Zinc finger protein Zfp335 controls early T-cell development and survival through β-selection-dependent and -independent mechanisms

T-cell development in the thymus undergoes the process of differentiation, selective proliferation, and survival from CD4−CD8− double negative (DN) stage to CD4+CD8+ double positive (DP) stage prior to the formation of CD4+ helper and CD8+ cytolytic T cells ready for circulation. Each developmental stage is tightly regulated by sequentially operating molecular networks, of which only limited numbers of transcription regulators have been deciphered. Here, we identified Zfp335 transcription factor as a new player in the regulatory network controlling thymocyte development in mice. We demonstrate that Zfp335 intrinsically controls DN to DP transition, as T-cell-specific deficiency in Zfp335 leads to a substantial accumulation of DN3 along with reduction of DP, CD4+, and CD8+ thymocytes. This developmental blockade at DN stage results from the impaired intracellular TCRβ (iTCRβ) expression as well as increased susceptibility to apoptosis in thymocytes. Transcriptomic and ChIP-seq analyses revealed a direct regulation of transcription factors Bcl6 and Rorc by Zfp335. Importantly, enhanced expression of TCRβ and Bcl6/Rorc restores the developmental defect during DN3 to DN4 transition and improves thymocytes survival, respectively. These findings identify a critical role of Zfp335 in controlling T-cell development by maintaining iTCRβ expression-mediated β-selection and independently activating cell survival signaling.

Introduction T-cell development proceeds in a series of developmental stages, which is precisely orchestrated by multiple signaling and molecular networks (Hosokawa and Rothenberg, 2021;López-Rodríguez et al., 2015;Rothenberg, 2014). Prethymic progenitor cells originated from bone marrow migrate into the thymus and sequentially differentiate into CD4 − CD8 − (DN), CD4 + CD8 + (DP), and the CD4 + or CD8 + (SP) stage. Based on the expression of CD44 and CD25, DN thymocytes are divided into several phenotypically distinct stages, including DN1 to DN4 (Rothenberg et al., 2008;Yang et al., 2010;Yui and Rothenberg, 2014;Kurd and Robey, 2016). In the presence of Notch signaling, early thymic progenitor (ETP)-DN1 cells transit into DN2a stage, initiating the T-cell lineage commitment, which is immediately accompanied by TCRβ gene arrangement. The majority of DN2 cells enter the DN3 stage with αβ lineage potential (Godfrey et al., 1993). Only DN3 cells with a complete pre-TCR complex, which consists of the functional TCRβ protein, pre-Tα (pTα) chain, and CD3 molecule, can successfully trigger the subsequent maturation into DN4 and DP thymocytes. Further differentiation into mature CD4 + or CD8 + T cells requires positive and negative selection at DP stage before they migrate to peripheral lymphoid organs (Dudley et al., 1994;Hoffman et al., 1996;von Boehmer and Fehling, 1997;Malissen et al., 1999).
Pre-TCR signals regulate thymocyte differentiation by mediating protection from apoptosis, stimulating proliferation, and inducing allelic exclusion at the TCRβ locus in post-β-selection DN3b cells and promoting DN to DP transition (Hoffman et al., 1996;Aifantis et al., 1997;Kruisbeek et al., 2000). Inactivation of pre-TCR components dampens T-lymphocyte development by arresting thymocytes at the DN3 stage and inducing apoptosis (Fehling et al., 1995;Malissen et al., 1995;Mombaerts et al., 1992a;Mombaerts et al., 1992b). Multiple transcription factors downstream of pre-TCR signaling are involved in T-cell differentiation and survival. The major pre-TCR signaling is conducted through the dose-dependent expression of Notch controlled by the Id3-E2A axis (Liu et al., 2021). Abrogation of either Notch or E2A expression may lead to the developmental block of thymocytes at multiple stages (Ikawa et al., 2006;Shah and Zúñiga-Pflücker, 2014;Belle and Zhuang, 2014). In addition, activation of NF-κB (Voll et al., 2000), Ets1 (Eyquem et al., 2004), and NFAT5 (Berga-Bolaños et al., 2013) by pre-TCR signals also contributes to the developmental block. The transcription factor T-cell factor 1 (TCF1), together with its downstream Bcl-11b, not only increases the potential to differentiate into T cells (Li et al., 2010;Ikawa et al., 2010), but also positively regulates thymocyte development via promoting TCRβ recombination and expression, as well as DP cell survival (Albu et al., 2007;Li et al., 2013). Apart from the essential role in the T follicular helper cell lineage commitment (Yu et al., 2009), Bcl6 induced by pre-TCR signals is also involved in the DN to DP transition and protection of DN4 cells from apoptosis (Solanki et al., 2020). Additionally, abrogation of Rorc expression in thymocytes leads to a decreased DP proportion and impaired DP survival in a Bcl-xl-dependent manner (Villey et al., 1999;Sun et al., 2000;Xi et al., 2006).
