8-/- BM chimeras versus Irf8 knockout mice.
Sichien et al., 2016, report on altered marker expression (CD11b, CD11c and MHC II), which for CD11b and CD11c was not very prominent. In our study on the lncIrf8 promoter KO we used immunophenotyping of CD11c+ CD11b- B220+ cells for in vitro pDC (Figure 2C and Figure 2—figure supplement 3) and CD45.2+ Gr1- Siglec H+ cells for pDC following transplantation (Figure 3A and D, G and J; Figure 3—figure supplement 1B). Thus, we are confident to correctly detect pDC.
A potentially altered DC marker expression on lncIrf8 promoter KO pDC cannot be measured because cells are absent in lncIrf8 promoter KO. Nevertheless, we visualized CD11c and CD11b expression on cells which are present by gating on single cells on day 7 and 9 of Flt3L directed DC differentiation in lncIrf8 promoter KO and control cultures. In lncIrf8 promoter KO cells CD11c expression was slightly reduced and CD11b expression was slightly increased (see Author response image 2). This indicates that lncIrf8 promoter KO does not have a major effect on the expression of CD11c and CD11b. Given this negative result we prefer not to include these data in the manuscript.

CD11c and CD11b median fluorescence intensity (MFI) of single cells in lncIrf8 promoter KO and Control cells.
Point 2: Deletion of the +32 kb enhancer region as defined in Durai et al. 2019.
We followed the request of the reviewers and deleted the +32 kb enhancer region by using the same gRNAs as described in Durai et al., Nat. Immunol., 2019, referred to as cDC1 specific +32 kb enhancer KO (see new Figure 2—figure supplement 4C, page 35). In parallel, we also designed a new pair of gRNAs to delete this +32 kb enhancer region. We followed the same procedure as in the lncIrf8 promoter KO experiments (Figure 2—figure supplement 1A) for both the Durai et al. gRNAs and our new self-designed gRNAs to obtain homozygous clones of cDC1 specific +32 kb enhancer KO. Five out of 165 single-cell clones were subjected to DC differentiation and further analyzed at day 7 and 9. Frequencies of pDC were unaffected by +32 kb enhancer KO at the end of culture (day 9). There was some effect at day 7, however this was not statistically significant. The cDC1 specific +32 kb enhancer KO compromised cDC1 development at day 7 and day 9, which is in line with the phenotypes described by Durai et al., Nat. Immunol., 2019 and Murakami et al., Nat. Immunol., 2021 in mice.
These new results are now shown in new Figure 2—figure supplement 4 in the revised manuscript (Results section, page 8, lines 235-249 and page 35). The Materials and methods section was also updated accordingly (page 18, lines 576-605). Source data of new Figure 2—figure supplement 4B are also provided and described on page 36 (Figure 2—figure supplement 4-source data 1 and source data 2)
In summary, our data show that deletion of cDC1 specific +32 kb enhancer affects cDC1 development while the lncIrf8 promoter KO affects both pDC and cDC1 development (Figure 2). This indicates that the lncIrf8 promoter element has further functions compared to the cDC1 specific +32 kb enhancer, e.g. to control pDC development. We have now indicated this point in the Discussion (Discussion section, page 13, lines 422-427).
3) Provide evidence that IRF8 binds in the lncIRF8 promoter. Authors showed show DNA binding motifs for Irf8 in the lncIrf8, but no evidence of actual binding. Perhaps the +32kb enhancer is also physically involved, but either way more evidence is needed to fully support the authors' models. Instead, IRF8 binds in the more 5' part of the +32kb enhancer identified by Durai 2019. How then is a putative IRF8 repressor complex linked to the lncIRF8 promoter as the authors propose? Moreover, the model does not appear to address why the IRF8 repressor complex binding to the lncIRF8 would be active in cDC1 and not pDC.
