Normal and Pathologic Functions of CTCF and Its Distinct Classes of DNA-targets

CTCF 的正常和病理功能及其不同类型的 DNA 靶标

基本信息

项目摘要

CTCF is a highly conserved, multi-functional nuclear factor involved both in global genome architecture and in many aspects of gene regulation, latter ranging from the direct gene repression/activation to enhancer blocking and hormone-facilitated silencing. CTCFs from evolutionary distant species all contain a central highly conserved 11 Zn-finger DNA-binding domain, which mediates the multiple sequence specificity of its DNA binding activity. Dimerization activity of DNA-bound CTCF may potentially be at the core of its activity as a versatile chromatin-bridging and chromatin-looping agent in most cell types, underlying its chromatin-insulator and heterochromatin-boundary functions. In the context of specific genes, CTCF may also functionally modulate transcriptional enhancers via chromatin-looping. Genome-wide mapping of tens of thousands of CTCF target sites (CTS) showed that CTCF can position nucleosomes around DNAse hypersensitive sites that landmark insulators, enhancers, and other regulatory sequences. By virtue of having so many vital functions CTCF became an essential gene in vertebrates, as CTCF-knockout mice are non-viable (lethality at the early embryonic stages). With respect to human disease, CTCF is a well-established tumor suppressor gene (TSG); several functional point mutations in the 11ZF DBD of CTCF have been characterized in primary cancers, in tumors initially characterized by the LOH of the CTCF locus. (1) In the past year, genome-wide analyses have led to significantly deeper understanding of the global role of CTCF in genomes of vertebrates. It is now clear that CTCF controls at least three major pathways: the global architecture of the genome, the chromatin-structure-mediated (loops formation, enhancer-blocker activity) regulation of gene expression (of both imprinted and imprinting-independent genes), and the direct regulation of gene expression (through interface with other transcription factors). We previously analyzed genome-wide CTCF targets for the first time (Cell 2007, vol. 128, pp1231-1245), and the fundamental roles of CTCF in cellular functions were validated by a strong correlation of CTCF target sites (CTS) with gene positions in human genome. Therefore, we developed a comprehensive program to expand genome-wide studies, in order to identify positive correlations and, eventually, functional significances of CTCF (and BORIS) binding at the individual chromosomal loci. We performed ChIP-chip analysis to map CTS, as well as the p300 coactivator, and compared them to histone modification patterns in 5 human cell lines: cervical carcinoma (HeLa), immortalized lymphoblast GM06690 (GM), leukaemia K562, embryonic stem cells (ES), and BMP4-induced ES cells (dES). We used the ENCODE microarrays (1% of the human genome) to identify putative CTCF-bound sites for these cell types and observed highly reproducible CTCF occupancy (in contrast to largely cell-type specific histone modifications and p300 binding), confirming our theory that the bulk of CTCF functions in the cell is cell-type independent. At the same time, a subset of the CTS that appeared to be cell-type-dependent, confirmes that in addition to its global function in the human genome, CTCF may be directly involved in the regulation of cell lineage, possibly through acting at few specific enhancers or at alternative (intragene) promoters. (2) We also conducted genome-wide DNA-binding analysis of Drosophila CTCF (DrCTCF), which we previously cloned and characterized. We identified more than 3561 strong DrCTS (as well as 8872 weaker ones, with two-fold lower enrichment) genome-wide. Whole-genome analysis showed that DrCTCF in general was often found to bind between genes that are closely positioned but differentially regulated. However, DrCTS were also highly enriched to the 5′ of genes, which did not have closely-positioned neighboring promoters. In contrast, distribution of predicted Su(Hw) insulator sites did not display any bias towards promoters. Therefore, it is likely that, in addition to its insulator function, DrCTCF binding upstream of promoters might have a more general role: either in direct regulation of transcription or/and in global genome organization of Drosophila genome. In a specific case, as a result of this genome-wide analysis, the Fab-6 insulator element from the Abd-B locus was identified as a new strong drCTCF binding site (with two CTS), and was shown (using specially designed transgenic and plasmid EB assays) to be a new CTCF-controlled EB element. Follow-up results indicate that DrCTCF is essential for the enhancer blocking activity of the Fab-6, in addition to the Fab-8 insulator, and that CTCF likely plays an important role in organizing the Abd-B locus. (3) A specific case of CTCF function as a direct regulator of gene expression and its interface with other (more specialized) transcription factors was revealed upon continuing analysis of CTCFs role in the regulation of human telomerase gene (hTERT). In our previous publications, we demonstrated that CTCF was essential for the repression of hTERT transcription in a variety of normal somatic cells, while CTCF downregulation (in specifically designed assays and in cancer cells) resulted in hTERT