Chromatin Structure In Regulation Of Mammalian Gene Expression

NLI/LDB1是一种广泛表达的核因子，我们已经证明它是β-珠蛋白LCR和β-珠蛋白启动子之间长距离相互作用所必需的。LDB1的C末端LIM相互作用域(LID)与LMO2相互作用，通过DNA结合伙伴GATA-1和TAL1提供LDB1与染色质的结合。已知LDB1的N-末端二聚结构域(DD)参与了体外蛋白质的同源二聚。我们认为LDB1的DD结构域在体内LCR和β-主要基因启动子之间的长距离相互作用中起关键作用。为了证实这一假设，通过从转基因构建物中表达标记的透明质酸来挽救MEL细胞中的LDB1基因敲除。转基因LDB1与LCR和β-珠蛋白启动子相互作用，挽救GATA1/TAL1/LMO2蛋白复合体结合，以及β-珠蛋白基因的表达。然后，在内源性LDB1敲除MEL细胞的背景下，表达了DD中缺失的LDB1短保守序列的几个缺失。这些突变蛋白大多能与LCR和β-主要基因启动子相互作用，但不能激活β-珠蛋白基因的表达。为了表明DD结构域在主要的β珠蛋白基因激活中的直接作用，在LDB1KD背景中表达了包含LMO2和LDB1的DD结构域的融合蛋白(LMO-DD)。LMO-DD融合蛋白可以与LCR和β-珠蛋白启动子相互作用，在激活β-珠蛋白基因表达的同时挽救GATA1/TAL1蛋白的结合。截短的只由DD结构域组成的LDB1蛋白与内源LDB1的免疫共沉淀证实了LDB1在细胞中的同源二聚是通过DD结构域之间的相互作用发生的。这些实验证明，LDB1的DD结构域是β-珠蛋白基因激活的必要条件和充分条件。 在其他实验中，我们探索了绝缘体阻断增强子的机制。我们发现，人β-珠蛋白HS5是依赖CTCF的鸡HS4绝缘体的同源物，具有内在的、可移植的增强子阻断活性，这通过染色质环的形成而明显。为了研究CTCF位点的环化活性是否是基因组中这些位点的一般特性，我们在11号染色体上超过2Mb的CTCF位点上进行了染色质构象捕获(3C)，包括β-珠蛋白基因座和嗅觉受体基因两侧。接下来，我们使用在红系细胞和非红系细胞中全面确定的绝缘蛋白CTCF的这些已建立的环相互作用来研究染色质的聚合物模型的三维折叠特性，在红系细胞中，该位点被活跃转录，在非红系细胞中，它是沉默的。我们的结果表明，在红系细胞中，CTCF介导的细胞类型特异的染色体相互作用频率在空间上被组织起来，以利于β-珠蛋白基因控制区(LCR)与基因之间的接触，这是已知发生在体内的。相比之下，非红系细胞中较少的显性相互作用被显示为特异性地将β-珠蛋白基因从LCR中驱离，这表明了一种转录控制的机制。因此，实验数据和适当的聚合物模型提供了对兆基尺度下染色质动力学的生理学意义的描述。特别是，我们假设β-珠蛋白基因座的生物学功能至少部分依赖于基于聚合物的生物物理机制，其中CTCF远程相互作用功能地影响控制元件和基因启动子之间的空间距离。

NLI/Ldb1, is a widely expressed nuclear factor that we have shown is required for long range interaction between the beta-globin LCR and beta-globin promoter. The C-terminal LIM-interaction domain (LID) of Ldb1 interacts with LMO2 which provides association of Ldb1 with chromatin through DNA-binding partners GATA-1 and TAL1. It is known that the N-terminal dimerization domain (DD) of Ldb1 participates in homodimerization of the protein in vitro. We proposed that the DD domain of Ldb1 plays a key role in long range interaction between the LCR and beta-major gene promoter in vivo. To confirm this hypothesis, Ldb1 knock down in MEL cells was rescued by expression of HA tagged Ldb1 from a transgenic construct. Transgenic Ldb1 interacts with the LCR and beta-globin promoter and rescues GATA1/TAL1/LMO2 protein complex binding, along with beta-globin gene expression. Then several deletions of Ldb1 missing short conserved sequences in DD were expressed in the background of endogenous Ldb1 knock down MEL cells. Most of these mutated proteins can interact with the LCR and beta-major gene promoter but are unable to activate beta-globin gene expression. To show a direct role of the DD domain in beta-major globin gene activation, a fusion protein that contains LMO2 and the DD domain of Ldb1 (LMO-DD) was expressed in the Ldb1 KD background. LMO-DD fusion protein can interact with the LCR and beta-globin promoter and rescue GATA1/TAL1 protein binding along with activation of beta-globin gene expression. Coimmunoprecipitation of a truncated Ldb1 protein consisting of only the DD domain with endogenous LDB1 confirms that homodimerization of LDB1 in cells occurs by interaction between DD domains. These experiments demonstrated that the DD domain of LDB1 is necessary and sufficient for beta-globin gene activation. In other experiments, we explored the mechanisms underlying enhancer blocking by insulators. We found that human beta-globin HS5, the orthologue of the CTCF dependent chicken HS4 insulator, has intrinsic, portable enhancer blocking activity that is manifest though chromatin loop formation. To investigate whether the looping activity of CTCF sites is a general property of these sites in the genome, we carried out chromatin conformation capture (3C) on CTCF sites over 2 Mb on chromosome 11 encompassing the beta-globin locus and flanking olfactory receptor genes. We next investigated the 3-dimensional folding properties of a polymer model of chromatin using these established looping interactions of the insulator protein CTCF comprehensively determined in erythroid cells, where the locus is actively transcribed, and in non-erythroid cells, where it is silent. Our results indicate that cell type specific chromosomal interaction frequencies mediated by CTCF in erythroid cells are spatially organized to favor contacts between the beta-globin locus control region (LCR) and genes, which are known to occur in vivo. By contrast, fewer dominant interactions in non-erythroid cells are shown to specifically drive the beta-globin genes away from the LCR, suggesting a mechanism for transcriptional control. Thus, experimental data and appropriate polymer models provide a physiological meaningful description of chromatin dynamics below the megabase scale. In particular, we posit that the biological function of the beta-globin locus relies, at least in part, on a polymer-based biophysical mechanism where CTCF long-range interactions functionally affect spatial distances between control elements and gene promoters.