We appreciate the reviewers’ point and can very well agree with the reviewers that the cDC1 specific +32 kb enhancer element by Durai et al. 2019 might also be involved in the negative feedback regulation of Irf8 during DC differentiation. The full +32 kb enhancer is delineated by the H3K27ac and H3K4me3 marks (Figure 2A and new Figure 2—figure supplement 4A) and comprises both the +32 kb enhancer element by Durai et al. 2019 and the lncIrf8 promoter described here. Both elements appear to be important for full Irf8 +32 kb enhancer function in DC differentiation: (i) Deletion of the lncIrf8 promoter compromised both pDC and cDC1 differentiation (Figure 2); (ii) Deletion of the +32 kb enhancer element by Durai et al. 2019 compromised cDC1 and to some extent also pDC differentiation (new Figure 2—figure supplement 4; although not statistical significant); (iii) positioning the dCas9-VP64 at the lncIrf8 promoter for CRISPR activation prominently enhanced cDC1 differentiation (Figure 5A, B and E) and (iv) positioning the dCas9-KRAB at the lncIrf8 promoter for CRISPR interference compromised both pDC and cDC1 differentiation (Figure 6A-E).
In our model we proposed that binding of the putative IRF8 repressor to the +32 kb enhancer is mainly in the 5’ part of the +32 kb enhancer, as demonstrated by the ChIP-seq of IRF8. There is a strong IRF8 binding signal detected in cDC and weak to no IRF8 binding signal in pDC (Figure 1A, Figure 1—figure supplement 1, Figure 2—figure supplement 2 and Figure 7B, ChIP-seq IRF8 signal).
Thus, we propose the putative IRF8 repressor complex is only active in cDC and not pDC (Figure 7B). We agree with the reviewers that at this stage of analysis we do not know why IRF8 binds to the 5’ part of the +32 kb enhancer in cDC but not in pDC. Addressing this point should be subject for further studies.
4) Authors should acknowledge/discuss clearly that one limitation of their study is that lncIRF8 might have other roles in DC/pDC biology which is yet unidentified in the current study.
We appreciate the suggestion made by the reviewer. We have now indicated that lncIrf8 might have other roles in DC biology which is yet unidentified in the current study (Discussion section, page 13, lines 435 and 436).
Reviewer #1 (Recommendations for the authors):
1. While refining the cross-talk between promoter and enhancers authors have completely overlooked the functional significance of lncIRF8 in pDCs. Authors have demonstrated the expression of lncIRF8 is specific to pDCs, it is not evident whether all pDCs express it or a fraction of the same.
a. Further authors should confirm the expression of lncRNA in ex vivo pDCs from various murine tissues. Is the expression of lncRNA also conserved in human pDCs?
The question by the reviewer on the expression of the novel lncIrf8 and Tcons_00190258 lncRNA in ex vivo pDC is well taken. We addressed this point be re-analyzing scRNA-seq data of BM and spleen pDC of GSE114313 (Rodrigues et al., Nat. Immunol., 2018) and bulk RNA-seq data of the splenic DC subsets pDC, cDC1 and cDC2 of GSE188992 (Pang et al., Front. Immunol., 2022).
The scRNA-seq data showed that lncIrf8 is expressed in BM and spleen pDC. Expression of the cDC1 specific Tcons_00190258 lncRNA in BM and spleen pDC is very low to absent. The bulk RNA-seq data fully support these results: lncIrf8 and Tcons_00190258 lncRNA are expressed in pDC and cDC1 of spleen, respectively, and the expression pattern is the same as in in vitro bone marrow DC.
These new data are in new Figure 1—figure supplement 3 (please see above our response to Essential Revision 1).
We also addressed the question on the conservation of the novel lncIrf8 across species. A lncRNA downstream of the human Irf8 gene was identified, referred to as LINC02132, but no lncRNA was observed downstream of zebrafish Irf8 gene (see Author response image 1A). By sequence alignment we demonstrate that the sequences of mouse lncIrf8 and human LINC02132 are not conserved (Author response image 1B; see also above our response above to Essential Revision 1 for further details).