expression activation, facilitating cell immortalization. The repressor activity of CTCF was not promoter-specific but was mediated by its binding to the first exons of the hTERT gene. In our recent work, we investigated what mechanism is involved in the atypical enhanced expression of hTERT in lymphoid cells. We uncovered that a transcription factor PAX5, which is specific for B-cell development and is essential for B-lymphocyte function, binds downstream of two CTCF-binding sites in hTERT and enables the derepression of the gene, overpowering the CTCF repressor activity (apparently without CTCF displacement). These results identify hTERT as a novel target of PAX5, which thus participates in cellular mechanisms underlying cell immortality. Furthermore, this finding reveals a novel pathway of CTCF involvement in the direct regulation of gene expression, possibly employing its interactions with a range of other cellular factors, including some that are cell-type specific. (4) Even more daring area of research was targeted with our investigation of the functions of CTCF outside of gene expression regulation. This subject is largely ignored in the CTCF literature, even though it is apparent that the bulk of cellular CTCF is bound to repeated/noncoding DNA. Nevertheless, such a localization pattern might indicate that the CTCFs function as a critical factor ensuring genome integrity and chromosome stability is largely mediated by these genomic loci. Indeed, our previous data on potential CTCFs functions in heterochromatin, as well as its roles in mitosis and meiosis, suggested a significant housekeeping role of CTCF in the organization of mammalian genome and in chromosome segregation. We focused our studies at one particular region of chromosomes the centromeric gamma satellite repeats residing in the heterochromatic regions flanking human centromeres. It was known that in hematopoetic cells these repeats are bound by Ikaros, a carrier of the heterochromatin barrier (or anti-silencing) activity of gamma-satellites. It was not understood, however, what proteins take the place of Ikaros binding in gamma-satellites in other cell types. Our data indicated that gamma-satellites could be bound by CTCF in vivo, and thus this binding could contribute to centromere function. Our data showing that CTCF-bound gamma-satellites serve as a heterochromatin barrier (protecting a transgene from silencing), indicate that the biological role of of human gamma-satellite chromatin may be to prevent spreading of pericentric hheterochromatin into gene-containing chromosomal zones.
CTCF是一种高度保守的多功能核因子,既参与全局基因组结构,又在基因调节的许多方面,后者从直接基因抑制/激活到增强子阻断和激素促成沉默。来自进化远处物种的CTCF都包含一个中央高度保守的11 Zn手指DNA结合结构域,该结构域介导了其DNA结合活性的多序列特异性。在大多数细胞类型中,DNA结合的CTCF的二聚化活性可能是其活性的核心,它是多功能染色质桥和染色质循环剂的核心,其基础是其染色质蛋白质蛋白质蛋白质素和异染色质 - 结合函数。在特定基因的背景下,CTCF还可以通过染色质循环在功能上调节转录增强子。数以万计的CTCF目标位点(CTS)的全基因组映射表明,CTCF可以将核小体定位在DNase超敏部位周围,以使绝缘剂,增强子和其他调节序列具有地标性过敏。由于具有如此多的重要功能,CTCF成为脊椎动物中必不可少的基因,因为CTCF-KNOCKOUT小鼠是不可行的(在早期胚胎阶段的致命性)。关于人类疾病,CTCF是一个公认的肿瘤抑制基因(TSG)。 CTCF的11ZF DBD中的几个功能点突变已在原发性癌症中表征,在最初以CTCF基因座的LOH为特征的肿瘤中。 (1)在过去的一年中,全基因组分析已使CTCF在脊椎动物基因组中的全球作用更加深入了解。现在很明显,CTCF控制至少三个主要途径:基因组的整体结构,染色质结构介导的(LOOP形成,增强子阻滞剂活性)调节基因表达(不含糊和不依赖性基因的基因和不依赖性基因)以及基因表达的直接调节(通过与其他转录因子的界面)。我们先前首次分析了全基因组CTCF靶标(Cell 2007,第128卷,PP1231-1245),并且通过CTCF靶位点(CTS)与人类基因组中的基因位置有很强的相关性,CTCF在细胞功能中的基本作用得到了验证。因此,我们开发了一个全面的计划来扩展全基因组研究,以确定CTCF(和Boris)结合在个体染色体基因座的正相关性,并最终是功能意义。 We performed ChIP-chip analysis to map CTS, as well as the p300 coactivator, and compared them to histone modification patterns in 5 human cell lines: cervical carcinoma (HeLa), immortalized lymphoblast GM06690 (GM), leukaemia K562, embryonic stem cells (ES), and BMP4-induced ES cells (dES).我们使用编码微阵列(占人类基因组的1%)来识别这些细胞类型的假定CTCF结合位点,并观察到高度可重复可重复的CTCF占用率(与很大程度上相比,与细胞类型的特定组蛋白修饰和p300结合相反),证实了我们的理论,即CTCF在细胞独立于Cell-typepe中起着CTCF的作用。同时,似乎是细胞类型依赖性的CTS子集证实,除了其在人类基因组中的全球功能外,CTCF还可能直接参与细胞谱系的调节,可能是通过在少数特定增强子或替代性(Intragene)启动子上作用的细胞谱系。 (2)我们还对果蝇CTCF(DRCTCF)进行了全基因组DNA结合分析,我们先前对其进行了克隆和表征。我们确定了超过3561个强的DRCT(以及8872个弱较弱的DRCT,较低的富集)全基因组。全基因组分析表明,通常发现DRCTCF在紧密位置但受差异调节的基因之间结合。然而,DRCT也高度富集到5'的基因,该基因没有紧密位置的邻近启动子。相比之下,预测的SU(HW)绝缘子位点的分布没有对启动子表现出任何偏见。因此,除了其绝缘函数外,启动子上游的DRCTCF结合可能具有更一般的作用:在果蝇基因组的全球基因组组织中,启动子的结合可能具有更一般的作用。在特定情况下,由于本基因组分析的结果,来自ABD-B基因座的FAB-6绝缘子元件被鉴定为一种新的强drctCF结合位点(具有两个CTS),并显示(使用特殊设计的转基因和质粒EB分析)是新的CTCF Controllolled EB元素。后续结果表明,除了FAB-8绝缘子外,DRCTCF对于FAB-6的增强子阻断活性至关重要,并且CTCF可能在组织ABD-B基因座中起重要作用。 (3)CTCF的特定病例作为基因表达的直接调节剂及其与其他(更专业的)转录因子的界面在继续分析CTCF在人类端粒酶基因(HTERT)调节中的作用后,揭示了CTCF。在我们以前的出版物中,我们证明了CTCF对于在多种正常体细胞中抑制HTERT转录至关重要,而CTCF下调(在专门设计的分析和癌细胞中)导致HTERT表达激活,促进细胞的永生。 CTCF的阻遏活性不是启动子特异性的,而是由其与HTERT基因的第一外显子结合而介导的。在我们最近的工作中,我们研究了淋巴样细胞中HTERT非典型增强表达的哪种机制。我们发现的是,转录因子PAX5是针对B细胞发育的特异性,对于B淋巴细胞功能至关重要,它结合了HTERT中两个CTCF结合位点的下游,并使基因的压抑,压倒了CTCF压抑物活性(显然没有CTCF替代)。这些结果将HTERT识别为PAX5的新靶标,因此参与了细胞永生的基础机制。此外,这一发现揭示了CTCF参与基因表达的直接调控的新途径,可能采用了其与其他一系列细胞因子的相互作用,包括一些细胞类型的特异性。 (4)我们研究了基因表达调节外的CTCF功能的针对更大的研究领域。在CTCF文献中,该主题在很大程度上被忽略了,即使显然细胞CTCF的大部分含义是重复/非编码DNA。然而,这样的定位模式可能表明CTCF是确保基因组完整性和染色体稳定性的关键因素,这些因素在很大程度上由这些基因组基因座介导。实际上,我们先前关于潜在CTCF在异染色质中起作用的数据及其在有丝分裂和减数分裂中的作用,这表明CTCF在哺乳动物基因组和染色体分离中的管家作用显着。我们将研究重点放在一个特定的染色体区域,centromeric伽玛卫星重复驻留在人类centromeres侧面的异质区域。众所周知,在造血细胞中,这些重复由Ikaros(γ-卫星的异染色质屏障(或抗沉积)活性的载体结合。然而,尚不清楚哪些蛋白在其他细胞类型中在伽马 - 卫星中结合了ikaros结合的位置。我们的数据表明,伽马 - 卫星可以在体内受到CTCF的约束,因此这种结合可能有助于中心粒功能。我们的数据表明,CTCF结合的伽马 - 卫星充当异染色质屏障(保护转基因免受沉默)表明,人γ-卫星染色质的生物学作用可能是防止丁香色质质质蛋白散布到伴有基因染色体的染色体Zone中。

