Regulation of DNA methylation by TETs and QSER1

这个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决定命运。在发育的早期，外胚层对胚芽的承诺 蛋鸡之后是决定血统命运的关键调控基因的激活。这些基因控制正常 人类先天缺陷的发生和发展是其遗传基础。我们已经研究过 Tet羟化酶家族的成员，调节DNA的去甲基化，或阻断活性 DNA甲基化，以控制基因表达。我们在早期发现了对Tet酶的需求 斑马鱼模型的发展，以及人类胚胎干细胞(HESCs)的祖细胞规格。 认识上的主要差距包括：一)控制去甲基化的共同或不同机制 来自不同胚层的祖细胞，II)不同的TET家族成员是否有不同的发育 计划，以及iii)TET如何针对二价启动子等受调控区域。怀疑这一点 需要Tets以外的额外蛋白质来靶向DNA去甲基化，我们进行了全基因组范围的 CRISPR筛选并发现了QSER1，一种以前未鉴定的染色质结合蛋白。我们展示了 QSER1是发育基因的二价启动子和稳定增强子的关键守护者，尤其是 这些人居住在DNA甲基化山谷，广泛分布在不同的细胞命运中。我们发现了生化和基因 QSER1和TETS之间的相互作用，表明它们协同保护转录和 甲基化所产生的发育程序。最近发现QSER1变异等位基因与冠状动脉相关 疾病，而QSER1同源基因PRR12的单倍性不足与多器官发育相关 出生缺陷综合症。我们建议充分探索沙门氏菌的遗传关系和下游网络 TET/QSER1(TQ)家族成员，包括它们如何控制甲基化和影响染色质 在两个相辅相成的模型系统--斑马鱼和人类胚胎干细胞--的背景下的结构。斑马鱼模型允许 对潜在补偿或合作的家庭成员(包括Tet1、TET2、Tet3、qser1、 和prr12)，在高度保守的发育程序的动物模型中。HESC模型提供了 突出的生化和组学能力，以及在培养人类祖细胞和分化方面的验证 细胞。多PI项目代表着调查人员之间的持续合作，具有互补性和 专业知识重叠，有很强的生产力记录。提出了确定相对目标的具体目标 这些基因对指导早期祖先命运的贡献，发现它们在其中的调控网络 功能，并测试相互作用因素作为连接TQ甲基化控制和染色质修饰的候选因素。 由于甲基化调控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.