Dynamic regulation of lineage-specific Polycomb repressive landscapes by pioneer and PRDM transcription factors

先锋和 PRDM 转录因子对谱系特异性 Polycomb 抑制景观的动态调节

• 批准号: 10438974
• 负责人: Makiko IWAFUCHI
• 金额: $ 33.39万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10438974
• 关键词: Binding; Binding Sites; Biological Assay; CRISPR interference; Cell Differentiation process; Cells; ChIP-seq; Chromatin; Complex; Data; Deposition; Development; Disease model; Doxycycline; Endoderm; Endoderm Cell; Epigenetic Process; Event; Fibroblasts; Future; Gene Activation; Gene Expression; Genes; Genetic Transcription; Genomics; Germ Cells; Goals; HNF4A gene; Hepatocyte; Human; Hybrid Cells; Impairment; Instruction; Knowledge; Longitudinal Studies; Mediating; Methods; Mus; Nuclear Extract; Nucleosomes; PRC1 Protein; PRDM1 gene; Play; Polycomb; Positioning Attribute; Process; Regenerative Medicine; Regulation; Repression; Role; Signal Transduction; Site; Technology; Testing; Therapeutic; Time; cell type; gene repression; genomic locus; human pluripotent stem cell; imprint; improved; insight; novel; pluripotency; programs; recruit; tool; transcription factor; transcriptome sequencing

Abstract The goal of this proposal is to determine how lineage-specific repressive epigenetic landscapes are established to safeguard cell identity. Current dogma is that pioneer TFs initiate lineage-specific activation processes by locally opening chromatin. In contrast, significantly less is known about how repressive epigenetic landscapes are dynamically reshaped during cell differentiation. This knowledge gap creates a major stumbling block in developing high-fidelity cell reprogramming technology. Hence, it is essential to define how alternative lineage programs are repressed and how this process can be manipulated. Intriguingly, our preliminary data unexpectedly revealed that pioneer TFs are involved in this process. We developed a new doxycycline (dox)- inducible CRISPR interference (CRISPRi) tool to target two pioneer TFs: OCT4 in human pluripotent stem cells (hPSCs) and FOXA1/A2/A3 (FOXA) in hPSC-derived endoderm differentiation. We found that OCT4 and FOXA play critical roles in repressing alternative-lineage programs. In fact, gene expression changes in OCT4/FOXA- CRISPRi cells correlate better with directional changes of the repressive H3K27me3 mark than with those of H3K4me1/3 active marks. To identify mechanisms underlying OCT4/FOXA-directed repression, we focused on the OCT4 and FOXA binding peaks that co-localize with the H3K27me3 domain. Interestingly, we found that these loci are highly enriched at key alternative-lineage genes, and unbiased motif analysis revealed significant enrichment for a PRDM14 (a key pluripotency TF) binding motif at OCT4 sites and a PRDM1 (a key germ-cell TF) binding motif at FOXA sites. This data provides the first indication that PRDM1 may play an important role in endoderm specification. Further, our ChIP-qPCR assays confirmed that OCT4-CRIPSRi hPSCs were impaired in recruiting PRDM14, PRC1/H2Aub, and PRC2 at OCT4-bound H3K27me3 sites. Intriguingly, our preliminary co-IP assay suggested that FOXA interacts with PRC1, and prior co-IP studies showed that OCT4 interacts with PRC1, and PRDM14 and PRDM1 interact with PRC25–9. Collectively, our findings led to our overarching novel hypothesis that pioneer TFs play a crucial role in establishing Polycomb repressive domains at key alternative- lineage genes by recruiting PRC1 and/or PRDM TFs, which in turn recruit PRC2. We will test this hypothesis in two Aims: Aim 1: Test the hypothesis that OCT4 and FOXA play instructive roles in recruiting PRCs and PRDM TFs to establish lineage-specific Polycomb repressive domains. Aim 2: Determine how PRDM1 impacts accurate lineage restriction during human endoderm specification. The successful completion of our Aims will provide valuable insights and reveal novel mechanisms underlying lineage-specific Polycomb repression to safeguard cell identity. In the long-term, this study will likely open new avenues to manipulate cell fate by selectively repressing alternative regulatory programs, with potential future therapeutic applications in regenerative medicine.

摘要 这个提议的目标是确定谱系特异性抑制性表观遗传景观是如何 以保护细胞身份。目前的教条是先驱TF启动谱系特异性激活 通过局部打开染色质进行加工。相比之下，关于抑制性表观遗传 景观在细胞分化过程中被动态地重塑。这种知识差距造成了一个重大的绊脚石 阻碍了高保真细胞重编程技术的发展。因此，必须确定替代方案 血统程序被压抑以及这个过程如何被操纵。有趣的是，我们的初步数据 出乎意料地揭示了先驱TF参与了这一过程。我们开发了一种新的强力霉素（dox）- 诱导型CRISPR干扰（CRISPRi）工具靶向两种先驱TF：人类多能干细胞中的OCT 4 F0 XA 1/A2/A3（F0 XA）在hPSC衍生的内胚层分化中的作用。我们发现OCT 4和FOXA 在抑制替代性血统程序中起着关键作用。事实上，OCT 4/FOXA基因表达的变化， CRISPRi细胞与抑制性H3 K27 me 3标记的方向性变化的相关性比CRISPRi细胞与抑制性H3 K27 me 3标记的方向性变化的相关性更好。 H3 K4 me 1/3有效标记。为了确定OCT 4/FOXA介导的抑制的潜在机制，我们重点研究了 与H3 K27 me 3结构域共定位的OCT 4和FOXA结合峰。有趣的是，我们发现， 这些位点在关键的替代谱系基因上高度富集，无偏基序分析显示， 在OCT 4位点富集PRDM 14（关键多能性TF）结合基序，并在OCT 4位点富集PRDM 1（关键生殖细胞因子）结合基序。 TF）结合基序。这一数据首次表明PRDM 1可能发挥重要作用 在内胚层规格中。此外，我们的ChIP-qPCR测定证实，OCT 4-CRIPSRi hPSC受损， 在OCT 4结合的H3 K27 me 3位点招募PRDM 14、PRC 1/H2 Aub和PRC 2。有趣的是，我们初步的 co-IP分析表明FOXA与PRC 1相互作用，先前的co-IP研究表明OCT 4与PRC 1相互作用。 PRC 1、PRDM 14和PRDM 1与PRC 25 -9相互作用。总的来说，我们的发现导致了我们的总体小说 假设先驱转录因子在关键替代区建立Polycomb抑制结构域中起着至关重要的作用- 通过募集PRC 1和/或PRDM TF，PRC 1和/或PRDM TF又募集PRC 2，来调节谱系基因。我们将测试这个假设， 两个目标： 目的1：检验OCT 4和FOXA在招募PRC和PRDM TF以 建立谱系特异性Polycomb抑制域。 目的2：确定PRDM 1如何影响人内胚层规范过程中的准确谱系限制。 我们的目标的成功完成将提供有价值的见解，并揭示新的机制， 谱系特异性Polycomb抑制以保护细胞身份。从长远来看，这项研究可能会开辟新的 通过选择性抑制替代调节程序来操纵细胞命运的途径，具有潜在的未来 再生医学中的治疗应用。