We conclude that the pDC specific lncIrf8 is not conserved across species. This is a common characteristic, which is very frequently found for lncRNA. We now include this information in the Results section, page 7, lines 200-202.
b. How is the expression of lncIRF8 modulated upon CpG stimulation/viral infection of pDCs?
This is an interesting question and we addressed this point by re-analyzing public RNA-seq data of FACS sorted pDC (CD3- CD19- CD11c+ CD11blow B220+ Siglec-H+ CD317+) from BM cells of in vitro Flt3L cultures (GSE170750, Mann-Nuettel et al., BMC Genom. Data, 2021). In this study pDC were stimulated by CpG 2216 for 0, 2, 6 and 12 hours and subjected to RNA-seq analysis. Our analysis showed that Irf8 expression was upregulated with time upon CpG stimulation, while lncIrf8 expression was downregulated (Author response image 3A). Obviously, pDC activation by CpG stimulation affects the expression of hundreds of transcription factors within a few hours (Author response image 3B, C taken from Mann-Nuettel et al., BMC Genom. Data, 2021), including IRF8. CpG stimulation of pDC occurs within hours and is different from pDC differentiation, which takes a few days.

Gene expression after pDC activation by CpG 2216 stimulation.
(A) Visualization of lncIrf8 and Irf8 expression in BM cell-derived pDC upon CpG 2216 stimulation after 0, 2, 6 and 12 hours recalculated from RNA-seq data of FACS sorted pDC (CD3- CD19- CD11c+ CD11blow B220+ Siglec-H+ CD317+; GSE170750 from Mann-Nuettel et al., BMC Genom. Data, 2021). (B and C) Differentially expressed transcription factors in CpG 2216 stimulated pDC. Panel (A) is our calculation, panels (B) and (C) are from Mann-Nüttel et al., BMC Genom. Data, 2021 Figure 2B and Figure 2F.
Therefore, it is not easy to explain the gene expression patterns of lncIrf8 and Irf8 in pDC upon CpG stimulation and a potential cross talk between lncIrf8 and Irf8 using our model that builds on DC differentiation. Given these rather different scenarios and the complexity of both systems, we prefer to not include these data in the paper.
c. Is there any significance of lncIRF8 in pDC functions (TLR stimulations, cytokine production, etc.) these aspects could be studied without much effort as authors already have systems that can knockout lncIRF8 promoter or overexpress lncIRF8.
We understand the reviewer’s point on the interest in a potential significance of lncIRF8 on pDC functions using knockout or overexpression studies. However this point is difficult to address: (i) The lncIrf8 promoter KO severely compromises pDC development and pDC are essentially absent in lncIrf8 promoter KO cultures. (ii) lncIRF8 overexpression is difficult, as overexpression of lncRNA in general, which is challenging. This is because lncRNA overexpression in lentivirus vectors is very different from protein overexpression. To limit lncIrf8 overexpression to the lncIrf8 sequence a polyA signal for transcription termination has to be inserted at the 3 ’end of lncIrf8 (Figure 4B). This polyA signal decrease the titer of lentivirus, which limits the overexpression capacity. We would expect to need a much higher lncIrf8 overexpression than the one achieved here (Figure 4 and Figure 4—figure supplement 1) to reveal a potential significance of lncIrf8 in pDC functions, such as TLR stimulations and/or cytokine production.
We are in accord with the reviewer that lncIrf8 might have other roles in DC biology than the one identified in our study. We now included a sentence on the potential other roles of lncIrf8 in DC biology, which are not yet identified in the current study (Discussion section, page 13, lines 435 and 436).
2. Though authors have identified that lncIRF8 is specifically expressed in pDC subtype but while understanding its significance and regulation, it was preferred to study bulk DC cultures overlooking the finding regarding specificity. Experiments performed by employing dCas9 VP64/KRAB-GFP constructs, do provide some direction in terms of mechanistic understanding of the cross-talk between the promoter of IRF8 and lncIRF8. This data needs to be further strengthened by sorting pDC, cDC1 and cDC2 populations and studying the effect of the genetic manipulations on IRF8 and lncRNA expression in each cell type.