Although pre-TCR signaling is crucial for the β-selection checkpoint, it is not sufficient for progression to the DN3 stage. Other pathways or transcription factors coupled with or independent of conventional pre-TCR signaling are found to play important roles in the process (Ciofani et al., 2004). The developmental blockade in Smarca5-or Nkap-deficient thymocytes is confirmed by intact pre-TCR signals in these mice (Pajerowski et al., 2009;Zikmund et al., 2019). Overall, it remains largely unknown which factors are crucial for T-cell development through mechanisms independent of pre-TCR signaling.
Zfp335, also known as the nuclear hormone receptor coregulator (NRC) -interacting factor 1 (NIF-1), is a zinc finger protein with a 13 C2H2 zinc finger repeating structure consisting of 1337 amino acids (Han et al., 2016). The C2H2-ZF family encodes more than 700 proteins in the human genome, some of which play important roles in ontogenesis, immune cell differentiation, and disease occurrence (Li et al., 2010;Heizmann et al., 2018), yet the biological characteristics and functions of most members are unclear (Stubbs et al., 2011;Emerson and Thomas, 2009). Zfp335 regulates gene transcription by recruiting H3K4 methyltransferase complexes, interacting with coactivators, or directly binding to certain gene promoters (Han et al., 2016;Yang et al., 2012;Mahajan et al., 2002;Wolfe et al., 2000). Zfp335 plays important regulatory roles in early embryonic development and neurogenesis (Yang et al., 2012). Germline knockout of Zfp335 is embryonic lethal, while deletion of Zfp335 gene in nerve cells impairs the proliferation and differentiation of nerve progenitor cells in mice, eventually leading to severe microcephaly (Yang et al., 2012). Function of Zfp335 in T-cell development has been observed in the study of the Zfp335bloto allele, a missense mutation derived from ENU mutagenesis. While thymocyte development is not significantly affected by this hypomorph mutation, there is a significant reduction in the number of peripheral T cells due to defects in the maturation and migration of thymocytes (Han et al., 2014). Without loss of function studies, it remains to be determined whether Zfp335 is required for intrathymic T-cell development.
In this study, we investigated Zfp335 expression during different thymocyte stages, as well as its function at the β-selection checkpoint and during DN to DP transition. We found that in the thymus, Zfp335 has the highest expression in DN3 thymocytes. Zfp335 is indispensable for thymocyte β-selection and supports the transition from DN to DP stage by maintaining intracellular TCRβ (iTCRβ) expression, as well as by promoting DN and DP thymocyte survival via directly regulating Bcl6 and Rorc expression.   The numbers of DN, DP, CD4, and CD8 thymocytes. (F-J) CD4 + and CD8 + cells in spleen and lymph nodes from WT and KO mice were measured by flow cytometry. (F) Representative FACS plots of CD4 + and CD8 + cells in spleen and lymph nodes. The percentage and number of CD4 + T cells (G) and CD8 + T cells (H) in the spleen from WT and KO mice. The percentage and number of CD4 + T cells (I) and CD8 + T cells (J) in the lymph nodes from WT and KO mice. Results represent three independent experiments. n = 4 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Figure 1D The percentages of DN, DP, CD4, and CD8 thymocytes from WT and KO mice.      Zfp335-deficient mice   To study the role of Zfp335 in T-cell development, we first assessed the expression of Zfp335 among  different thymocyte subsets, including DN3, DN4, DP, CD4, CD8, NKT, and γδ T cells. We found that DN3 cells displayed a relatively high level of Zfp335 mRNA expression ( Figure 1A). Flow cytometry analysis also revealed that Zfp335 protein had the highest expression in DN3 thymocytes ( Figure 1-figure supplement 1A, B). Consistently, microarray data from ImmGen showed higher expression of Zfp335 specifically at the DN3a stage during T-cell development from ETP to CD4/ CD8 SP (Figure 1-figure supplement 2A). Although RNA-seq data from ImmGen exhibited the highest expression of Zfp335 in DP thymocytes, a gradually increased expression was observed from ETP to DN3 (Figure 1-figure supplement 2B). Given the importance of DN3 stage during β-selection checkpoint, we obtained T-cell-specific Zfp335 mice by crossing Zfp335 fl/fl strain with Lck-Cre strain ( Figure 1-figure supplement 3A). Zfp335 deletion was confirmed by real-time PCR (qPCR) analysis in DN4 cells (Figure 1-figure supplement 3B). Strikingly, LckCre + Zfp335 fl/fl (KO) mice exhibited significantly smaller thymi and drastically decreased thymocyte numbers than WT control ( Figure 1B). Further analysis showed that both percentages and numbers of DP cells, as well as CD4 SP and CD8 SP cells, were considerably reduced in KO mice ( Figure 1C-E). Conversely, the percentage of DN cells was increased by nearly 10-15-folds, although the total number was decreased ( Figure 1C-E). In secondary lymphoid organs, we also observed reduced CD4 + and CD8 + cells in the spleen and lymph nodes ( Figure 1F-J). Thus, Zfp335 is essential for the development of αβT cells in the thymus.

