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

• 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
• 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

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，是染色质结构的全球组织者。 CTCF参与了转录激活和抑制作用，基因的烙印，细胞增殖和凋亡的控制，染色质分区 - 重新化，X染色体灭活，预防三核苷酸重肽的扩张以及其他染色质居民过程。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存的“多价11 ZF DBD结合多种DNA序列的能力，以及与伴侣蛋白质通过组合使用DNA contant和prots Zontations Zontanting＆Protanting Zontanting和protains的固有能力，以及其本质上的能力。去年，果蝇（HW）因子中的另一个聚-ZF阵列显示了类似的多价。随着下一代测序技术的出现，CTCF结合位点在跨越蝇，小鼠和人类基因组之间被鉴定出来。反映CTCF函数的众多，发现成千上万的非同源CTS序列与参与远程染色质相互作用的基因组区域有关，包括增强子，启动子，启动子和基因间边界元素。但是，关于任何给定CT的特定DNA序列与同一位点的特定CTCF函数如何相关，这仍然是晦涩的。今年，我们已经取得了进一步的进步，以理解通过DNA接触手指的不同组合形成的不同CTCF/DNA复合物的多重功能。通过绘制同时绘制CTCF和Boris占用基因组的占用，我们发现了一类新的CTCF结合区域，这些区域在功能上进行了预先编程和进化为Epegenet，我们发现CTCF结合区域中有70％的CTCF结合区域包含一个CTCF结合点，由单个CTCF结合部位，AKA“ AKA” 1xcts，而其他30％的ctff-bindsys sege syessysseys syessysseysseys”，同时30％CTC。实际上，由于单个峰被证明包含双CTCF结合位点，也就是二进制的“ 2xctses”。二进制2xcts-区域内相邻CTS的占用率限制了2种相邻的CTCF蛋白在正常体细胞中形成同型二聚体，或者在胚芽中同一DNA的CTCF+ Boris共同组装的异二聚体，而癌细胞在CTCF顶部共表达Boris。最近发现的2xcts-Regions的突破性发现，与任何ChIP-Seq峰峰值呼叫算法无法避免的相邻区域使我们首次使我们能够解决一个长期以来的问题，以解决CTCF-BIN如何在与核心的范围内保持在核心方面的范围，以使其在范围内保持不变，并在范围内保持范围，以使其在范围内保持范围，并在范围内保持范围的范围。转录活动。实际上，所有CTCF结合区域中只有20％位于任何给定的细胞类型的启动子区域中，而其余的CTSE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本身就是DNA序列本身和/或在这两种类型的位点的染色质差异身份。在我们的研究中，我们提供了全基因组的证据，表明两种类型的CTCF目标位点的DNA序列在结构上是不同的。 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在成熟的人和小鼠精子中保留沿单倍体epi基因组的修饰组蛋白。在一个鲜明的对比中，具有一个或多个基于1XCTS的CTCF峰的基因间和内在基因组区域，其名称为5'-CCC（C/T）CT（A/G）-3'基序通常受到疾病相关的SNP的击中，该组织通常受到与基本自我启动的SNP相关的SNP，从而受到了cacksy c的自我启动的snp，c-ccc-ccc（a/t）tobif ti Indersal condefient conteffience confimential conteffience confimential conteffience confimentials confsient snp。 CTCF/DNA复合物参与特定于位点的DI-/多合并，通过粘蛋白保留稳定。在遗传负担的人类受试者中发现的与CTCF +/-单倍不使言语的显着联系可能会在临床上导向的CTCF研究中开辟一个新颖的AVENENUE，与具有CTCF结合的CHIP-seq峰的临床导向的CTCF研究与异常的组蛋白/DNA-甲基化相关，该研究与H3K27Ac-Sarked-benderant interhant-by seqs equalenth chip-seq-seq-noundhention themant-by sequrantherations ctcf-2xcts elements in thement-nbound-bound seq。表达与以前发现在与西雅图的弗雷德·哈钦森癌症中心合作分析的CTCF +/-小鼠背景下作用的方式。 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.在CTCF（1996）中首先表征的多价11 ZNF CTCF DBD中的取代，后来在（2002年）上发现了CTCF衍生的paralog中，名为“ Boris”（名为“ Boris”（“印刷状态的常规兄弟”））。接下来，我们对二进制2xcts代码的发现和进一步的研究开始挑战当前文献中的广泛误解，声称所有CTCF站点彼此相等，并且单个CTCF分子在单个CTS序列上绑定的单个CTS序列具有单个或两种元素的事实，该事实可能会与不同的基因组合元素相比，这可能会构成不同的基因组合元素。 CTCF基序没有任何基于可靠基序的预测所需的同源性。 POL2结合增强子/启动子相关的2XCTS元素的功能和结构表观特征与在内在和基因间非编码区域内仅由CTCF仅由CTCF的单体绑定的含1xcts的区域的相同特征不同。具有两个（而不是一个）紧密间隔的CTCF基序（又称“ 2xcts”）的CTCF结合区域的先前被忽视的类别具有非常独特的作用，在调节基于各种染色质的现象中，包括。癌细胞和正常生殖细胞中可遗传的表观遗传调节。例如，发现精子核小体的非随机保留是通过含2xcts reg的特定NT上下文预先确定的。DNA元素通常由CTCF＆Boris在后期的精子中共表达。此外，我们最新的“自然通讯”论文在线可在线提供，网址为https://www.nature.com/articles/s41467-021-24140-6-介绍了将CTCF与单倍型与鲍里斯（Boris）与鲍里斯（Boris）与鲍里斯（Boris-Null Genot）相结合的CTCF HaploSufficied在Gress-Null基因型中的基本鼠标的不可预测的协同效应，该效果完全揭示了CTC Fors the hy the CTCF+精子发生和生育能力。此外，CTCF和粘着蛋白现在被广泛认为是所有哺乳动物细胞中3D基因组结构中的关键参与者。这两种蛋白质不仅在科学界广为人知，而且最近进入了流行媒体和媒体。综上所述，我们的数据允许开发染色质动力学的全球视图，并为研究不同细胞谱系中基因表达的长期表观遗传控制提供了独特的资源。最后，协同的DKO效应使我们有了惊人的理解，即只有母亲才被CTCF挑出，不仅在所有体细胞类型的DNA上都在DNA上充当一个真正的普遍和不可逆的表观遗传标记，而且在无休止的再生回合后和之后，在所有体细胞类型中，而且在成熟的精子中也是如此。与选择用于免疫疗法试验的其他CTA不同，只有鲍里斯可以重新激活所有其他已知的CTA基因。因此，看到鲍里斯（Boris）提升到翻译研究的顶端也就不足为奇了。

项目成果

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

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

• DOI: 10.1038/srep41279
• 发表时间: 2017-02-01
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Rivero-Hinojosa S;Kang S;Lobanenkov VV;Zentner GE
• 通讯作者: Zentner GE

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

Discovering a binary CTCF code with a little help from BORIS.

• DOI: 10.1080/19491034.2017.1394536
• 发表时间: 2018-01-01
• 期刊: Nucleus (Austin, Tex.)
• 作者: Lobanenkov VV;Zentner GE
• 通讯作者: Zentner GE