We appreciate the reviewer’s suggestion on studying the effects of dCas9-VP64/KRAB-GFP constructs targeting the lncIrf8 and Irf8 promoters in sorted pDC, cDC1 and cDC2 populations. However, this is difficult and in some cases not possible, since the dCas9-VP64/KRAB-GFP constructs are introduced in HoxB8 multipotent progenitors (HoxB8 MPP) (Figure 5—figure supplement 1A) and following Flt3L directed DC differentiation some DC subsets are very low in abundance to even absent (Figures 5 and 6). For example, in the dCas9-KRAB experiment in Figure 6A-E, frequencies of cDC1 and pDC are very low to absent. In the dCas9-VP64 experiment in Figure 5 cDC1 and pDC are there, but the impact of targeted CRISPR activation on the lncIrf8 promoter is mainly seen in HoxB8 MPP at day 0 of DC differentiation (Figure 5C) and the activation effect is largely lost in the differentiated cells at later time points. Thus, studying the impact of targeted CRISPR activation and interference on IRF8 and lncRNA expression in DC subsets should include also MPP/CDP and pre-DC, and should be ideally performed by scRNA-seq and scATAC-seq. This analysis is beyond the scope of the present study and will be subject of our future work.
3. One of the most important findings of the submitted study is to demonstrate that knocking out the lncIRF8 promoter also diminishes the expression of IRF8, pDC as well as cDC1 development (Figure 2). Once the IRF8 expression is suppressed the analysis of pDC and cDC1 differentiation is redundant. It is previously reported that the deletion of +32 Kb enhancer (Durai et al. 2019) in a mouse model did not affect pDC but specifically depleted cDC1 development. Authors should examine the deletion of only the +32 kb enhancer region in their culture system to sort out the hierarchy between the +32 Kb enhancer and +32 Kb lncRNA promoter in terms of DC subtype development (cDC1 Vs pDC). Does the deletion of the lncIRF8 promoter lead to epigenetic changes at the overall +32 region? It is expected that due to interactions and chromatin loop formation between IRF8 promoter with various enhancers elements; deletion of lncIRF8 promoter might play an important role in defining these interactions in a cell type-specific manner. These interactions become more valuable by the fact that activation of the lncIRF8 promoter leads to enhanced cDC1 differentiation. Does this simply mean more open chromatin conformation around the cDC1-specific +32 Kb region rather than correlating with the lncIRF8?
We agree with the reviewer that deletion of the lncIrf8 promoter might lead to epigenetic changes in the entire +32 region and might have multiple effects, such as affecting transcription factor binding, histone modifications, open chromatin regions and chromatin interactions. Similarly, targeting dCas9-VP64 to the lncIrf8 promoter for CRISPR activation caused more cDC1 differentiation, which might be also due to multiple effects, such as changes in transcription factor binding, chromatin interactions and/or chromatin conformation. Here CRISPR activation of the lncIrf8 promoter might cause +32 kb enhancer activation and as a consequence IRF8 activation and more cDC1 (Figure 5A-E).
We now followed the request of the reviewer and deleted the +32 kb enhancer region with the four 4 AICE elements as described by Durai et al., Nat. Immunol., 2019 by using the same gRNAs as described in Durai et al., in the following referred to as cDC1 specific +32 kb enhancer KO. In parallel, we also designed a novel pair of gRNAs to delete this +32 kb enhancer region. We followed the same procedure as the lncIrf8 promoter KO experiments (Figure 2—figure supplement 1A) to obtain homozygous clones of cDC1 specific +32 kb enhancer KO. Five out of 165 single-cell clones were subjected to DC differentiation and further analyzed. cDC1 specific +32 kb enhancer KO did not affect pDC differentiation but compromised cDC1 development, which is in line with the phenotypes described by Durai et al., Nat. Immunol., 2019 and Murakami et al., Nat. Immunol., 2021 in mice.