项目成果

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Victor Lobanenkov其他文献

Victor Lobanenkov的其他文献

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{{ truncateString('Victor Lobanenkov', 18)}}的其他基金

Regulation of CTCF Functions and Target Sites by Cancer/Testis-specific CTCF Like BORIS Factor
癌症/睾丸特异性 CTCF 样 BORIS 因子对 CTCF 功能和靶位点的调节
  • 批准号:
    10272128
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Regulation of CTCF Functions and Target Sites by Cancer/Testis-specific CTCF Like BORIS Factor
癌症/睾丸特异性 CTCF 样 BORIS 因子对 CTCF 功能和靶位点的调节
  • 批准号:
    10692106
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Regulation of CTCF Functions and Target Sites by Cancer/Testis-specific CTCF Like BORIS Factor
癌症/睾丸特异性 CTCF 样 BORIS 因子对 CTCF 功能和靶位点的调节
  • 批准号:
    10927815
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS
BORIS 对正常和病理 CTCF 功能的表观遗传调控
  • 批准号:
    8336243
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS
BORIS 对正常和病理 CTCF 功能的表观遗传调控
  • 批准号:
    8946422
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS
BORIS 对正常和病理 CTCF 功能的表观遗传调控
  • 批准号:
    9354824
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Normal and Pathologic Functions of CTCF and Its Distinct Classes of DNA-targets
CTCF 的正常和病理功能及其不同类型的 DNA 靶标
  • 批准号:
    8745378
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS
BORIS 对正常和病理 CTCF 功能的表观遗传调控
  • 批准号:
    8745467
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:
Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS
BORIS 对正常和病理 CTCF 功能的表观遗传调控
  • 批准号:
    7964638
  • 财政年份:
  • 资助金额:
    $ 57.84万
  • 项目类别:

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