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Deciphering CTCF code in mammalian host and viral epigenomes

破译哺乳动物宿主和病毒表观基因组中的 CTCF 代码

基本信息

批准号：
10927769
负责人：
Victor Lobanenkov
金额：
$ 164.75万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS 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/10927769
关键词：
16q22 3-Dimensional Address Affect Algorithms Animals Apoptosis Architecture Binding Binding Sites Biological Boundary Elements Brothers CCCTC-binding factor Cancer Center Cell Lineage Cell Nucleus Cell Proliferation Cells Cessation of life ChIP-seq Chromatin Chromatin Loop Chromosomes Clinical Code Codon Nucleotides Collaborations Communication Communities Complex DNA DNA Binding Domain DNA Methylation DNA Polymerase II DNA Sequence DNA-Binding Proteins DNase-I Footprinting Data Data Set Development Disease Distal Distant Drosophila genus Elements Enhancers Epigenetic Process Eukaryota Evolution Fertility Fertilization Fingers Future Gene Expression Genes Genetic Code Genetic Diseases Genetic Transcription Genome Genomic Segment Genomics Genotype Germ Cells Goals Haploidy Heritability Histones Human Human Genetics Human Genome Individual Length Letters Link Literature Mammalian Cell Maps Meiosis Messenger RNA Modification Molecular Biology Mothers Mus Mutate Mutation Names National Institute of Allergy and Infectious Disease Nature Nucleosomes Nucleotides Open Reading Frames Organism Paper Pathology Persuasive Communication Phylogenetic Analysis Play Prevention Process Promoter Regions Protamines Proteins Reproduction Resources Ribosomes Role Sagittaria Site Somatic Cell Spermatids Spermatogenesis Spottings Techniques Time Transcription Initiation Site Transcriptional Activation Transcriptional Regulation Translational Research Translations Trinucleotide Repeat Expansion Untranslated RNA Viral Vocabulary X Inactivation Zinc Fingers acronyms base cancer cell cancer/testis antigen cell type cohesin combinatorial dosage encryption epigenetic regulation epigenome falls fly gene regulatory network gene repression genetic information genome-wide human subject immunotherapy trials imprint in vivo monomer mouse genetics mouse genome mutant next generation sequencing novel paralogous gene postmitotic promoter sperm cell transcription factor transcription regulatory network tumor

