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，是染色质结构的全局组织者。CTCF参与调节转录激活和抑制、基因印迹、细胞增殖和凋亡的控制、染色质结构域绝缘、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的多功能性是基于其结合广泛的不同DNA序列的能力以及通过11个C2 H2锌指（11 ZF）的组合使用与伴侣蛋白相互作用的内在能力。 随着下一代测序技术的发展，CTCF结合位点已经在果蝇、小鼠和人类基因组中被鉴定。CTCF靶位点（CTSes）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、绝缘子和边界元件，反映了CTCF的多种功能。然而，给定CTSes的DNA序列如何与这些位点的特定CTCF功能相关仍然不清楚。今年，我们在理解CTCF多功能性方面取得了进展。通过对CTCF和BORIS的全基因组定位，我们发现了两类CTCF结合区域，它们在DNA序列中是预编程和进化保守的。我们发现，70%的CTCF结合区域包含单个CTCF结合位点，也称为“1xCTSes”，而ChIP-seq检测到的其他30%的CTCF结合区域为单峰，实际上包含双重CTCF结合位点，又名“2xCTS”。在二分的2xCTS区域内占据两个DNA位点限制了2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CTCF和BORIS的异源二聚体，所述异源二聚体在相同的DNA位点处共结合。最近的突破性发现的2xCTS-区域（未解决的标准CTCF ChIP Seq单独），我们第一次，以解决长期存在的问题，CTCF如何可以作为一个真正的转录因子在相同的细胞核的背景下，同时保持大量存在于假定的绝缘子/边界位点，没有转录活性的迹象。事实上，在任何给定的细胞类型中，所有CTCF结合区中只有20%位于启动子区中，而其余的CTSes与转录起始位点无关。 这种不同功能作用的决定因素的明显候选者是DNA序列本身和/或这两种类型位点上染色质的差异性。在我们的研究中，我们提出了全基因组证据，表明两种类型的CTCF靶位点的DNA序列在结构上不同。两类CTCF结合位点之间的结构差异与功能差异有关：2xCTSes优先位于活性启动子和增强子处，并与人类和小鼠精子中保留的组蛋白相关，与包含单个CTCF结合位点的基因组区域形成鲜明对比。 我们的研究是挑战的看法，在目前的文献中，所有的CTCF网站是平等的，其特征在于一个单一的CTCF基序。接下来，在人类胚胎干（ES）细胞和四个人类ES细胞衍生谱系中绘制全基因组染色质相互作用，我们发现了谱系特化过程中广泛的染色质重组。我们观察到，虽然自缔合染色质结构域在分化过程中是稳定的，但结构域内和结构域之间的染色质相互作用以惊人的方式变化，改变了整个基因组中36%的活性和非活性染色体区室。通过将染色质相互作用图谱与单倍型分辨表观基因组和转录组数据集相结合，我们发现基因表达中广泛的等位基因偏倚与连锁启动子和远端增强子的等位基因偏倚染色质状态相关。我们的研究结果提供了一个染色质动力学的全局视图，并为研究不同人类细胞谱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 advance 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 the genomic regions engaged in the 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 multiple functionality. By mapping of 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) has unable us, for the first time, to address the long-standing question 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.