Zfp335 intrinsically regulates T-cell development in the thymus
To address whether Zfp335 deletion intrinsically affects T-cell development, Lin − CD25 + CD44 − DN3 cells from WT or KO mice were harvested and plated with OP9-DL1 cells in the presence of Flt3L and IL-7, an in vitro model for T-cell development ( Figure 2A; Kondo et al., 2017). On both days 2 and 4, KO group produced fewer DP cells than WT control ( Figure Figure 3A, B), in which the numbers of both DN3 and DN4 thymocytes were decreased ( Figure 3C). The developmental blockade from DN3 to DN4 in Zfp335-deficient cells was verified by in vitro coculture assays on day 2 using OP9-DL1 cultured with WT and KO DN3 cells, respectively ( Figure 3D, E) or mixed at a 1:4 ratio ( Figure 3F, G) as described above. The in vivo bone marrow chimera models further confirmed the development block at the DN3 stage ( Figure 3H-K), indicating that Zfp335 is indispensable for DN3 to DN4 transition during early-stage differentiation.

Ablation of Zfp335 promotes apoptosis in thymocytes
During the β-selection, efficient proliferation of pre-T cells is necessary for DN to DP progression (Kreslavsky et al., 2012), during which the pre-TCR signal functions as a positive regulator of thymocyte survival, allowing for differentiation from pre-T cells into DP thymocytes. We sought to examine whether the defect in Zfp335 KO thymocyte development is due to impaired proliferation or survival. The in vivo BrdU incorporation assay showed comparable or even higher percentages of BrdU + DN3 and DN4 cells ( Figure 4A, B) as well as Ki67 + DN3 and DN4 cells ( indicating that Zfp335 regulates thymocyte apoptosis in a TCR-independent manner. Moreover, in mixed bone marrow chimeras, Zfp335-deficient DN3 and DN4 cells also displayed significantly higher Annexin V + cells ( Figure 4G, H), indicating an intrinsic role of Zfp355 in regulating thymocyte survival.
The online version of this article includes the following source data and figure supplement(s) for figure 2: Source data 1. Figure 2C, D. The percentages of DN, DP, CD4 + , and CD8 + thymocytes 2 and 4 days post separate culture in vitro.      Figure 5-figure supplement 3). Thus, the reduced iTCRβ expression may be a consequence of protein degradation. Given that Zfp355 deficiency led to diminished iTCRβ expression in DN4 cells, we next investigated the effect of TCR overexpression on aberrant thymocyte development caused by Zfp335 deficiency. Offspring (LckCre + Zfp335 fl/fl OT1 + ) of LckCre + Zfp335 fl/fl mice crossed to OT1 transgenic (Tg) mice was generated to constitutively express Tcra-V2 and Tcrb-V5 Tg gene (OT1 Tg KO). Notably, forced expression of αβTCR successfully restored the decreased percentage of iTCRβ + DN4 cells in the KO mice, despite with little impact on the number of iTCRβ + DN4 cells ( Figure 5G-I). Importantly, developmental arrest at the DN3 stage in Zfp335-deficient mice was fully rescued by OT1 transgene ( Figure 5J-L). Unfortunately, DN3 and DN4 cells from OT1 Tg KO mice still exhibited a similar degree of apoptosis with Zfp335-deficient cells ( Figure 5-figure supplement 4), suggesting Zfp335 affects thymocyte apoptosis in a TCR-independent manner. Consistently, the proportions of DN, DP, CD4, and CD8 were still comparable in KO and OT1 Tg KO mice ( Figure    The ratio of DN3 to DN4 thymocytes. (J, K) Full chimeric mice were generated by transplanting a mixed population of WT (CD45.1 + ) and KO (CD45.2 + ) bone marrow progenitor cells at a 1:4 ratio into lethally irradiated (8.5 Gy) WT recipient mice (CD45.1 + CD45.2 + ). Six weeks after transplantation, thymi from recipient mice were harvested. The expression of CD44 versus CD25 was measured by flow cytometry (n = 4). (J) Representative FACS plots of DN3 and DN4 thymocytes. (K) The ratio of DN3 to DN4 thymocytes. Results represent three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.