项目摘要

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-chromosome inactivation, prevention of tri-nucleotide-repeat's expansions, and other chromatin-resident processes. It took us over 20 years of CTCF studies to persuade others that the multi-functionality of CTCF is indeed based on the ability of a highly-conserved 'multivalent 11 ZF DBD to bind a wide range of diverse DNA sequences, as well as on its intrinsic capacity to interact with a partner-proteins through the combinatorial usage of DNA-contating and protein-contacting ZFs. Last year, a similar multivalency was shown for another poly-ZF-array in the 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 CTS sequences were found to be associated with genomic regions engaged in long-range chromatin interactions, including enhancers, promoters, and inter-genic boundary elements. It remained obscure, however, as to how a particular DNA sequence of any given CTS is related to specific CTCF functions at the same site. This year, we have made additional advances towards understanding multiple functionality of distinct CTCF/DNA-complexes formed via different combinations of DNA-contacting fingers. By mapping simultaneous CTCF & BORIS occupancy genome-wide, we uncovered a new class of CTCF binding regions that are functionally pre-programmed and evolutionary conserved to serve as epigenet 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 of CTCF. The recent breakthrough discovery of 2xCTS-regions w/ adjacent CTCF motifs unresolvable by any ChIP-Seq peak-calling algorithms enabled us, for the first time, to address the long-standing question as to how CTCF-bin 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 their functional differences: 2xCTSes are preferentially located at H4K27ac-marked promoters and enhancers co-bound by Pol II, and the same 2xCTS elements are found to be associated with normal CTCF-BORIS-heterodimers in post-meiotic spermatids wherein BORIS marks the future protamine-free DNA zones that retain modified histones along haploid epi-genome in mature human and mouse spermatozoa. In a stark contrast, intergenic and intronic genomic regions harboring one or more 1xCTS-based CTCF peaks with the name-giving 5'-CCC(C/t)CT(a/g)-3' motif which is often hit by a disease-associated SNP affecting three-dimensional organization imprinted upon essential self-interactions among sticky C-termini and DNA-free ZF-subsets from distal CTCF/DNA complexes engaged into site-specific di-/multi-merization stabilized by cohesin retention. A remarkable link with CTCF +/- haplo-insufficiency found in genetically burdened human subjects might open up a novel avenenue in a clinically-oriented CTCF studies associated with aberrant histone/DNA-methylation encompassing CTCF-bound ChIP-Seq peaks with 2xCTS elements in H3K27ac-marked Pol2-bound promoter-enhancer pairs capable of altering gene expression in the same way that we had previously found to act in context of Ctcf+/- mice analyzed in collaboration with Fred Hutchinson Cancer Center in Seattle. Therefore, similar pathology-associated mechanisms seem to underlie both human and mouse genetic disorders caused by insufficient CTCF dosage exclusive of additional ZnF mutations which, even in tumors with 16q22/CTCF LOH, would cause a complete CTCF loss leading to death rather than a partial loss of DNA-CTCF interactions caused by in vivo selection of viable single a.a. substitutions within the multivalent 11 ZnF CTCF DBD that were characterized first in CTCF (1996) and found later on (2002) to be recapitulated in the CTCF-derived paralog named "BORIS" (an acronym for "Brother Of the Regular of Imprinted States"). Next, our discovery and further studies of the binary 2xCTS code begun to challenge a widespread misconception in the current literature claiming that all CTCF sites are equivalent to each other, with a single CTCF molecule bound at a single CTS sequence in spite of the fact that CTS elements with different genomic coordinates may contain either one or two adjacent DNase I footprints over single or dual CTCF motifs without any homologies necessary for reliable motif-based predictions. The functional and structural epigenetic features of Pol2-bound enhancer/promoter-associated 2xCTS-elements are distinct from the same features of 1xCTS-containing regions bound by CTCF-only monomers within intronic and intergenic non-coding regions. The previously overlooked class of CTCF binding regions with two (rather than one) closely-spaced CTCF motifs (aka "2xCTS") has a very distinct role in regulating diverse chromatin-based phenomena, incl. heritable epigenetic regulation in cancer cells and in normal germ cells. For instance, non-random retention of sperm nucleosomes was found to be predetermined by specific nt context of 2xCTS-containing reg.DNA elements that are normally co-bound by both CTCF & BORIS co-expressed together in late round spermatids. Moreover, our latest "Nature Communications" paper available online at https://www.nature.com/articles/s41467-021-24140-6 has described an unpredictable synergistic effect of combining Ctcf haploinsufficiency with Boris-/- null genotype in the novel mouse DKO strain which revealed that CTCF+BORIS heterodimers are absolutely essential for spermatogenesis and fertility. Moreover, CTCF and the cohesin are widely recognized now as the key players in 3D genome architecture in all mammalian cells. These 2 proteins are not just well known in the scientific community but have recently entered popular press and media. Taken together, our data allowed to develop a global view of chromatin dynamics and provided unique resources for studying long-range epigenetic control of gene expression in distinct cell lineages. Finally, synergistic DKO effects led us towards a stunning understanding that only CTCF has been singled out by the Mother Nature to serve as a truly universal and irreversible epigenetic mark present on DNA not only in all somatic cell types but also in mature spermatozoa before and after fertilization upon endless reproduction rounds. Unlike other CTA selected for immunotherapy trials, only BORIS can re-activate all other known CTA genes. Hence, it's not surprising to see BORIS raising to the top of translational research.

