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

CTCF是一个高度保守的多功能核因子，既参与全球基因组结构，也参与基因调控的许多方面，后者从直接的基因抑制/激活到增强子阻断和激素促进的沉默。来自进化遥远物种的ctcf都含有一个中心高度保守的11锌指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。dna结合CTCF的二聚化活性可能是其在大多数细胞类型中作为多用途染色质桥接和染色质环连接剂活性的核心，是其染色质绝缘体和异染色质边界功能的基础。在特定基因的背景下，CTCF也可能通过染色质环对转录增强子进行功能性调节。对成千上万个CTCF靶点（CTS）的全基因组定位表明，CTCF可以将核小体定位在dna酶敏感位点周围，这些位点标志着绝缘子、增强子和其他调控序列。由于CTCF具有如此多的重要功能，CTCF成为脊椎动物的重要基因，因为CTCF敲除小鼠是不可存活的（在早期胚胎阶段是致命的）。就人类疾病而言，CTCF是一种公认的肿瘤抑制基因（TSG）；CTCF的11ZF DBD的几个功能点突变在原发性癌症中被表征，在最初以CTCF位点LOH为特征的肿瘤中。

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.