Epigenetic and Developmental Regulation of Mammalian Genes

Ldb 1/NLI复合物，包括加塔-1和TAL 1，介导成年小鼠红系细胞中β-珠蛋白基因座控制区（LCR）和基因之间的长程相互作用，但该复合物是否介导其他发育阶段或人类细胞中的染色质相互作用尚不清楚。胎儿γ-珠蛋白基因可以在成人红系细胞中被细胞因子稳健地再激活。我们研究了激活剂，阻遏物和共阻遏物的占用率和染色质构象的β-珠蛋白基因座在这些细胞中，当γ-珠蛋白被抑制或重新激活。在主要转录β-珠蛋白的细胞中，γ-珠蛋白阻遏物BCL 11 A占据A-γ珠蛋白基因下游的位点，该位点在基因间RNA转录物BGL 3的序列内。Ldb 1/NLI复合物还占据BGL 3序列以及LCR和辅阻遏物ETO 2。在这些条件下，观察到LCR和β-珠蛋白基因之间的长程相互作用。相反，当γ-珠蛋白被重新激活时，BGL 3处的BCL 11 A占据丢失，BGL 3和γ-珠蛋白都被转录。ETO 2不再参与Ldb 1/NLI复合物，γ-珠蛋白/BGL 3区域与LCR之间的接近性得以确立。这些结果暗示了不同的Ldb 1/NLI复合物介导γ-珠蛋白的转录或沉默，通过涉及非编码RNA转录的基因间位点的长距离LCR相互作用。此外，ETO 2与γ-珠蛋白阻遏物BCL 11 A一起作为治疗靶点，以改善镰状细胞病和β-地中海贫血。 单个珠蛋白基因如何建立阶段特异性增强子通讯尚不清楚，大多数研究都是在非染色体环境中进行的。我们正在使用小鼠ES细胞中的同源重组来解决这个问题。我们靶向了小鼠胚胎干细胞基因上游的一个区域和ES细胞中β-major基因上游的第二个区域，然后使用重组酶介导的盒交换将鸡HS 4绝缘子插入这两个位置，这两个位置是（1）LCR和珠蛋白基因之间以及（2）胚胎和成体基因之间。我们用促红细胞生成素沿着红系分化ES细胞，然后监测珠蛋白基因表达。在LCR和下游基因之间插入cHS 4减少了胚胎ey、β-h1和β主基因的转录，并导致非基因转录物在绝缘子中积累。目前，我们正在研究本地化的RNA聚合酶II和其他因素，如GATA 1和Ldb 1从LCR到3 HS 1使用染色质免疫沉淀，以了解绝缘的分子机制。此外，我们计划监测ES细胞的珠蛋白基因转录，这些ES细胞在胚胎珠蛋白基因和β主基因之间具有cHS 4。此外，我们正在使用我们的ES细胞克隆的胚泡注射到在β-珠蛋白基因座的这两个位置插入cHS 4的小鼠品系中。这些实验是新颖的，因为它们改变了转录因子的招聘和染色质组织在正常的染色体设置，并将提供信息的LCR和珠蛋白基因如何在体内通信过程中的发展和珠蛋白基因开关从胚胎到成人类型。

The Ldb1/NLI 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, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. Fetal gamma-globin genes can be robustly re-activated in adult human erythroid cells by cytokines. We investigated activator, repressor and co-repressor occupancy and chromatin conformation of the beta-globin locus in these cells when gamma-globin is either repressed or re-activated. In cells transcribing primarily beta-globin, the gamma-globin repressor BCL11A occupies a site downstream of the A-gamma globin gene, which is within sequences of BGL3, an intergenic RNA transcript. The Ldb1/NLI complex also occupies BGL3 sequences as well as the LCR together with the co-repressor ETO2. In these conditions, long range interactions between the LCR and the beta-globin gene are observed. In contrast, when gamma-globin is re-activated, BCL11A occupancy at BGL3 is lost and both BGL3 and gamma-globin are transcribed. ETO2 no longer participates in the Ldb1/NLI complex and proximity between the gamma-globin/BGL3 region and LCR is established. These results implicate diverse Ldb1/NLI complexes in mediating gamma-globin transcription or silencing through long range LCR interactions involving an intergenic site of non-coding RNA transcription. Furthermore, ETO2 joins gamma-globin repressor BCL11A as a therapeutic target to ameliorate Sickle Cell Disease and beta-thalassemia. How individual globin genes establish stage specific enhancer communication is unknown and most studies have been performed in a non-chromosomal environment. We are using homologous recombination in mouse ES cells to address this issue. We targeted a region upstream of the mouse embryonic epsilon y gene and a second region upstream of beta-major in ES cells and then used recombinase mediated cassette exchange to insert the chicken HS4 insulator in these two positions which are (1) between the LCR and the globin genes and (2) between the embryonic and adult genes. We differentiated ES cells with erythropoietin along erythroid lines and then monitored globin gene expression. The cHS4 insertion between the LCR and downstream genes reduced transcription of the embryonic ey, beta-h1 and beta major genes and resulted in non-genic transcript accumulation in the insulator. Currently, we are investigating localization of RNA polymerase II and other factors such as GATA1 and Ldb1 from the LCR to 3HS1 using chromatin immunoprecipitation to understand the molecular mechanism underlying insulation. In addition, we plan to monitor globin gene transcription of ES cells that have cHS4 between the embryonic globin genes and the beta major gene. Furthermore, we are using blastocyst injection of our ES cell clones to mouse lines with insertions of cHS4 at these two positions in the beta-globin locus. These experiments are novel since they alter transcription factor recruitment and chromatin organization in a normal chromosomal setting and will provide information on how the LCR and globin genes communicate in vivo during development and during globin gene switches from embryonic to adult types.