The online version of this article includes the following source data for figure 3: Source data 1. Figure 3B, C. The percentages and numbers of DN3 and DN4 thymocytes from WT and KO mice. supplement 5), demonstrating that the aberrant DP development has not yet been restored by αβTCR overexpression.
The online version of this article includes the following source data and figure supplement(s) for figure 5: Source data 1. Figure 5B. The percentages and numbers of iTCRβ + thymocytes in DN3 and DN4 cells from WT and KO mice.   Figure 6B). Prominently downregulated genes associated with lymphocyte differentiation and apoptosis were summarized in the heatmap ( Figure 6C). To further determine genes directly regulated by Zfp335, we analyzed Zfp335 by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). We screened a total of 2797 Zfp335-binding sites (Supplementary file 2) and the prevalence of binding peaks across genomic regions was displayed as a pie chart ( Figure 6-figure supplement 1). To identify the Zfp335-targeting candidates, 119 profoundly downregulated genes were further selected out by a cutoff of twofold change, and 22 genes were overlapped with Zfp335-targeting genes from ChIP-seq data ( Figure 6-figure supplement 2A,  Supplementary file 3). Among these genes, top 10 genes were listed based upon their expression level from RNA-seq result ( Figure 6-figure supplement 2B). qPCR analysis was performed to confirm their downregulation in KO DN4 cells ( Figure 6-figure supplement 2C). Next, we focused on genes related to lymphocyte differentiation and apoptosis. In line with RNA-seq results ( Figure 6C), qPCR analysis verified that Bcl6 and Rorc were significantly downregulated in KO DN4 cells ( Figure 6D). Importantly, Zfp335 directly targeted the promoter regions of Bcl6 and Rorc in ChIP-seq analysis ( Figure 6E), which were further verified by luciferase assay ( Figure 6F). Taken together, in depth genomic analysis of DN thymocytes supports that Zfp335 directly regulates the transcription of Bcl6 and Rorc.

Defects in thymocyte development caused by Zfp335 deficiency can be rescued by Bcl6 and Rorc
To determine whether Bcl6 and Rorc participate in the regulation of thymocyte development downstream of Zfp335, overexpression of Bcl6 and Rorc was conducted in vitro in the thymocyte development model ( Figure 7A). DN3 cells from KO mice were cocultured with OP9-DL1 cells and transduced with retrovirus encoding Mock-GFP, Zfp335-GFP, Bcl6-GFP, or Rorc-GFP. After 3.5 days, overexpression of Bcl6 and Rorc resulted in a substantial DP generation, particularly in the Bcl6 group, resulting in a similar DP proportion to that in Zfp335-overexpressing cells ( Figure 7B, C). Of note, overexpression of Psmg2, Dctn1, Ankle2, Cep76, Fgf13, and Ddx31 identified in the qPCR results ( Figure 6-figure supplement 2B, C) in KO DN3 cells did not restore DP generation in vitro (Figure 7-figure supplement 1). Importantly, enhanced expression of Bcl6 in DN3 cells rescued DN thymocyte apoptosis ( Figure 7D, E), while overexpression of both Bcl6 and Rorc rescued DP thymocytes from enhanced apoptosis ( Figure 7F, G). p53, negatively regulated by Bcl6, is involved in lymphocyte apoptosis (Haks et al., 1999;Guidos et al., 1996;Mombaerts et al., 1995). Thus, we crossed Zfp335 KO strain with Trp53 KO strain to obtain double knockout mice, in which Trp53 deletion resulted in a partial recovery of DP percentage, but not cell number (Figure 7-figure supplement 2), further supporting the role of Bcl6 in Zfp335-controlled thymocyte survival. Together, these data demonstrate that Zfp335 controls DN thymocyte survival through direct regulation of Bcl6 and Rorc expression.

