Regulation of DNA methylation by TETs and QSER1

该项目的目标是发现基本的表观基因组调控机制，使细胞 在胚胎发育的早期阶段确定命运。在发育早期，外胚层向胚细胞的定向分化 层之后是驱动谱系命运的关键调控基因的激活。这些基因控制着正常的 发育和基础广泛的人类结构性出生缺陷的遗传基础。我们研究了 羟基化酶泰特家族的成员，其调节DNA的去甲基化或阻断活性 DNA甲基化，控制基因表达。我们发现了在早期阶段对泰特酶的需求， 在斑马鱼模型中的发育，以及从人胚胎干细胞（hESC）的祖细胞特化。 理解上的主要差距包括：i）是否共同或不同的机制控制脱甲基化， 来自不同胚层的祖细胞，ii）不同的泰特家族成员是否区分发育的 程序，和iii）TcB如何靶向调节区如二价启动子。怀疑 除了TcB之外，还需要其他蛋白质来靶向DNA去甲基化，我们进行了全基因组的 CRISPR筛选并发现了QSER 1，这是一种以前未表征的染色质结合蛋白。我们展示 QSER 1是二价启动子和发育基因增强子的关键监护人， 那些存在于DNA甲基化谷中的基因，广泛分布于不同的细胞命运中。我们发现了生物化学和遗传学 QSER 1和TbR之间的相互作用，表明它们合作保护转录和 甲基化的发育程序。QSER 1变异等位基因最近与冠状动脉 疾病，而QSER 1 Parkinson，PRR 12的单倍不足与多器官发育相关。 出生缺陷综合症我们建议充分探索遗传关系和下游网络， 泰特/QSER 1（TQ）家族成员，包括它们如何控制甲基化和影响染色质 结构的背景下，两个互补的模型系统，斑马鱼和人胚胎干细胞。斑马鱼模型允许 对潜在的补偿或合作家族成员（包括Tet 1，Tet 2，Tet 3，QSER 1， 和PRR 12）。hESC模型提供了 杰出的生物化学和“组学”能力，并在开发人类祖细胞和分化 细胞多PI项目代表了研究者之间的持续合作， 重叠的专业知识，具有良好的生产力记录。具体目标是确定相对 这些基因对指导早期祖细胞命运的贡献，发现了它们在其中的调控网络， 功能，并测试相互作用的因素作为候选人连接TQ甲基化控制染色质修饰。 因为甲基化的调节是决定祖细胞命运的基本步骤，我们的结果将是 与器官发生和结构性出生缺陷广泛相关。

The goal of this project is to discover fundamental epigenomic regulatory mechanisms that commit cells to defined fates during early stages of embryogenesis. Early in development, commitment of the epiblast to germ layers is followed by activation of key regulatory genes that drive lineage fate. These genes control normal development and underlie the genetic basis for a broad range of human structural birth defects. We have studied members of the TET family of hydroxylation enzymes, which regulate the demethylation of DNA, or block active methylation of DNA, to control gene expression. We discovered requirements for TET enzymes during early development in the zebrafish model, and for progenitor specification from human embryonic stem cells (hESCs). Major gaps in understanding include: i) whether common or distinct mechanisms control demethylation for progenitors from different germ layers, ii) whether different TET family members distinguish developmental programs, and iii) how TETs are targeted to regulatory regions such as bivalent promoters. Suspecting that additional proteins beyond TETs are needed to target DNA demethylation, we carried out a genome-wide CRISPR screen and discovered QSER1, a previously uncharacterized chromatin-binding protein. We showed that QSER1 is a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys, broadly across different cell fates. We found biochemical and genetic interactions between QSER1 and TETs, suggesting that they cooperate to safeguard transcriptional and developmental programs from methylation. QSER1 variant alleles were recently linked to coronary artery disease, while haploinsufficiency of a QSER1 paralog, PRR12, is associated with multi-organ developmental birth defect syndromes. We propose to fully explore the genetic relationships and downstream networks of TET/QSER1 (TQ) family members, including how they function to control methylation and impact chromatin structure in the context of two complementary model systems, zebrafish and hESCs. The zebrafish model allows full genetic analysis of potentially compensatory or cooperating family members (including tet1, tet2, tet3, qser1, and prr12), in an animal model with highly conserved developmental programs. The hESC model provides outstanding biochemical and “omics” capacity, and validation in developing human progenitor and differentiated cells. The multi-PI project represents a continued collaboration among investigators with complementary and overlapping expertise, with a strong record of productivity. Specific Aims are proposed to determine the relative contribution of these genes for directing early progenitor fate, discover the regulatory networks in which they function, and to test interacting factors as candidates for linking TQ methylation control to chromatin modification. Because regulation of methylation is a fundamental step of progenitor fate determination, our results will be broadly relevant to organogenesis and structural birth defects.