These new results are now shown in new Figure 2—figure supplement 4 in the revised manuscript (Results section, page 8, lines 235-249 and page 35). The Materials and methods section was also updated accordingly (page 18, lines 576-605). Please see above our response to Essential Revision 2.
Can authors analyze the epigenetic changes by performing similar experiments in some of the available pDC and cDC1 cell lines? As these cell lines are likely to have active IRF8 transcription and it would help to measure the effect of lncIRF8 promoter deletion/mutation/activation/repression on IRF8 expression and specific DC subtype maintenance.
We thank the reviewer for the suggestion to study the epigenetic changes reported here in some of the available pDC and cDC1 cell lines. However, we hesitate to do so, since established cell lines have inevitably suffered from mutation and multiple alterations, which are often unknown and/or hard to control. We rather have built our study on conditional immortalization of mouse bone marrow cells by HoxB8 (referred to as HoxB8 MPP; Figure 1—figure supplement 2A).
These conditionally immortalized HoxB8 MPP exhibit an essentially unlimited lifespan when grown with estrogen (E2), when HoxB8 is active (Xu et al., Eur. J. Immunol., 2022). Additionally, they stop proliferating when E2 is removed and faithfully recapitulate hematopoietic stem cell commitment, DC differentiation and allow genetic manipulations by CRISPR/Cas tools. Thus, we consider HoxB8 MPP a particularly versatile and useful model to study gene regulations during DC differentiation. Repeating the study in pDC and cDC1 cell lines will, in our opinion, only provide limited new information but entails the risk of picking up aberrant phenomena, which are inherent to studies in cell lines.
4. Based on the various earlier published reports and results from the current study authors have shown a schematic representation of the pDC and cDC1-specific interactions. Can these interactions be validated in primary/ex-vivo DCs and be correlated with lncIRF8 as well as IRF8 expression? Will it be possible for authors to validate these interactions in light of the lncIRF8 promoter deletion/mutation/activation/repression? Authors may be careful to perform these experiments in purified pDC and cDC1 fractions to get meaningful data.
In case it is not possible to perform experiments with primary DCs/genetically modified HOXB8 cultures; if required, some of these experiments may be performed in the cell lines wherein overall interaction between various enhancer elements and promoters can be validated.
We agree with the reviewer that validating the pDC and cDC1-specific interactions mediated by lncIrf8 and Irf8 in primary/ex-vivo DC should be interesting. In this context we now provide data that ex-vivo pDC express lncIrf8 similar to in vitro BM pDC (new Figure 1—figure supplement 3), which indicates that the gene regulation patterns we found in our in vitro systems also exist in vivo.
Primary/ex-vivo DC have a limited lifespan in vitro, thus it will be challenging to impossible to generate genetically modified cells for studying gene regulations in primary cells. In particular, deletions of cis-regulatory elements (such as enhancer or promoter sequences) that require single-cell clones, are not possible with primary mouse cells due to lifespan restrictions. These limitations made us to develop the conditional immortalized mouse bone marrow HoxB8 MPP system (Figure 1—figure supplement 2A; Xu et al., Eur. J. Immunol., 2022). Experimental details of the HoxB8 MPP-DC differentiation system are now included in a Guideline paper by Lutz et al., Eur. J. Immunol., 2022. We added this reference to the reference list and cite this paper along with the Xu et al., 2022 paper as indicated.
5. Authors have also identified a TCONS_00190258 which is specifically expressed in the cDC1 subtype. Why not look at the expression levels of this transcript in the various experiments in the current study as enhancer element crosstalk will help in cell type-specific transcription of IRF8, lncIRF8, and TCONS_00190258?
We really appreciate the suggestion made by the reviewer. We have now provided evidence that Tcons_00190258 is also express in ex-vivo tissue (new Figure 1—figure supplement 3C). Studying the impact of Tcons_00190258 on DC differentiation and its potential crosstalk with lncIrf8/Irf8 both in vitro and in vivo will be subject of our future work and described elsewhere.
Reviewer #2 (Recommendations for the authors):
I find the quality and interpretation of the genomics data to be excellent and I have no major concerns with that data. I do have some specific comments around the interpretation of the cellular data and the model presented.
Specific comments
1. The authors conclude that lncIRF8 promoter KO hematopoietic progenitors are not capable of differentiating to pDCs (Figure 2C, Figure 2 Suppl 3D). However, it is now understood that Irf8 is not essential for pDC development (see Sichien Immunity 2016 where they study WT:IRF8-/- BM chimeras) and that Irf8 deregulates some key markers, including CD11b. The authors need to account for this discrepancy. Are pDCs with aberrant marker expression produced in the lncIRF8 promoter KO, and if not, why does this phenotype differ from the Irf8 KO?
We appreciate the point made by the reviewer. In our study on DC differentiation of lncIrf8 promoter KO in vitro and following transplantation into sublethally irradiated mice in vivo, we observed low to absent CD11c+ CD11b- B220+ pDC (Figure 2C, Figure 2—figure supplement 3) or Gr1- Siglec H+ pDC (Figure 3A and D, G and J), respectively.
We know the work by Sichien et al., 2016 on WT:Irf8-/- BM chimeras and the conclusions reached from their work that IRF8 is dispensable for pDC development. The results in Sichien et al., 2016 are based on WT:Irf8-/- BM chimeras upon transplantation in lethally irradiated mice and thus the divergent results compared to the classical Irf8-/- mice might be due to the experimental set-up WT:Irf8-/- BM chimeras versus Irf8 knockout mice (see also our response above to Essential revisions 2).
We now followed the advice of the reviewer and checked whether the lncIrf8 promoter KO had an impact on DC marker genes, such as CD11c and CD11b. We visualized CD11c and CD11b expression (gating on single cells) on day 7 and 9 of Flt3L directed DC differentiation in lncIrf8 promoter KO and control cells. In lncIrf8 promoter KO cells CD11c expression was slightly reduced and CD11b expression was slightly increased (see above Author response image 2 and our response to Essential revisions 2).
This indicates that lncIrf8 promoter KO does not have a major effect on CD11c and CD11b expression. Given this negative result we prefer not to include these data in the manuscript.
2. There does not appear to be compelling evidence that IRF8 binds in the lncIRF8 promoter in cDC1. Instead, IRF8 binds in the more 5' part of the +32kb enhancer identified by Durai 2019. How then is a putative IRF8 repressor complex linked to the lncIRF8 promoter as the authors propose? Moreover, the model does not appear to address why the IRF8 repressor complex binding to the IRF8 repressor complex would be active in cDC1 and not pDC.
The point made by the reviewer is well taken. In our model we proposed that binding of the putative IRF8 repressor to the +32 kb enhancer is mainly in the 5’ part of the +32 kb enhancer, as demonstrated by the ChIP-seq of IRF8. There is a strong IRF8 binding signal detected in cDC and weak to no IRF8 binding signal in pDC (Figure 1A, Figure 1—figure supplement 1, Figure 2—figure supplement 2 and Figure 7B, ChIP-seq IRF8 signal).
Thus, we propose the putative IRF8 repressor complex is only active in cDC and not pDC (Figure 7B). We agree with the reviewer that at this stage of analysis we do not know why IRF8 binds to the 5’ part of the +32 kb enhancer in cDC but not in pDC. Addressing this point should be subject for further studies. See also our response above to Essential revisions 3.
3. The authors use 2 versions of the HoxB8 MPP differentiation system (lines 207-234). Firstly, it is unclear, why two similar approaches are used, but more importantly, the second system outlined in figure 2 Suppl 3 shows that the lncIRF8 promoter is also required for cDC2 differentiation. This is a concern, as IRF8 is not expressed in cDC2, and Irf8-/- mice produce cDC2.
In the present study we used two approaches to differentiate the lncIrf8 promoter KO cells, thereby following the DC differentiation systems described in Xu et al., Eur. J. Immunol., 2022:
The first system is spontaneous DC differentiation, where we just remove estrogen (E2) and keep SCF, Flt3L (low concentration), hyper-IL6 and IGF1 in culture. Under these conditions most of the lncIrf8 promoter KO cells moved to Gr1+ monocytes (Figure 2E). This spontaneous DC differentiation mimics in vitro the differentiation potential of these cells in vivo upon cell transplantation, where most of the lncIrf8 promoter KO cells also differentiated into Gr1+ monocytes (Figure 3B and H).
The second system is Flt3L directed DC differentiation, where estrogen (E2) and the growth promoting cytokines SCF, Flt3L (low concentration), hyper-IL6 and IGF1 are removed and high Flt3L concentration is applied to direct HoxB8 MPP differentiation towards DC, and all DC subsets (cDC1, cDC2 and pDC) are obtained. In the manuscript we used both approaches, spontaneous DC differentiation (-E2) and Flt3L directed DC differentiation, to support the conclusions reached.
Concerning the reduced cDC2 population in Figure 2—figure supplement 3G, this is because the cDC2 population was gated from CD11c+ cells and calculated based on living single cells. In the lncIrf8 promoter KO most cells differentiated into Gr1+ monocytes (Figure 2E) and thus CD11c+ cDC2 were much lower in the lncIrf8 promoter KO compared to control.
We now also calculated cDC2 based on CD11c+ cells and show these data as new Figure 2—figure supplement 3H. We did not observe significant differences between lncIrf8 promoter KO and control cells on day 8 and 10. On day 6 the spontaneous DC differentiation of control cells progressed slower than the lncIrf8 promoter KO cells, thus lower frequency of cDC2 was observed. We added this information to the legend for Figure 2—figure supplement 3 (page 34, lines 1214-1217).
https://doi.org/10.7554/eLife.83342.sa2Article and author information
Author details
Funding
German Research Foundation
- Martin Zenke
German Ministry of Science and Technology (Fibromap)
- Ivan G Costa
Interdisciplinary Center for Clinical Research Aachen
- Ivan G Costa
- Martin Zenke
China Scholarship Council (202008080170)
- Huaming Xu
CAPES-Alexander von Humboldt Foundation (99999.001703/2014-05)
- Marcelo AS de Toledo
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Acknowledgements
We acknowledge the support of the Interdisciplinary Center for Clinical Research Aachen (IZKF Aachen) FACS Core Facility and Genomics Facility. We thank Magdalena Karpinska and Marieke Oudelaar for help with the Capture-C experiments and analysis, Susanne Schmitz and Paul Wanek for assistance, Stefan Rose-John for hyper-IL-6, Lisa Weixler and Carmen Schalla for help with enzyme purification, and Thomas Hieronymus for valuable discussion and suggestions. Part of this work was supported in part by funds from German Research Foundation (DFG) to MZ and from the Germany Ministry of Science and Technology (BMBF - Fibromap) and the Interdisciplinary Center for Clinical Research Aachen (IZKF Aachen) from the RWTH Medical Faculty to IC. HX was supported by a fellowship of China Scholarship Council (CSC) (Grant number 202008080170). MAST was funded by CAPES-Alexander von Humboldt postdoctoral fellowship (99999.001703/2014–05) and donation by U Lehmann.
Ethics
All the animal experiments were approved by the local authorities of the German Federal State North Rhine-Westphalia, Germany according to the German animal protection law (reference number 81-02.04.2018.A228).
Senior Editor
- Satyajit Rath, Indian Institute of Science Education and Research (IISER), India
Reviewing Editor
- Florent Ginhoux, Agency for Science Technology and Research, Singapore
Reviewer
- Stephen L Nutt, Walter and Eliza Hall Institute of Medical Research, Australia
Version history
- Preprint posted: August 12, 2022 (view preprint)
- Received: September 8, 2022
- Accepted: March 13, 2023
- Accepted Manuscript published: March 14, 2023 (version 1)
- Version of Record published: March 27, 2023 (version 2)
Copyright
© 2023, Xu et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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