Normal and Pathologic Functions of CTCF and Its Distinct Classes of DNA-targets

CTCF是一种高度保守的DNA结合蛋白，是染色质结构的全球组织者。CTCF参与转录激活和抑制、基因印迹、控制细胞增殖和凋亡、染色质结构域隔离、X染色体失活、防止寡核苷酸重复扩增和其他染色质驻留过程。CTCF的多重功能是基于其结合多种不同DNA序列的能力，以及通过组合使用11个C2H2锌指(11个锌)与伴侣蛋白质相互作用的内在能力。 随着下一代测序技术的出现，已经在苍蝇、小鼠和人类基因组中鉴定了CTCF结合位点。CTCF靶点(CTS)被发现与参与染色质长程相互作用的基因组区域相关，包括增强子、启动子、绝缘体和边界元件，反映了CTCF的多种功能。然而，给定CTS的DNA序列如何与这些位点的特定CTCF功能相关仍不清楚。今年，我们在理解CTCF多功能方面取得了进展。通过对CTCF和Boris占位的全基因组作图，我们发现了两类在DNA序列中预先编程和进化保守的CTCF结合区。我们发现，70%的CTCF结合区包含一个CTCF结合位点，也就是“1xCTSes”，而CHIP-SEQ检测到的其他30%的CTCF结合区实际上包含两个CTCF结合位点，也就是“2xCTSes”。 在二分的2xCTS-区域内的两个DNA位点的占据限制了两个相邻的CTCF蛋白在正常体细胞中形成同源二聚体，并在表达Boris的生殖细胞和癌细胞中组装共结合在同一DNA点的CTCF和Boris的异源二聚体。最近2xCTS区域的突破性发现(仅通过标准的CTCF芯片-seq无法解决)使我们第一次解决了长期存在的问题，即CTCF如何在同一核的背景下作为真正的转录因子服务，同时保持在假定的绝缘体/边界位置的大量存在，没有转录活性的迹象。事实上，在任何给定的细胞类型中，只有20%的CTCF结合区位于启动子区域，而其余的CTS与转录起始点无关。 这种不同功能角色的决定因素显然是DNA序列本身和/或染色质在这两种类型的位点上的不同同一性。在我们的研究中，我们提出了全基因组的证据，表明两种类型的CTCF靶点下的DNA序列在结构上是不同的。两类CTCF结合位点之间的结构差异与功能差异有关：2xCTS优先定位于活性启动子和增强子，并与人和小鼠精子中保留的组蛋白相关，这与只有一个CTCF结合位点的基因组区域形成鲜明对比。 我们的研究挑战了目前文献中认为所有CTCF位点是相等的，并且具有单一CTCF基序的看法。下一步，我们绘制了人类胚胎干细胞和四个人类胚胎干细胞来源的谱系中全基因组染色质的相互作用图，揭示了在谱系指定过程中广泛的染色质重组。我们观察到，尽管自结合的染色质结构域在分化过程中是稳定的，但结构域内部和结构域之间的染色质相互作用发生了显著的变化，改变了整个基因组中36%的活跃和不活跃的染色体区段。通过将染色质相互作用图与单倍型解析的表观基因组和转录组数据集相结合，我们发现基因表达中普遍存在的等位基因偏见与连锁启动子和远端增强子的染色质状态相关。我们的结果提供了染色质动力学的全球视角，并为研究不同人类细胞系中基因表达的远程控制提供了资源。

CTCF, a highly conserved DNA binding protein, serves as a global organizer of chromatin architecture. CTCF is involved in the regulation of transcriptional activation and repression, gene imprinting, control of cell proliferation and apoptosis, chromatin domain insulation, X-chromosome inactivation, prevention of oligonucleotide repeat expansion, and other chromatin resident processes. The multiple functionality of CTCF is based on its ability to bind a wide range of diverse DNA sequences as well as on the intrinsic capacity to interact with a partner protein through the combinatorial usage of eleven C2H2 Zinc Fingers (11 ZFs). With the advent of next generation sequencing techniques, CTCF binding sites have been identified across fly, mouse, and human genomes. Reflecting the multitude of CTCF functions, CTCF Target Sites (CTSes) were found to be associated with genomic regions engaged in long-range chromatin interactions, including enhancers, promoters, insulators and boundary elements. It remains obscure, however, how the DNA sequences of given CTSes are related to the specific CTCF functions at these sites. This year we have made advances in the direction of understanding of CTCF multifunctionality. By mapping CTCF and BORIS occupancy genome-wide, we uncovered two classes of CTCF binding regions that are pre-programmed and evolutionary conserved in DNA sequence. We found that 70% of CTCF bound regions enclose a single CTCF binding sites, aka "1xCTSes" while other 30% of CTCF-binding regions detected by ChIP-seq as single peaks are, in fact, shown to contain the dual CTCF binding sites, aka "2xCTSes". Occupancy of both DNA sites within bipartite 2xCTS-regions constrains 2 adjacent CTCF proteins to form homodimers in normal somatic cells and to assemble heterodimers of CTCF and BORIS co-bound at the same DNA spot in germ and cancer cells expressing BORIS. The recent breakthrough discovery of 2xCTS-regions (unresolved by a standard CTCF ChIP-seq alone) enabled us, for the first time, to address the long-standing question as to how CTCF can serve in the context of the same nucleus as a bona fide transcription factor, while maintaining a substantial presence at putative insulator/boundary sites that bear no indications of transcriptional activity. Indeed, only 20% of all CTCF binding regions are located in promoter regions in any given cell type, while the rest of CTSes are not associated with transcriptional start sites. The obvious candidates for the determinants of such distinct functional roles would be DNA sequences themselves and/or differential identity of chromatin at these two types of sites. In our study we presented genome-wide evidence that DNA sequences underlying the two types of CTCF target sites are structurally different. The structural difference between two classes of CTCF binding sites is connected to the functional difference: 2xCTSes are preferentially located at active promoters and enhancers, and are associated with retained histones in human and mouse sperm, in stark contrast to genomic regions harboring a single CTCF binding site. Our study is challenging the perception in the current literature that all CTCF sites are equal and characterized by a single CTCF motif. Next, mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncovered extensive chromatin reorganization during lineage specification. We observed that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we found that widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results provided a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.