Epigenetic and Developmental Regulation of Mammalian Genes

LDB1复合体，包括GATA-1和TAL1，在成年小鼠红系细胞中介导了β-珠蛋白基因控制区(LCR)和基因之间的长距离相互作用。在一个人类红系细胞模型中，胎儿的伽马珠蛋白基因可以被细胞因子强有力地重新激活，我们证明了LDB1复合体通过与基因的第3位点介导了伽马珠蛋白/LCR的接近。这些基因的重新激活对β-地中海贫血和镰状细胞疾病是有治疗作用的，了解其潜在的机制至关重要。结果表明，当ETO2与LDB1复合体结合时，γ-珠蛋白/LCR的相互作用和表达减少。为了确定ETO2下游可能调节从伽马到β-珠蛋白转录转换的因子，我们在对照和ETO2耗竭的人红系细胞中进行了RNA-Seq。我们确定并正在研究400多个基因(Padj&lt；0.05)，这些基因在没有ETO2的情况下发生了错误的调节。为了确定血红蛋白转换是否需要ETO2，我们在CD34+脐带血细胞中建立了这一过程的模型。在这些细胞分化过程中ETO2的下调支持了ETO2是伽马珠蛋白表达的抑制因子和人类血红蛋白转换的调节因子的观点。此外，RNA-Seq实验确定了近4000个基因(Padj&lt；0.01)在转换过程中受到差异调控。在这些基因中，超过200个是已知的红系指纹基因，正在研究中。 参与LCR环的伽马珠蛋白基因的LDB1位点3位于BGL3的序列中，BGL3是一种非编码的RNA转录本。在不同的红系细胞模型中，BGL3的转录水平与伽马珠蛋白基因相似，尽管水平要低得多。我们正在研究BGL3在血红蛋白转换过程中的作用。我们正在通过RT-PCR和RNA-FISH跟踪BGL3和伽玛珠蛋白的转录本。为了研究伽马珠蛋白的表达是否依赖于BGL3转录本或转录本身，我们正在敲除或过度表达BGL3，并检测其对伽马珠蛋白长程LCR相互作用和转录的影响。我们还瞄准了BGL3序列的缺失，并研究了它的蛋白质相互作用组。 我们正在利用小鼠ES细胞中的同源重组来研究单个珠蛋白基因如何建立阶段特异性增强子通信。我们通过同源重组将小鼠胚胎epsilon y基因上游的一个区域和成年β-主要基因上游的第二个区域定位于ES细胞中的一个等位基因。用重组酶介导盒交换在这两个位置插入染色质绝缘子人HS5或转录终止子。在ES细胞分化过程中，转录终止子或hHS5在LCR和下游基因之间的插入会以等位基因特异性的方式抑制胚胎ey基因的激活，但这两种插入都不会影响成年β-珠蛋白基因的表达。我们已经成功地将我们的靶向ES细胞注射到了胚泡中。我们正在生产的小鼠品系将被用来了解在红系发育过程中，β-珠蛋白基因的长距离相互作用和基因转录是如何受到插入的干扰的。这些实验是新颖的，因为它们改变了内源性基因座中的染色质组织。

The Ldb1 complex, including GATA-1 and TAL1, mediates long range interaction between the beta-globin locus control region (LCR) and gene in adult mouse erythroid cells. In a human erythroid cell model in which fetal gamma-globin genes can be robustly re-activated by cytokines, we showed that the Ldb1 complex mediates gamma-globin/LCR proximity through a site 3 to the genes. Reactivation of these genes is therapeutic in beta-thalassemia and sickle cell disease and understanding the underlying mechanisms is critical. The results suggested that when Eto2 associates with the Ldb1 complex gamma-globin/LCR interactions and expression are reduced. To identify factors functioning downstream of Eto2 that may regulate switching from gamma to beta-globin transcription, we performed RNA-Seq in control and Eto2 depleted human erythroid cells. We identified and are studying over 400 genes (Padj < 0.05) that were mis-regulated in the absence of Eto2. To determine if Eto2 is required for hemoglobin switching, we established a model of this process in CD34+ umbilical cord blood cells. Knock down of ETO2 during differentiation of these cells supports the idea that Eto2 is a repressor of gamma-globin expression and a regulator of human hemoglobin switching. In addition, RNA-Seq experiments identified nearly 4,000 genes (Padj < 0.01) that were differentially regulated during switching. Of these, over 200 are known erythroid fingerprint genes and are under study. The Ldb1 site 3 to the gamma-globin genes that is involved in LCR looping is within sequences of BGL3, a non-coding RNA transcript. BGL3 transcription parallels that of the gamma-globin genes in various erythroid cell models although at a much lower level. We are studying the function of BGL3 during hemoglobin switching. We are following BGL3 and gamma-globin transcripts by RT-PCR and RNA-FISH. To investigate whether the expression of gamma-globin depends on the BGL3 transcript or transcription per se, we are knocking down or over-expressing BGL3 and examining the effect on gamma-globin long range LCR interactions and transcription. We are also targeting deletion of BGL3 sequences and investigating its protein interactome. We are using homologous recombination in mouse ES cells study how individual globin gene establish stage specific enhancer communication. We targeted a region upstream of the mouse embryonic epsilon y gene and a second region upstream of the adult beta-major gene on one allele in ES cells by homologous recombination. Recombinase mediated cassette exchange was used to insert chromatin insulator human HS5 or a transcription terminator in these two positions. In differentiating ES cells, insertion of the transcription terminator or hHS5 between the LCR and downstream genes inhibits embryonic ey gene activation in an allele-specific fashion but neither insertion affects adult beta-globin gene expression. We have undertaken blastocyst injection of our successfully targeted ES cells. The mouse lines we are producing will be used to understand how long range interactions and gene transcription in the beta-globin locus are perturbed by the insertions during erythroid development. These experiments are novel since they alter chromatin organization in an endogenous locus.