CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells [ChIP-Seq]. Homo sapiens

CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human HSPC hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone 3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. These genomic data support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. Overall design: CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO. The cells were further cultured in the presence of EPO, and formaldehyde crosslinked chromatin was isolated after cells differentiated into R3/R4 nucleated erythroid cells. Chromatin Immunoprecipitation followed by sequencing (chIP-seq) was performed using antibodies against CTCF, Cohesin SA1 and H3K27me3, along with a total input control. Two replicates for CTCF and Cohesin SA1 were obtained.

CTCF与黏连蛋白SA-1（cohesin SA-1）是一类参与多种关键细胞进程的调控蛋白，涵盖转录、染色质域结构维持以及绝缘子功能。为探究红细胞生成过程中CTCF与黏连蛋白SA-1相互作用组的变化，研究团队在来自单个供体的原代人造血干细胞与祖细胞（hematopoietic stem and progenitor cells，HSPC）以及原代人红细胞中，开展了结合高通量测序的染色质免疫沉淀（chromatin immunoprecipitation coupled with high-throughput sequencing，ChIP-seq）实验，以及基于RNA测序（RNA-seq）的mRNA转录组分析。与仅结合单个CTCF或黏连蛋白的位点相比，HSPC与红细胞中CTCF和黏连蛋白SA-1共占据的染色质位点在基因启动子区域呈现富集特征。红细胞内的细胞类型特异性CTCF位点与高表达基因存在关联，而HSPC中则呈现完全相反的分布模式。研究人员通过靶向组蛋白3赖氨酸27三甲基化（trimethylated lysine 27 histone 3，H3K27me3）的抗体进行ChIP-seq以鉴定染色质域，该组蛋白修饰与染色质沉默功能密切相关。造血进程中，沉默型染色质域的数量与规模均出现扩增，且红细胞内的沉默型染色质域数量远多于HSPC。CTCF与黏连蛋白SA-1可通过细胞类型特异性的方式，标记这些沉默型染色质域的边界区域。上述基因组数据支持如下假说：CTCF与黏连蛋白SA-1在红细胞生成过程的基因表达调控中发挥多重功能，包括基因启动子处的转录调控以及染色质三维结构的维持。总体实验设计：经CD34+分选的造血干祖细胞在不含促红细胞生成素（erythropoietin，EPO）的培养基中扩增3天，随后转移至添加EPO的培养基中继续培养，待细胞分化为R3/R4阶段有核红细胞后，分离甲醛交联的染色质。采用针对CTCF、黏连蛋白SA-1及H3K27me3的抗体开展染色质免疫沉淀测序（ChIP-seq）实验，并设置总输入对照。本研究获得了CTCF与黏连蛋白SA-1的两组生物学重复样本。