Chromatin Structure, Epigenetic and Developmental Regulation of Mammalian Gene

真核生物基因组在不同水平上组织成染色体区域、转录区室和拓扑关联结构域（TADs），这些结构特征在很大程度上在不同细胞类型和跨物种之间共享。相反，在TADs中，染色质环连接增强子及其靶基因，以建立独特的转录组，将细胞和组织彼此区分开来，并成为发育和分化的基础。我们一直在研究广泛表达的Lim结构域结合1蛋白LDB1，我们已经证明LDB1通过形成具有红系细胞类型特异性的复合物在造血过程中连接增强子和基因中起着关键作用。 GATA1、TAL1和LMO2是红系细胞转录因子，有助于LDB1复合物的染色质成环和转录激活特性。我们最近使用红系祖细胞和胎肝细胞的工作表明，阻遏蛋白ETO 2和激活蛋白hemogen各自参与LDB1复合物，并招募相反作用的辅助调节因子来进行LDB1结合增强子的表观遗传调节。ETO 2募集抑制性NuRD复合物，而血红素募集SWI/SNF重塑复合物。这两个转录因子产生了一个拨动开关，以调节红细胞生成过程中基因表达中LDB 1复合体的功能。我们还进行了全基因组CRISPR敲除筛选（GecKO），以鉴定红细胞生成所需的表观遗传新因子和机制。组蛋白伴侣HIRA是一个突出的候选人，我们正在积极调查。 LDB 1是一种从蠕虫到人类广泛表达的高度保守蛋白。我们最近发现LDB1在胚胎干细胞中的功能非常有限，但随着细胞向造血分化而变得重要。这为在ESC分化为造血细胞期间启用红系增强子库打开了一扇窗。长期以来，人们一直认为LDB 1在其他发育途径中发挥重要作用，如神经发生、心脏发生和视网膜发生，但对其作用的机制缺乏了解。通过在肝脏中有条件地敲除LDB1，我们已经揭示了LDB1在调节代谢基因表达中的作用。在人肝细胞系中的机制研究进一步揭示了LDB 1与已知对肝基因调控重要的转录因子组的协作。 我们以前观察到，增强子RNA（eRNA），BGLT 3，在人类β-珠蛋白基因座内编码，有助于LDB 1在基因座中的长距离染色质环，这激活珠蛋白基因转录。我们现在已经进行了荟萃分析，以确定全球eRNA在红系细胞中从事与基因启动子的长距离相互作用。我们将使用候选人来解决这个问题，是否是eRNA本身，基因座或转录的基因座，在染色质循环和基因表达的功能性作用。总的来说，我们的研究旨在广泛深入地了解LDB1和辅助调节因子如何协调健康和疾病中不同细胞类型的染色质拓扑结构的变化。

The eukaryotic genome is organized at varying levels into chromosome territories, transcriptional compartments and topologically associating domains (TADs), which are architectural features largely shared between different cell types and across species. In contrast, within TADs, chromatin loops connect enhancers and their target genes to establish unique transcriptomes that distinguish cells and tissues from each other and underlie development and differentiation. We have been studying the widely expressed Lim domain binding 1 protein, LDB1, which we have shown plays a critical role in connecting enhancers and genes during hematopoiesis by forming a complex with erythroid cell-type specificity. GATA1, TAL1 and LMO2 are erythroid transcription factors that contribute to the chromatin looping and transcription activation properties of the LDB1 complex. Our recent work using erythroid progenitor cells and fetal liver cells shows that a repressor protein, ETO2, and an activator protein, hemogen, each participate in the LDB1 complex and recruit oppositely acting co-regulators to carry out epigenetic modulation of LDB1-binding enhancer. ETO2 recruits the repressive NuRD complex and hemogen recruits SWI/SNF remodeling complex. These two transcription factors create a toggle switch to regulate LDB1 complex function in gene expression during erythropoiesis. We have also carried out a genome wide CRISPR knock out screen (GecKO) to identify epigenetic novel factors and mechanisms required for erythropoiesis. The histone chaperone HIRA is a prominent candidate that we are actively investigating. LDB1 is a widely expressed and highly conserved protein from worms to humans. We recently found that LDB1 functions in embryonic stem cells are very limited but become important as cells are differentiated towards hematopoiesis. This has opened a window on the commissioning of the erythroid enhancer repertoire during differentiation of ESC to hematopoietic cells. LDB1 has long been known to have important roles in other developmental pathways such as neurogenesis, cardiogenesis and retinogenesis, although a mechanistic understanding of its role is lacking. By conditional knock out of LDB1 in liver we have uncovered a role for LDB1 in regulation of metabolic gene expression. Mechanistic studies in a human liver cell line further reveal collaboration of LDB1 with a cohort of transcription factors known to be important for liver gene regulation. We had previously observed that an enhancer RNA (eRNA), BGLT3, encoded within the human beta-globin locus, contributes to LDB1 long range chromatin looping in the locus, which activates globin gene transcription. We have now carried out a meta-analysis to identify globally eRNAs in erythroid cells that engage in long range interactions with gene promoters. We will use the candidates to address the question whether it is the eRNA per se, the locus or transcription of the locus that has a functional role in chromatin looping and gene expression. Overall, our studies are aimed at broadly and deeply understanding how LDB1 and co-regulators orchestrate changes in chromatin topology in diverse cell types in health and disease.