Discussion
In this study, we reveal that Zfp335 is essential for thymocyte development, particularly during DN to DP transition. Zfp335 deficiency in T cells led to a significant loss of DP, CD4 SP, and CD8 SP cells while an accumulation of DN3 cells. Mechanistically, the developmental blockade is attributed to both impaired pre-TCR signal and increased susceptibility to apoptosis. Serving as a transcription factor,     Source data 1. Figure 7C. The percentages of DP cells differentiated from KO DN3 thymocytes transduced with Mock, or Zfp335, Bcl6, and Rorc genes.

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Zfp335 directly promotes Bcl6 and Rorc expression in DN thymocytes to ensure their survival during early development. Zfp335 was previously demonstrated to be crucial for early embryonic development as homozygous deletion of this gene resulted in neonatal death (Garapaty et al., 2009). Conditional knockout of Zfp335 in neural system led to severely reduced cortical size and impaired neurogenesis. Mechanistically, Zfp335 was required for neural progenitor cell self-renewal and proliferation, and neuronal differentiation (Yang et al., 2012) and neuronal morphogenesis (Zhao et al., 2015). Besides, deficiency of naive T cells in mice carrying a hypomorph allele of Zfp335 (Zfp335 bloto ) uncovered its role in immune system (Han et al., 2014). So far, there is still very limited information about the functions of Zfp335 in other aspects of immune system. Here, we found that Zfp335 is absolutely required for multiple steps of early T-cell development. Of note, it will be worth investigating whether and how Zfp335 is involved in the regulation of mature T-cell differentiation and functions under static and immunized conditions in future.
We have shown that Zfp335 expression was upregulated specifically in DN3 thymocytes and significantly decreased in the subsequent stages, suggesting a critical role at the DN3 stage. Of note, loss of Zfp335 led to a dramatic reduction in both thymus size and thymocyte number. The accumulation of DN3 cells results from an intrinsic mechanism that hinders the transition from DN3 to DN4 stage, leading to the reduction of DP thymocytes and mature T cells in the periphery. These data are in line with another recent study reporting that Zfp335 mutation led to a reduction in peripheral T cells as a result of defective naive T cells and SP thymocytes (Han et al., 2014). However, given the intact thymic selection with Zfp335 mutation, the report was inconsistent with our observation of decreased β-selection with Zfp335 deficiency. The discrepancy is likely due to the different approaches used to disrupt Zfp335 function since a single-nucleotide missense mutation of Zfp335 may affect its function differently. Nevertheless, by knocking out the entire Zfp335 protein, we provide evidence that Zfp335 is indispensable for early thymocyte development.
Thymocyte β-selection is a critical developmental checkpoint allowing for the progression from DN3 to DN4 stage and the maintenance of DP cell numbers, which is primarily dependent on TCRβ and pre-TCR signals constituted with a functional iTCRβ paired with a pTα chain (Yamasaki and Saito, 2007). Pre-TCR signaling regulates thymocytes differentiation, proliferation, and survival in the full developmental process (Koch and Radtke, 2011). In addition, there are reports that other pathways or transcription factors coupled with or independent of conventional pre-TCR signaling are found to play important roles in the process (Ciofani et al., 2004;Pajerowski et al., 2009;Zikmund et al., 2019). While Zfp335-deficient DN4 cells exhibited no defects in the rearrangement of TCRβ chain genes and pTα gene expression, our results clearly demonstrated that Zfp335 deficiency markedly impaired iTCRβ expression and led to an unbiased reduction of the majority of Vβ genes in DN4 populations. Future studies will investigate the mechanisms how Zfp335 regulates iTCRβ expression. Importantly, forced iTCRβ expression in DN3 and DN4 cells by transduction of OT1-TCR completely rescued the developmental impairment during the DN3-DN4 transition in Zfp335-deficient mice despite the failure to rescue the DN3, DN4, and DP population size. This suggests that Zfp335 controls the DN3-DN4 transition dependent on pre-TCR signals, but other mechanisms may also regulate the DP population size.