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参与转录激活和抑制的调控、基因印迹、细胞增殖和凋亡的控制、染色质区隔化、x染色体失活、防止三核苷酸重复扩增以及其他染色质驻留过程。我们花了20多年的时间来研究CTCF，以说服其他人CTCF的多功能性确实是基于高度保守的“多价11 ZF DBD”结合多种不同DNA序列的能力，以及通过组合使用DNA连接和蛋白质连接ZF与伴侣蛋白相互作用的内在能力。去年，果蝇Su（Hw）因子的另一个poly- zf阵列显示了类似的多价性。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出来。数以千计的非同源CTS序列被发现与参与远程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，这反映了CTCF的多种功能。然而，对于任何给定CTS的特定DNA序列如何与同一位点的特定CTCF功能相关，仍然是模糊的。今年，我们在了解通过dna接触手指的不同组合形成的不同CTCF/ dna复合物的多种功能方面取得了额外的进展。通过在全基因组范围内同时绘制CTCF和BORIS占用图谱，我们发现了一类新的CTCF结合区域，它们具有功能预编程和进化保守性，可作为表观基因。我们发现70%的CTCF结合区域包含单个CTCF结合位点，即“1xCTSes”，而ChIP-seq检测到的其他30%的CTCF结合区域实际上包含双CTCF结合位点，即二元“2xCTSes”。相邻CTSes在二元2xcts区域内的占用限制了2个相邻CTCF蛋白在正常体细胞中形成同型二聚体，或在CTCF上共表达BORIS的生殖细胞和癌细胞中在同一DNA位点组装CTCF+ BORIS共结合的异源二聚体。最近的突破性发现是，任何ChIP-Seq峰值调用算法都无法解析具有相邻CTCF基序的2xcts区域，这使我们首次能够解决长期存在的问题，即CTCF-bin如何在同一细胞核中作为真正的转录因子服务，同时在假定的绝缘子/边界位点保持大量存在，而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结合区域位于启动子区域，而其余的ctse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将是DNA序列本身和/或在这两种类型的位点染色质的不同身份。在我们的研究中，我们提出了全基因组证据，证明两种类型的CTCF靶点的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其功能差异有关：2xctse优先位于Pol II共同结合的h4k27acs标记的启动子和增强子上，并且在减数分裂后的精子中发现相同的2xCTS元件与正常的CTCF-BORIS异源二聚体相关，其中BORIS标记了成熟的人类和小鼠精子中单倍体外基因组中保留修饰组蛋白的未来无蛋白DNA区。与此形成鲜明对比的是，基因间和内含子基因组区域包含一个或多个基于1xcts的CTCF峰，具有命名的5‘- ccc (C/t)CT(a/g)-3’基序，该基序经常受到影响三维组织的疾病相关SNP的打击，这些SNP影响着粘性C端和来自远端CTCF/DNA复合物的无DNA zf亚群之间的基本自相互作用，参与由黏结蛋白保留稳定的位点特异性二/多聚合。在遗传负担的人类受试者中发现的与CTCF +/-单倍体缺陷的显著联系，可能为临床导向的CTCF研究开辟了一条新的途径，CTCF研究与异常组蛋白/ dna甲基化相关，包括h3k27ac标记的pol2结合启动子-增强子对中的CTCF结合ChIP-Seq峰和2xCTS元素，这些甲基化能够以与我们之前在与Fred Hutchinson癌症合作分析的CTCF +/-小鼠中发现的相同的方式改变基因表达在西雅图的中心。因此，类似的病理相关机制似乎是CTCF剂量不足引起的人类和小鼠遗传疾病的基础，不包括额外的ZnF突变，即使在具有16q22/CTCF LOH的肿瘤中，会导致CTCF的完全丧失，而不是DNA-CTCF相互作用的部分丧失，而DNA-CTCF相互作用是由多价11 ZnF CTCF DBD中有活力的单个a.a.取代的体内选择引起的，这种选择首先在CTCF中被描述（1996年），后来（2002年）发现，在CTCF衍生的“BORIS”（“印迹状态规则的兄弟”的首字母缩写）中进行了概述。接下来，我们对二进制2xCTS代码的发现和进一步研究开始挑战当前文献中普遍存在的误解，即所有CTCF位点彼此等效，单个CTCF分子结合在单个CTS序列上，尽管具有不同基因组坐标的CTS元件可能在单个或双CTCF基序上包含一个或两个相邻的DNase I足迹，而无需可靠的基于基序的预测所需的任何同源性。pol2结合的增强子/启动子相关2xcts元件的功能和结构表观遗传特征与内含子和基因间非编码区内仅ctcf单体结合的含有1xcts的区域的相同特征不同。以前被忽视的一类CTCF结合区具有两个（而不是一个）紧密间隔的CTCF基序（又称“2xCTS”），在调节多种基于染色质的现象中具有非常独特的作用，包括癌细胞和正常生殖细胞中的可遗传表观遗传调节。例如，发现精子核小体的非随机保留是由含有2xcts的reg的特定nt环境预先决定的。通常由CTCF和BORIS共同结合的DNA元件在晚期圆形精子中共同表达。此外，我们在《自然通讯》（Nature Communications）网站https://www.nature.com/articles/s41467-021-24140-6上发表的最新论文描述了一种不可预测的协同效应，即Ctcf单倍不全与Boris-/-零基因型在新型小鼠DKO菌株中结合，这表明Ctcf + Boris异源二聚体对精子发生和生育是绝对必要的。此外，CTCF和内聚蛋白被广泛认为是所有哺乳动物细胞三维基因组结构的关键参与者。这两种蛋白质不仅在科学界广为人知，最近还进入了大众媒体和媒体。综上所述，我们的数据允许开发染色质动力学的全局视图，并为研究不同细胞系中基因表达的远程表观遗传控制提供了独特的资源。最后，协同DKO效应使我们有了一个惊人的认识，即只有CTCF被大自然挑选出来，作为一个真正普遍的、不可逆的表观遗传标记，不仅存在于所有体细胞类型的DNA上，而且存在于受精前后的成熟精子中。与其他用于免疫治疗试验的CTA不同，只有BORIS可以重新激活所有其他已知的CTA基因。因此，鲍里斯升到翻译研究的顶峰也就不足为奇了。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line.

