Deciphering novel binary CTCF code encrypted in Host and Proviral Epigenomes by Distinct Classes of CTCF & BORIS Binding Sites

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参与调控转录激活和抑制、基因印迹、细胞增殖和凋亡的控制、染色质区室化、X-chr失活、防止3-nt重复扩增和其他染色质驻留过程。我们花了20多年的CTCF研究来说服其他人，CTCF的多功能性确实是基于高度保守的“多价11锌指DBD”结合广泛的不同DNA序列的能力，以及其通过组合使用DNA接触与蛋白质接触指与伴侣蛋白相互作用的内在能力。去年，在果蝇Su（Hw）因子中的另一个poly-ZF阵列已经证明了类似的多价性。随着下一代测序技术的出现，CTCF结合位点已经在果蝇、小鼠和人类基因组中被鉴定。反映了CTCF功能的多样性，发现数千个非同源CTSequences与参与长距离染色质相互作用的基因组区域相关，包括增强子、启动子和基因间边界元件。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/DNA复合物的多功能性方面取得了进展。通过在全基因组范围内同时定位CTCF和BORIS占用，我们发现了两类CTCF结合区域，它们在DNA序列中是预编程的和进化保守的。我们发现，70%的CTCF结合区域包含单个CTCF结合位点，也称为“1xCTSes”，而通过ChIP-seq检测为单峰的其他30%的CTCF结合区域实际上显示含有双重CTCF结合位点，也称为二元“2xCTSes”。在二元2xCTS区域内相邻CTS的占据限制了2个相邻CTCF蛋白在正常体细胞中形成同源二聚体，或在顶部CTCF上共表达BORIS的生殖细胞和癌细胞中组装在相同DNA点处共结合的CTCF+ BORIS的异源二聚体。最近对2xCTS区域的突破性发现（无法通过标准CTCF ChIP-seq解析）使我们能够首次解决长期存在的问题，即CTCF如何在与真正的转录因子相同的细胞核中发挥作用，同时在假定的绝缘子/边界位点保持大量存在，这些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，只有20%的CTCF结合区位于启动子区，而其余的CTSes与转录起始位点无关。 这种不同的功能作用的决定因素的明显候选者将是DNA序列本身和/或在这两种类型的网站染色质的差异身份。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靶位点的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：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。 2017年8月报告的一项关于在显著的人类CTCF+/-受试者中发现的病理学的新发现强烈表明，CTCF单倍不足可能诱导CTCF结合位点的异常甲基化（改变基因表达等）。类似于我们先前在Ctcf+/-小鼠中的结果（肯普、Lobanenkov和Filippova）。因此，CTCF缺失引起的疾病可能存在共同的潜在病理机制，与我们在2000年代初首次报道的功能完全丧失不同。因此，类似的病理机制似乎是由CTCF剂量不足引起的人类和小鼠遗传性疾病的基础，不包括额外的ZnF突变，即使在肿瘤w/16 q22洛中，ZnF突变也会导致与“多价CTCF”的DNA相互作用的完全而不是部分丧失。 接下来，我们对二进制2xCTS代码的研究挑战了当前文献中普遍存在的一种看法，即所有CTCF位点都是等效的，单个CTCF分子结合在单个CTS上，尽管它们可能包含两个相邻的DNA酶I足迹。最后，发现选择性地置于无鱼精蛋白DNA区中的精子核小体的非随机目的地是由相同的含有2xCTS的CTCF元件的nt背景预先确定的，所述CTCF元件通常由CTCF和BORIS 11 ZF旁系同源物共同结合，所述CTCF和BORIS 11 ZF旁系同源物在成年减数分裂后的圆形精子细胞中共表达。 总之，我们的研究结果提供了染色质动力学的全局视图和研究不同人类细胞谱系中基因表达的远程控制的资源，以及解释为什么从众多的transFactors中，只有CTCF被认为是一种普遍的表观遗传标记，存在于所有细胞类型中类似于修饰的组蛋白和DNA的功能不同区域。

CTCF, a highly conserved DNA binding protein, serves as a global organizer of chromatin architecture. CTCF is involved in regulation of transcriptional activation and repression, gene imprinting, control of cell proliferation and apoptosis, chromatin compartmentali-zation, X-chr inactivation, prevention of the 3-nt repeat expansions, and other chromatin resident processes. It took us over 20 years of CTCF studies to persuade others that multiple functionality of CTCF is indeed based on the ability of a highly-conserved 'multivalent 11 Zn Finger DBD" to bind a wide range of diverse DNA sequences as well as on its intrinsic capacity to interact with a partner protein through the combinatorial usage of DNA-contating vs Protein-contacting Fingers. Last year, a similar multivalency has been proven for another poly-ZF array in Drosophila Su(Hw) factor. 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, many thousands of non-homologous CTSequences were found to be associated with genomic regions engaged in long-range chromatin interactions, including enhancers, promoters, and inter-genic 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 multi functionality of CTCF/DNA-complexes. By mapping simultaneous CTCF & BORIS occupancy genomewide, 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 site, 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 binary "2xCTSes". Occupancy of adjacent CTSes within binary 2xCTS-regions constrains 2 adjacent CTCF proteins to form homodimers in normal somatic cells, or to assemble heterodimers of CTCF+ BORIS co-bound at the same DNA spot in germ and cancer cells co-expressing BORIS on top CTCF. The recent breakthrough discovery of 2xCTS-regions (unresolvable by a standard CTCF ChIP-seq) 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 remaining 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. A new finding reported in August 2017 on pathologies found in remarkable human CTCF+/- subjects strongly suggested that CTCF haploinsufficiency might induce aberrant methylation at CTCF binding sites (altering gene expression, etc.) similar to our previous results in Ctcf+/- mice (Kemp, Lobanenkov, and Filippova). Hence, it is possible that there is a common underlying patho-mechanism for the disorders caused by CTCF deletions distinct from a complete loss of function reported by us in the early 00's for the first time. Therefore, similar patho-mechanisms seem to underlie both human and mouse genetic disorders caused by insufficient CTCF dosage exclusive of additional ZnF mutations that even in tumors w/16q22 LOH would cause a complete rather than partial loss of DNA interactions with the "multivalent CTCF". Next, our studies of the binary 2xCTS code challenge a perception prevalent in the current literature that all CTCF sites are equivalent, with a single CTCF molecule bound at a single CTS, despite the fact that they may contain two adjacent DNase I footprints. Finally, Non-random Destination of Sperm nucleosomes placed selectively into protamine-free DNA Zones was found to be pre-determined by nt context of the same 2xCTS-containing CTCF elements that are normally co-bound by both CTCF & BORIS 11 ZF paralogs co-expressed together in adult post-meiotic round spermatids. Taken together, our results provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages, as well as explain why from a multitude of TransFactors, only CTCF has been recognized as a universal epigenetic mark that is present in all cell types at functionally distinct regions similar to modified histones and DNA.