Transcription factor antagonism regulates heterogeneity in embryonic stem cell states.

Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development.

ZNF281 enhances cardiac reprogramming by modulating cardiac and inflammatory gene expression.

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.

ZFP281 controls transcriptional and epigenetic changes promoting mouse pluripotent state transitions via DNMT3 and TET1.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

ZFP281 coordinates DNMT3 and TET1 for transcriptional and epigenetic control in pluripotent state transitions.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naïive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

Zfp281 Inhibits the Pluripotent-to-Totipotent State Transition in Mouse Embryonic Stem Cells.

The cell-fate transition between pluripotent and totipotent states determines embryonic development and the first cell-lineage segregation. However, limited by the scarcity of totipotent embryos, regulators on this transition remain largely elusive. A novel model to study the transition has been recently established, named the 2-cell-like (2C-like) model. The 2C-like cells are rare totipotent-like cells in the mouse embryonic stem cell (mESC) culture. Pluripotent mESCs can spontaneously transit into and out of the 2C-like state. We previously dissected the transcriptional roadmap of the transition. In this study, we revealed that Zfp281 is a novel regulator for the pluripotent-to-totipotent transition in mESCs. Zfp281 is a transcriptional factor involved in the cell-fate transition. Our study shows that Zfp281 represses transcripts upregulated during the 2C-like transition via Tet1 and consequentially inhibits mESCs from transiting into the 2C-like state. Interestingly, we found that the inhibitory effect of Zfp281 on the 2C-like transition leads to an impaired 2C-like-transition ability in primed-state mESCs. Altogether, our study reveals a novel mediator for the pluripotent-to-totipotent state transition in mESCs and provides insights into the dynamic transcriptional control of the transition.

ZNF281/Zfp281 is a target of miR-1 and counteracts muscle differentiation.

Defects in achieving a fully differentiated state and aberrant expression of genes and microRNAs (miRs) involved in differentiation are common to virtually all tumor types. Here, we demonstrate that the zinc finger transcription factor ZNF281/Zfp281 is down-regulated during epithelial, muscle, and granulocytic differentiation in vitro. The expression of this gene is absent in terminally differentiated human tissues, in contrast to the elevated expression in proliferating/differentiating ones. Analysis of the 3'UTR of ZNF281/Zfp281 revealed the presence of numerous previously undescribed miR binding sites that were proved to be functional for miR-mediated post-transcriptional regulation. Many of these miRs are involved in differentiation pathways of distinct cell lineages. Of interest, ZNF281/Zfp281 is able to inhibit muscle differentiation promoted by miR-1, of which ZNF281/Zfp281 is a direct target. These data suggest that down-regulation of ZNF281/Zfp281 during differentiation in various cell types may occur through specific miRs whose expression is tissue-restricted. In addition, we found that in rhabdomyosarcoma and leiomyosarcoma tumors, the expression of ZNF281/Zfp281 is significantly higher compared with normal counterparts. We extended our analysis to other human soft tissue sarcomas, in which the expression of ZNF281 is associated with a worse prognosis. In summary, we highlight here a new role of ZNF281/Zfp281 in counteracting muscle differentiation; its down-regulation is at least in part mediated by miR-1. The elevated expression of ZNF281/Zfp281 in soft tissue sarcomas warrants further analysis for its possible exploitation as a prognostic marker in this class of tumors.

The transcription factor Zfp281 sustains CD4+ T lymphocyte activation through directly repressing Ctla-4 transcription.

The expression of coinhibitory receptors, such as CTLA-4, on effector T cells is a key mechanism for the negative regulation of T-cell activation. However, the transcriptional regulation of CTLA-4 is not well understood. Zfp281, a C2H2 zinc finger protein, is a negative regulator of pluripotency maintenance of embryonic stem cells. Nevertheless, the function of Zfp281 in differentiated cells has not been studied. We generated Zfp281 conditional knockout mice in which the function of the Zfp281 gene was conditionally disrupted by the Cd4Cre transgene to study its impact on T cell function. Zfp281 had no effect on T-cell development, but CD4+ T cell activation and cytokine production were impaired due to diminished T-cell receptor signaling. Furthermore, Zfp281 deficiency inhibited in vivo T cell responses to Listeria monocytogenes infection. Using genome-wide expression profiling assays, we determined that Zfp281 repressed Ctla-4 expression by directly binding to GC-rich sites in its promoter, which inhibited the negative feedback of T cell activation. In line with this result, CTLA-4 blockade and shRNA knockdown partly rescued the reduced cytokine production caused by Zfp281 deficiency. These findings indicate that Zfp281 sustains CD4+ T lymphocyte activation by directly repressing Ctla-4 transcription.

ZFP281 Recruits MYC to Active Promoters in Regulating Transcriptional Initiation and Elongation.

The roles of the MYC transcription factor in transcriptional regulation have been studied intensively. However, the general mechanism underlying the recruitment of MYC to chromatin is less clear. Here, we found that the Krüppel-like transcription factor ZFP281 plays important roles in recruiting MYC to active promoters in mouse embryonic stem cells. At the genome scale, ZFP281 is broadly associated with MYC, and the depletion of ZFP281 significantly reduces the levels of MYC and RNA polymerase II at the ZFP281- and MYC-cobound genes. Specially, we found that recruitment is required for the regulation of the Lin28a oncogene and pri-let-7 transcription. Our results therefore suggest a major role of ZFP281 in recruiting MYC to chromatin and the integration of ZFP281 and the MYC/LIN28A/Let-7 loop into a multilevel circuit.

Zfp281 Shapes the Transcriptome of Trophoblast Stem Cells and Is Essential for Placental Development.

Placental development is a key event in mammalian reproduction and embryogenesis. However, the molecular basis underlying placental development is not fully understood. Here, we conduct a forward genetic screen to identify regulators for extraembryonic development and identify Zfp281 as a key factor. Zfp281 overexpression in mouse embryonic stem cells facilitates the induction of trophoblast stem-like cells. Zfp281 is preferentially expressed in the undifferentiated trophoblast stem cell population in an FGF-dependent manner, and disruption of Zfp281 in mice causes severe defects in early placental development. Consistently, Zfp281-depleted trophoblast stem cells exhibit defects in maintaining the transcriptome and differentiation capacity. Mechanistically, Zfp281 interacts with MLL or COMPASS subunits and occupies the promoters of its target genes. Importantly, ZNF281, the human ortholog of this factor, is required to stabilize the undifferentiated status of human trophoblast stem cells. Thus, we identify Zfp281 as a conserved factor for the maintenance of trophoblast stem cell plasticity.