The large population of DP thymocytes is maintained by both cell proliferation and survival mechanisms. Zfp335-deficient DN3 and DN4 cells showed slightly higher or unchanged incorporation of BrdU, suggesting that cell proliferation was not affected. However, our data revealed a significant increase in apoptosis in Zfp335-deficient DN3 and DN4 thymocytes. Transcriptomic analysis (RNA-seq      and qPCR) unveiled the downregulation of Bcl6 and Rorc signaling which are critically involved in thymocyte apoptosis (Solanki et al., 2020;Xi et al., 2006). Indeed, we have demonstrated that Zfp335, a transcription factor, direct bound to the promoter regions of Bcl6 and Rorc genes. More importantly, enhanced expression of Bcl6 and Rorc could improve thymocyte survival and substantially restore the DP thymocyte population. Trp53 deletion resulted in a partial recovery of DP cells, further supporting the role of Bcl6 in Zfp335-controlled thymocyte survival. Of note, Zfp335 may also control thymocyte survival through directly regulating other targets.
In our study, Zfp335 is indispensable for thymocyte β-selection given that T-cell-specific deficiency in Zfp335 leads to impaired iTCRβ expression, blockade of thymocytes at DN stage as well as a substantial DN cell apoptosis. Though enhanced expression of TCRβ restores the developmental defect during DN3 to DN4 transition, it had little impact on the population size of DN3, DN4, and DP cells, suggesting the regulation of Zfp335 on DN cell apoptosis through mechanisms more than β-selection. Indeed, we provided the evidence that Zfp335-controlled DN cell survival through regulating Bcl6 and Rorc expression. Moreover, Zfp335 regulates TCRβ expression independent on Bcl6 and Rorc since overexpression of neither Bcl6 or Rorc in Zfp335-deficient DN3 thymocytes could restore the decreased iTCRβ expression (Figure 7-figure supplement 3).
Several key factors have been shown to involve in the regulation of T-cell developmental process from DN3 to DP stages. Notch signaling is required for early T-cell commitment and β-selection (Radtke et al., 2010;Ciofani and Zúñiga-Pflücker, 2005;Hosokawa and Rothenberg, 2018), which is subsequently weaken by Bcl6 repression for the differentiation from DN to DP stage development (Solanki et al., 2020). Consistently, our results found that Zfp335 could directly target Bcl6, and Zfp335 deficiency led to decreased expression of Bcl6 and upregulation of Notch target genes such as Dtx1 and Notch1 (data not shown), which collectively contributing to the developmental block from DN to DP stage. In addition, Tcf1 plays a vital role in T-cell lineage commitment since Tcf1 −/− DN3 thymocytes failed to progress to DN4 and subsequent DP stage through regulating Bcl11b and Gata3 expression (Garcia-Perez et al., 2020). Bcl11b and Gata3 also differentially regulate the differentiation and survival of thymocytes at DN3, DN4, and subsequent ISP stages via TCR or/and survival signals (Inoue et al., 2006;Pai et al., 2003). However, we did not detect reduced expression of Tcf1, Bcl11b, or Gata3 in Zfp335-deficient DN4 cells, suggesting that Zfp335 regulates DN3 to DP thymocyte development independent on these molecules.
In conclusion, our study reveals that the C2H2 zinc finger protein Zfp335 plays a novel and crucial role during thymocyte development, specifically during the transition from DN to DP stage. Mechanistically, Zfp335 promotes Bcl6 and Rorc signaling to prevent thymocytes apoptosis and ensure the survival and differentiation of thymocytes. Collectively, we provide evidence that Zfp335 is essential for thymocyte development through both pre-TCR-dependent and -independent mechanisms.

Antibodies and reagents
The following antibodies and kits were purchased from Biolegend (

Quantitative RT-PCR
Cell lysis was performed with RNA extraction and cDNA synthesis using Quick-RNA Microprep Kit (Zymo Research) and ReverTra Ace qPCR RT Master Mix Kit (TOYOBO), respectively. The qRT-PCR reactions were carried out using StepOnePlus Real-Time PCR Systems (ABI) with SYBR mixture (Genstar) to determine relative gene expression. The sequences for the primers are list in Supplementary file 4.
Zfp335-binding genes from ChIP-seq data.
• Supplementary file 4. Primers used for quantitative PCR.
• Transparent reporting form

Data availability
The sequencing data presented in this paper are available for download on GEO data repository with accession numbers GSE184532 and GSE184705. Source data files have been provided for relevant figures.
The following datasets were generated: Author (