DOI：
10.1073/pnas.2204071119
发表时间：
2022-10-04
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
通讯作者：

An updated catalog of CTCF variants associated with neurodevelopmental disorder phenotypes.

DOI：
10.3389/fnmol.2023.1185796
发表时间：
2023
期刊：
FRONTIERS IN MOLECULAR NEUROSCIENCE
影响因子：
4.8
作者：
Price, Emma;Fedida, Liron M.;Pugacheva, Elena M.;Ji, Yon J.;Loukinov, Dmitri;Lobanenkov, Victor V.
通讯作者：
Lobanenkov, Victor V.

Expression of a testis-specific form of Gal3st1 (CST), a gene essential for spermatogenesis, is regulated by the CTCF paralogous gene BORIS.

Gal3st1 (CST) 是精子发生所必需的基因，其睾丸特异性形式的表达受 CTCF 旁系同源基因 BORIS 的调节。

DOI：
10.1128/mcb.01093-09
发表时间：
2010
期刊：
Molecular and cellular biology
影响因子：
5.3
作者：
Suzuki,Teruhiko;Kosaka-Suzuki,Natsuki;Pack,Svetlana;Shin,Dong-Mi;Yoon,Jeongheon;Abdullaev,Ziedulla;Pugacheva,Elena;Morse3rd,HerbertC;Loukinov,Dmitri;Lobanenkov,Victor
通讯作者：
Lobanenkov,Victor

Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells.