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Chromatin Structure in Regulation of Mammalian Gene Expression

哺乳动物基因表达调节中的染色质结构

基本信息

批准号：
9553219
负责人：
Ann Dean
金额：
$ 89.77万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9553219
关键词：
Address Affect Alleles Architecture Binding Binding Sites CCCTC-binding factor CRISPR/Cas technology Cells ChIP-seq Chromatin Chromatin Loop Chromatin Structure Chromosomes Chromosomes, Human, Pair 11 Communication Complex Development Dimerization Disease Distant Elements Embryo Enhancers Erythroid Frequencies GATA1 gene Gene Activation Gene Expression Gene Expression Regulation Gene Family Gene Targeting Genes Genome Geometry Globin Goals HDAC1 gene Health Human Human Development Individual K-562 K562 Cells KDM1A gene Knock-out LMO2 gene Link Locus Control Region Mediating Modeling Molecular Nuclear Nucleic Acid Regulatory Sequences Null Lymphocytes Pattern Proteins Recruitment Activity Regulation Reporting Research Sickle Cell Anemia Site Specificity TAL1 gene TRIM Gene Technology Testing Therapeutic Tissues Transcription Repressor/Corepressor Upstream Enhancer Variant Western Blotting Zinc Fingers beta Globin beta Thalassemia carbonate dehydratase cohesin design dimer embryonic stem cell enhancer binding protein fetal gamma Globin gene repression genome editing genome-wide genome-wide analysis histone modification in vivo novel promoter protein complex therapeutic target transcription factor transcriptome sequencing

项目摘要

Understanding the complex regulation of beta-globin genes is critically important to design therapeutic approaches to beta-thalassemia and sickle cell disease. We have described an erythroid-specific protein complex containing LDB1/GATA-1/TAL1/LMO2 (LDB1 complex). that is directly involved in activation of globin gene expression. Through dimerization of LDB1, the complex mediates long-range interaction between the beta-globin LCR enhancer and globin gene promoters. Genome-wide studies show that the LDB1 complex regulates erythroid enhancers, more generally. However, some gene targets of LDB1 enhancers are not occupied by LDB1, raising the question of additional mechanisms for looping by LDB1. We find that a novel LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Genome-wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression. Cohesin is a frequent partner of CTCF genome-wide and has been reported to participate directly in enhancer-gene contacts in embryonic stem cells. However, we find that cohesin is almost completely absent from LDB1-regulated erythroid enhancer-gene pairs, whether or not they involved CTCF. Enhancer-gene pairs are primarily located within topologically associated domain, or TADs, that are thought to be looped domains created by interactions between CTCF sites at the TAD borders. Do such indirect loops favor the internal enhancer-gene interations or is the reverse true? To address this issue in vivo, we used CRISPR/Cas9 technology to delete CTCF sites flanking the beta-globin locus over 1 Mb of chromosome 11 in human erythroid K562 and non-erythroid 293T cells. Bi-allelic deletions were validated by PCR. None of the deletions affected CTCF levels in cells according to Western blots and each deletion resulted in loss of CTCF binding uniquely at that site according to ChIP analysis. Histone modifications in the vicinity of each deletion are being analyzed by CHIP-seq. Globin gene expression and expression of Trim genes adjacent to the globin locus were affected in different ways by deletion of individual CTCF sites. To further understand how CTCF binding site deletion alters chromosome architecture and local gene expression, we are performing CTCF-capture Hi-C. Transcriptional factor ETO2 can participate in the LDB1 complex and this confers negative function on the complex but the mechanisms involved are not known. ETO2 can recruit HDACs to target genes through its dimerization domain and zinc finger region, which may underlie the repressor function. We knocked out ETO2 in K562 cells using CRISPR-Cas9 technology and performed RNA-seq to determine differentially regulated genes. Embryonic and fetal gamma globin genes as well as erythroid specific transcription factors were up-regulated in the absence of ETO2. In addition, loss of ETO2 caused significant decrease of HDAC1 and LSD1 occupancy and significant increase of H3K27ac in the beta-globin LCR region and increased H3K9ac and H3K4me2 at the gamma-globin gene promoter. Furthermore, ETO2 null cells showed higher interaction frequency between the LCR and gamma-globin promoter than WT cells and increased loading of LDB1 complex, which mediates loop formation, at the LCR. We next expressed ETO2 variant proteins missing either the TAF110, dimerization and zinc finger domains in the ETO2 null background. In summary, we found that all three domains are required for ETO2 to function as a transcriptional repressor in gamma globin gene regulation, suggesting potential therapeutic targets for sickle cell disease and beta-thalassemia.

了解β-珠蛋白基因的复杂调控对于设计β-地中海贫血和镰状细胞病的治疗方法至关重要。我们已经描述了一种含有LDB 1/加塔-1/TAL 1/LMO 2的红系特异性蛋白复合物（LDB 1复合物）。直接参与珠蛋白基因表达的激活。通过LDB 1的二聚化，该复合物介导β-珠蛋白LCR增强子和珠蛋白基因启动子之间的长程相互作用。全基因组研究表明，LDB 1复合物更普遍地调节红细胞增强子。然而，LDB 1增强子的一些基因靶不被LDB 1占据，这就提出了LDB 1成环的其他机制的问题。我们发现，一种新的LDB 1结合增强子上游的碳酸酐酶2（Car 2）激活其表达直接与CTCF在基因启动子相互作用。全基因组研究和CRISPR/Cas9基因组编辑表明，LDB 1-CTCF增强子环是大部分红细胞基因激活的基础。我们的研究结果提供了一种机制，通过这种机制，可以定制建筑蛋白CTCF的长程相互作用，以实现组织限制的染色质环和基因表达模式。黏连蛋白是CTCF基因组内的一个常见的伴侣，并已被报道直接参与胚胎干细胞中增强子-基因的接触。然而，我们发现，无论是否涉及CTCF，LDB 1调节的红系增强子-基因对中几乎完全不存在粘附素。增强子-基因对主要位于拓扑相关结构域或TADs内，其被认为是由CTCF位点之间在边缘处的相互作用产生的环状结构域。这样的间接环有利于内部增强子-基因相互作用，还是相反？为了在体内解决这个问题，我们使用CRISPR/Cas9技术来删除人红细胞K562和非红细胞293 T细胞中11号染色体的1 Mb以上的β-珠蛋白基因座侧翼的CTCF位点。通过PCR验证双等位基因缺失。根据蛋白质印迹，没有一个缺失影响细胞中的CTCF水平，并且根据ChIP分析，每个缺失导致在该位点独特的CTCF结合丧失。通过CHIP-seq分析每个缺失附近的组蛋白修饰。珠蛋白基因表达和邻近珠蛋白基因座的Trim基因表达以不同的方式受到单个CTCF位点缺失的影响。为了进一步了解CTCF结合位点缺失如何改变染色体结构和局部基因表达，我们正在进行CTCF捕获Hi-C。转录因子ETO 2可以参与LDB 1复合物，这赋予复合物负功能，但所涉及的机制尚不清楚。ETO 2可以通过其二聚化结构域和锌指区将HDAC募集到靶基因，这可能是阻遏物功能的基础。我们使用CRISPR-Cas9技术敲除K562细胞中的ETO 2，并进行RNA-seq以确定差异调节的基因。胚胎和胎儿γ珠蛋白基因以及红细胞特异性转录因子在ETO 2不存在的情况下上调。此外，ETO 2的缺失导致HDAC 1和LSD 1占据率显着降低，β-球蛋白LCR区域中H3 K27 ac显着增加，以及γ-球蛋白基因启动子处H3 K9 ac和H3 K4 me 2的增加。此外，ETO 2空细胞显示LCR和γ-珠蛋白启动子之间的相互作用频率高于WT细胞，并且在LCR处介导环形成的LDB 1复合物的负载增加。我们接下来表达了在ETO 2空白背景中缺失TAF 110、二聚化和锌指结构域的ETO 2变体蛋白。总之，我们发现，所有这三个域所需的ETO 2作为一个转录抑制因子在γ珠蛋白基因调控，这表明镰状细胞病和β地中海贫血的潜在治疗靶点。