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、基因印记、细胞增殖和凋亡控制、染色质区室化、X 染色体失活、三核苷酸重复扩增的预防以及其他染色质驻留过程的调节。我们花了 20 多年的 CTCF 研究时间才说服其他人，CTCF 的多功能性确实基于高度保守的“多价 11 ZF DBD”结合多种不同 DNA 序列的能力，以及它的通过组合使用 DNA 接触和蛋白质接触 ZF 与伴侣蛋白质相互作用的内在能力。去年，果蝇 Su(Hw) 因子中的另一个多 ZF 阵列显示出类似的多价性。随着下一代测序技术的出现，CTCF 结合位点已在果蝇、小鼠和人类基因组中得到鉴定。数千个非同源 CTS 序列被发现与参与长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，任何给定 CTS 的特定 DNA 序列如何与同一位点的特定 CTCF 功能相关仍然不清楚。今年，我们在了解通过 DNA 接触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更多进展。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，这些区域在功能上已预先编程且在进化上保守，可用作表观基因。我们发现 70% 的 CTCF 结合区域包含单个 CTCF 结合位点，即“1xCTSes”，而通过 ChIP-seq 检测为单峰的其他 30% CTCF 结合区域实际上显示包含双 CTCF 结合位点，又名二进制“2xCTSes”。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二聚体，或在 CTCF 顶部共表达 BORIS 的生殖细胞和癌细胞中组装在同一 DNA 点上共结合的 CTCF+ BORIS 异二聚体。最近突破性的发现，任何 ChIP-Seq 峰识别算法都无法解析带有相邻 CTCF 基序的 2xCTS 区域，这使我们第一次能够解决长期存在的问题，即 CTCF-bin 如何在以下情况下发挥作用：与真正的转录因子具有相同的核，同时在没有转录活性迹象的推定绝缘体/边界位点上保持大量存在。事实上，在任何给定的细胞类型中，所有 CTCF 结合区中只有 20% 位于启动子区域，而其余的 CTS 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/或这两类位点染色质的差异身份。在我们的研究中，我们提供了全基因组证据，表明两种 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其功能差异有关：2xCTS 优先位于与 Pol II 共同结合的 H4K27ac 标记的启动子和增强子处，并且发现相同的 2xCTS 元件与正常 CTCF-BORIS 相关。 -减数分裂后精细胞中的异二聚体，其中 BORIS 标记了未来的无鱼精蛋白 DNA 区域，该区域在成熟人类和人类中沿单倍体表观基因组保留修饰的组蛋白小鼠精子。形成鲜明对比的是，基因间和内含子基因组区域含有一个或多个基于 1xCTS 的 CTCF 峰，其名称为 5'-CCC(C/t)CT(a/g)-3' 基序，该基序经常受到疾病的影响-影响三维组织的相关SNP印记在粘性C末端和来自参与位点特异性的远端CTCF/DNA复合物的无DNA ZF子集之间的基本自相互作用上通过粘连蛋白保留来稳定二聚/多聚。在有遗传负担的人类受试者中发现的与 CTCF +/- 单倍体不足的显着联系可能会在临床导向的 CTCF 研究中开辟一条新途径，该研究与异常组蛋白/DNA 甲基化相关，包括 CTCF 结合的 ChIP-Seq 峰，其中包含 2xCTS 元件H3K27ac 标记的 Pol2 结合启动子-增强子对能够以与我们之前发现的作用方式相同的方式改变基因表达与西雅图 Fred Hutchinson 癌症中心合作对 Ctcf+/- 小鼠进行了分析。因此，类似的病理相关机制似乎是由 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，即使在具有 16q22/CTCF LOH 的肿瘤中，也会导致 CTCF 完全丧失而不是部分死亡。由于体内选择可行的单一氨基酸而导致 DNA-CTCF 相互作用丧失。多价 11 ZnF CTCF DBD 中的取代首先在 CTCF (1996) 中被表征，后来 (2002) 被发现在 CTCF 衍生的旁系同源物中重述，名为“BORIS”（“Brother Of the Regular of Imprinted States”的缩写））。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错误观念，即所有 CTCF 位点彼此等效，单个 CTCF 分子结合在单个 CTS 序列上，尽管事实上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，而没有任何基于基序的可靠预测所需的同源性。 Pol2 结合增强子/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域内仅 CTCF 单体结合的包含 1xCTS 的区域的相同特征不同。先前被忽视的一类具有两个（而不是一个）紧密间隔的 CTCF 基序（又名“2xCTS”）的 CTCF 结合区域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遗传表观遗传调控。例如，发现精子核小体的非随机保留是由包含 2xCTS 的 reg.DNA 元件的特定 nt 上下文预先确定的，这些元件通常由晚轮精子细胞中共同表达的 CTCF 和 BORIS 共同结合。此外，我们最新的《自然通讯》论文（https://www.nature.com/articles/s41467-021-24140-6）描述了将 Ctcf 单倍体不足与 Boris-/- null 基因型相结合的不可预测的协同效应新型小鼠 DKO 品系揭示了 CTCF+BORIS 异二聚体对于精子发生和生育能力绝对重要。此外，CTCF 和粘连蛋白现在被广泛认为是所有哺乳动物细胞 3D 基因组结构中的关键角色。这两种蛋白质不仅在科学界众所周知，而且最近还进入了大众媒体。总而言之，我们的数据可以形成染色质动力学的全局视图，并为研究不同细胞谱系中基因表达的远程表观遗传控制提供独特的资源。最后，协同 DKO 效应使我们获得了惊人的认识，即只有 CTCF 被大自然挑选出来作为真正普遍且不可逆的表观遗传标记，不仅存在于所有体细胞类型中，而且存在于成熟精子之前和之后的 DNA 中。在无尽的繁殖轮次中受精。与选择用于免疫治疗试验的其他 CTA 不同，只有 BORIS 可以重新激活所有其他已知的 CTA 基因。因此，鲍里斯登上转化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）

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.

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：

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.