Rtf1-dependent transcriptional pausing regulates cardiogenesis
- Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, United States
Figures
Figure 1
Generation of zebrafish rtf1 null mutants with CRISPR/Cas9.
(a) Schematic of CRISPR/Cas9 mutagenesis of the rtf1 gene. Two mutant alleles, rtf1LA2678 and rtf1LA2679, were recovered after targeting of rtf1 exon 3. Both alleles are predicted to disrupt translation of the Rtf1 protein and eliminate the histone modification domain (HMD), Plus3, polymerase II (Pol II) interaction, and Polymerase Associated Factor 1 Complex (PAF1C) interaction domains. (b) Agarose gel electrophoresis results of genotyping rtf1 mutants by PCR. Deletions in rtf1LA2678 and rtf1LA2679 alleles can be distinguished from wild-type allele using primers rtf1-e3-F and rtf1-e3-R. (c) Western blot detecting Rtf1 and β-actin (loading control) proteins in lysates of wild-type and rtf1 mutant embryos. The image is representative of two independent experiments.
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Figure 1—source data 1
PDF file containing original agarose gel image for Figure 1b, indicating the relevant bands and genotypes.
- https://cdn.elifesciences.org/articles/94524/elife-94524-fig1-data1-v1.zip
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Figure 1—source data 2
Original file for agarose gel image displayed in Figure 1b.
- https://cdn.elifesciences.org/articles/94524/elife-94524-fig1-data2-v1.zip
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Figure 1—source data 3
PDF file containing original western blots for Figure 1c, indicating the relevant bands and genotypes.
- https://cdn.elifesciences.org/articles/94524/elife-94524-fig1-data3-v1.zip
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Figure 1—source data 4
Original files for western blots displayed in Figure 1c.
- https://cdn.elifesciences.org/articles/94524/elife-94524-fig1-data4-v1.zip
Figure 2 with 1 supplement
Characterization of early cardiac development in Rtf1-deficient embryos.
(a) Representative images of RNA in situ hybridization detecting myl7 expression in 24 hpf zebrafish embryos. (b) Quantification of myl7 signal intensity in control and Rtf1-deficient embryos at 24 hpf. Numbers on bars indicate the number of embryos analyzed. (c) Representative images of RNA in situ hybridization detecting mef2ca expression in 8 somite stage zebrafish embryos. (d) Quantification of mef2ca signal intensity in control and Rtf1-deficient embryos at the 8 somite stage. Numbers on bars indicate the number of embryos analyzed. (e) Representative images of RNA in situ hybridization detecting nkx2.5 expression in 8 somite stage zebrafish embryos. (f) Quantification of nkx2.5 signal intensity in control and Rtf1-deficient embryos at the 8 somite stage. Numbers on bars indicate the number of embryos analyzed. (g) Representative images of RNA in situ hybridization detecting tbx5a expression in 8 somite stage zebrafish embryos. (h) Quantification of tbx5a signal intensity in control and Rtf1-deficient embryos at the 8 somite stage. Numbers on bars indicate the number of embryos analyzed. (i) Representative images of RNA in situ hybridization detecting tbx20 expression in 8 somite stage zebrafish embryos. (j) Quantification of tbx20 signal intensity in control and Rtf1-deficient embryos at the 8 somite stage. Numbers on bars indicate the number of embryos analyzed. Scale bars in a, c, e, g, and i represent 0.1 mm. *: p<0.05, **: p<0.01, ***: p<0.001 based on Fisher’s exact test.
Figure 2—figure supplement 1
Bright-field microscopic images of 2 days post-fertilization (d2) wild-type (a) and rtf1LA2679 mutant (b) embryos.
Figure 3
Failure of cardiac differentiation upon loss of Rtf1 in the mammalian cardiac mesoderm.
(a) Diagram of generation of cardiac mesoderm-specific Rtf1 knockout mouse embryos. A Mesp1:Cre insertion allele was bred into an Rtf1 exon 3 floxed background. Rtf1flox/+ mice with Mesp1Cre/+ were bred to homozygous Rtf1flox/flox females, resulting in one quarter of embryos lacking Rtf1 activity in the cardiac mesoderm (Rtf1 CKO). (b) Representative images of RNA in situ hybridization detecting Nkx2.5 (wild type n=7, Rtf1 CKO n=5) and Tbx20 (wild type n=16, Rtf1 CKO n=4) gene expression in Cre-negative (wild-type siblings) and Cre-positive Rtf1flox/flox (Rtf1 CKO) embryos at E8.5. (c) Quantification of Nkx2.5 and Tbx20 signal intensity in Cre-negative (wild-type siblings) and Cre-positive Rtf1flox/flox (Rtf1 CKO) embryos at E8.5. Numbers on bars indicate the number of embryos analyzed. *: p<0.05, ***: p<0.001 based on Fisher’s exact test.(a) created with BioRender.com.
Figure 4
Failure of cardiac differentiation from Rtf1 knockdown mouse embryonic stem cells (mESCs).
(a) Diagram of short hairpin RNA (shRNA) knockdown of Rtf1 in mESCs and differentiation of plated embryoid bodies (EBs). mESCs were transduced with non-target control or Rtf1 shRNA lentivirus and selected with puromycin. Transduced mESCs were grown into EBs using the hanging drop method, which were then plated to examine differentiation into cell types, including beating cardiomyocyte clusters. (b) Western blot verifying reduction of Rtf1 protein in Rtf1 shRNA mESCs (~70% reduced based on densitometry) compared to unchanged level of the loading control protein β-actin. Image is representative of three independent experiments. (c) qPCR analysis of Myh6, Nkx2-5, and Nppa expression in non-target control (NT) and Rtf1 shRNA plated EBs. Data are normalized to the mean expression level in NT shRNA samples, and error bars indicate the standard error of the mean. (d) Mean percentage (± standard error) of plated EBs exhibiting beating cardiomyocyte differentiation (cEBs) in NT and Rtf1 shRNA plated EBs. (e) qPCR analysis of Brachyury expression in non-target control (NT) and Rtf1 shRNA plated EBs. (f) qPCR analysis of Afp expression in non-target control (NT) and Rtf1 shRNA plated EBs. (g) qPCR analysis of Ncam1 expression in non-target control (NT) and Rtf1 shRNA plated EBs. Data in e, f, and g are normalized to the mean expression level in NT shRNA samples at day 0 (D0), and error bars indicate the standard error of the mean. *: p<0.05, ***: p<0.001. (a) created with BioRender.com.
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Figure 4—source data 1
PDF file containing original western blot for Figure 4b, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/94524/elife-94524-fig4-data1-v1.zip
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Figure 4—source data 2
Original file for the western blot displayed in Figure 4b.
- https://cdn.elifesciences.org/articles/94524/elife-94524-fig4-data2-v1.zip
Figure 5 with 1 supplement
Dysregulation of cardiac gene expression in the Rtf1-deficient lateral plate mesoderm.
(a,b) Projections of confocal z-stacks of hand2:GFP signal in 11 somite stage uninjected control (a) and rtf1 morphant (b) embryos. Scale bars represent 100 µm. (c) Diagram of lateral plate mesoderm (LPM) FACS RNA-seq experiment. Transgenic hand2:GFP embryos were injected at the one-cell stage with rtf1 morpholino and grown to the 10–12 somite stage prior to dissociation and sorting based on GFP expression. RNA isolated from GFP-positive LPM cells was subjected to RNA-seq and compared to RNA from uninjected embryo LPM cells. (d) Heatmap of scaled expression levels of the top 40 genes most significantly differentially expressed (based on p-value) between uninjected control and rtf1 morphant hand2:GFP-positive LPM. High and low z-scores represent high and low gene expression, respectively. (e) Hierarchical clustering of Biological Process gene ontology (GO) terms based on semantic similarity for the genes most significantly downregulated in the rtf1 morphant hand2:GFP-positive LPM. (c) created with BioRender.com.
Figure 5—figure supplement 1
Gating strategy for 10–12 somite stage zebrafish hand2:GFP FACS.
Abnormal cells or multiplets were excluded by gating out events with high side scatter (SSC) and forward scatter (FSC). Live hand2:GFP-positive cells (pink cells in panel d) were sorted based on high GFP and low DAPI signal. (a) Gating control experiment using unstained cells from non-transgenic embryos. (b) Gating control experiment using unstained cells from hand2:GFP transgenic embryos. (c) Gating control experiment using DAPI-stained cells from non-transgenic embryos. (d) Gating of sorting sample for analysis using DAPI-stained cells from hand2:GFP transgenic embryos. 0.6% of the total cells were sorted as live GFP-positive cells. Plots are representative of 4 independent experiments.
Figure 6 with 5 supplements
Rtf1 is required for transcriptional progression of cardiac precursors.
(a–c) UMAP plots of anterior lateral plate mesoderm (ALPM) and derivatives from merged and integrated single-cell RNA-seq datasets of 11–12 somite stage uninjected control and rtf1 morphant embryos. Cells in plots are colored by cell type (a), sample (b), and pseudotime (c). (d,f) Expression dynamics plots displaying expression levels (y-axis) for selected genes in uninjected control (d) and rtf1 morphant (f) cells over the pseudotime trajectory (x-axis) from ALPM (root) to cardiac precursor state shown in (c). (e,g) UMAP plots of uninjected control (e) and rtf1 morphant (g) cells colored by gene expression levels.
Figure 6—figure supplement 1
UMAP plots of 11–12 somite stage control and rtf1 morphant embryo integrated multimodal (ATAC+Gene Expression) single-cell sequencing datasets split based on sample (control on left vs. rtf1 morphant on right).
Cells are colored based on Seurat cluster. Predicted identities of each cell type are based on manual inspection of marker gene expression.
Figure 6—figure supplement 2
Clustered dot plot displaying expression of marker genes in cells belonging to each Seurat cluster identified in the integrated multimodal single-cell sequencing analysis of 11–12 somite stage control and rtf1 morphant embryos.
Each cluster is further split by sample (control vs. rtf1 morphant).
Figure 6—figure supplement 3
Comparison of proportions of cells belonging to each Seurat cluster identified in the integrated multimodal single-cell sequencing analysis of 11–12 somite stage control and rtf1 morphant embryos.
Predicted identities of each cell type based on marker gene expression are displayed to the left of the Seurat cluster IDs. Cell-type names are colored based on whether they are significantly expanded (pink) or significantly reduced (light blue) in rtf1 morphants based on Fisher’s exact test with an adjusted p-value cutoff of 0.05.
Figure 6—figure supplement 4
Rtf1 deficiency impacts differentiation of the anterior lateral plate mesoderm.
Pie charts displaying the abundances of cell types identified in analysis of re-clustered anterior lateral plate mesoderm (ALPM) and derivatives from merged and integrated single-cell RNA-seq datasets of 11–12 somite stage uninjected control (a) and rtf1 morphant (b) embryos.
Figure 6—figure supplement 5
Genes relevant to the development of the lateral plate mesoderm and its derivatives are dysregulated in Rtf1-deficient embryos.
(a–z) Violin plots showing the expression of genes relevant to the development of the lateral plate mesoderm and its derivatives. Each dot represents the expression level of a gene in a single cell. Violin-shaped areas are color-coded based on cell types identified by re-clustering anterior lateral plate mesoderm (ALPM) and derivative cells from merged and integrated single-cell RNA-seq datasets of 11–12 somite stage uninjected control and rtf1 morphant embryos. Shaded violins are not present when statistically significant expression of a particular gene was not detected.
Figure 7
Rtf1’s Plus3 domain is necessary for cardiac progenitor formation.
(a) Schematics of Rtf1 wild-type (wt*) and Rtf1 mutant constructs (ΔHMD and ΔPlus3). Domains are indicated by colored boxes (histone modification domain [HMD]: red, Plus3: blue, polymerase II [Pol II], and Polymerase Associated Factor 1 Complex [PAF1C] interaction: gray). Deleted regions are represented by lines. Numbers refer to the amino acid positions in wild-type Rtf1. Rtf1 wt*, ΔHMD, and ΔPlus3 also harbor 8 silent mutations that alter the rtf1 translation blocking morpholino binding site. (b) Projections of confocal z-stacks of whole-mount immunostaining detecting N-terminal FLAG-tagged Rtf1 constructs (red) expressed in 75% epiboly zebrafish embryos. Nuclei are labeled by DAPI staining (blue). Scale bars represent 20 µm. (c) Representative images of RNA in situ hybridization detecting nkx2.5 expression in 10 somite stage (10S) zebrafish embryos co-injected with rtf1 morpholino and mRNA encoding Rtf1 wild-type (wt*) or mutant (ΔHMD and ΔPlus3) proteins. Scale bars represent 0.1 mm.
Figure 8 with 1 supplement
Rtf1-dependent transcriptional pausing regulates cardiogenesis.
(a) Cumulative frequency plot of pause release ratios (PRRs) of 6078 genes with substantial RNA Polymerase II (RNA Pol II) signal. Both control (red) and flavopiridol-treated rtf1 morphant (light blue) samples displayed significantly different PRR frequencies compared to untreated rtf1 morphants (blue). ***: p<2.2 × 10–16; Welch’s paired two-tailed t-test. The median PRR of control samples is indicated by a vertical gray dashed line. (b) Dot plot comparing PRRs in controls and rtf1 morphants. Each dot represents the PRR values for a single gene that differ significantly (colored point) or are not significantly different (gray point) between controls and rtf1 morphants. (c) Dot plot comparing PRRs in rtf1 morphants and flavopiridol-treated rtf1 morphants. Each dot represents the PRR values for a single gene that differ significantly (colored point) or are not significantly different (gray point) between rtf1 morphants and flavopiridol-treated rtf1 morphants. Dot colors in (b) and (c) are based on the density of points, with lighter colors indicating more dense points. (d) Representative images of RNA in situ hybridization detecting myl7 expression in 24 hpf control and Rtf1-deficient (±flavopiridol) zebrafish embryos. (e) Quantification of myl7 signal intensity in control and Rtf1-deficient (±flavopiridol) embryos at 24 hpf. Numbers on bars indicate the number of embryos analyzed. ***: p<0.001. (f) Representative images of RNA in situ hybridization detecting myl7 expression in 24 hpf control and Rtf1-deficient (±cdk9 morpholino) zebrafish embryos. (g) Quantification of myl7 signal intensity in control and Rtf1-deficient (±cdk9 morpholino) embryos at 24 hpf. Numbers on bars indicate the number of embryos analyzed. ***: p<0.001. (h) Venn diagram of significantly altered gene expression in 8–9 somite stage rtf1 morphant embryos (±flavopiridol treatment). Shaded circles represent genes that are significantly: downregulated in rtf1 morphants vs. uninjected controls (blue), upregulated in rtf1 morphants vs. uninjected controls (yellow), downregulated in flavopiridol-treated rtf1 morphants vs. vehicle-treated (DMSO) rtf1 morphants (green), and upregulated in flavopiridol-treated rtf1 morphants vs. vehicle-treated rtf1 morphants (pink). Overlapping regions with thick black outlines represent the 1447 genes (*) with expression levels that were significantly rescued in 8–9 somite stage rtf1 morphants by flavopiridol treatment. (i) Plot of enriched Biological Process gene ontology terms for the set of genes that were significantly rescued in rtf1 morphants by flavopiridol treatment. Dot sizes indicate the number of genes associated with a given ontology term. Dot color indicates the significance level (adjusted p-value). (j) Selected list of genes that are critical to cardiac development and were significantly rescued in rtf1 morphants by flavopiridol treatment. Fold-changes and adjusted p-values were calculated with DESeq2. Scale bars in panels d and f represent 0.1 mm.
Figure 8—figure supplement 1
Promoter-proximal pausing of RNA Polymerase II is diminished at some cardiac mesoderm-related genes in Rtf1-deficient embryos.
Representative ChIP-seq tracks displaying RNA Polymerase II (RNA Pol II) read densities (y-axis) for control (red), rtf1 morphant (blue), and flavopiridol-treated rtf1 morphant (light blue) embryos at cardiac mesoderm-related genes, including hand2 (a), gata5 (b), aplnrb (c), bmp4 (d), nkx2.7 (e), and rbfox1l (f).
Additional files
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Supplementary file 1
Sequences of primers used for quantitative real-time PCR analysis of gene expression in mouse embryonic stem cells.
- https://cdn.elifesciences.org/articles/94524/elife-94524-supp1-v1.xlsx
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Supplementary file 2
Predicted identities of cell types belonging to each Seurat cluster identified in analysis of 11–12 somite stage control and rtf1 morphant embryo integrated multimodal (ATAC +Gene Expression) single-cell sequencing datasets.
Predicted identities were based on manual inspection of marker gene expression.
- https://cdn.elifesciences.org/articles/94524/elife-94524-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/94524/elife-94524-mdarchecklist1-v1.pdf
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Source data 1
DESeq2 results file containing differential gene expression analysis data comparing FACS-isolated hand2:GFP-positive lateral plate mesoderm (LPM) from control and rtf1 morphant 10–12 somite stage embryos.
- https://cdn.elifesciences.org/articles/94524/elife-94524-data1-v1.xlsx
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Source data 2
Significantly restricted marker genes for Seurat cell clusters from integrated multimodal (ATAC+Gene Expression) single-cell sequencing dataset from control and rtf1 morphant embryos at the 11–12 somite stage.
- https://cdn.elifesciences.org/articles/94524/elife-94524-data2-v1.xlsx
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Rtf1-dependent transcriptional pausing regulates cardiogenesis
eLife 13:RP94524.
https://doi.org/10.7554/eLife.94524.3
