CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF-DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization

Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene

Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5′-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression

Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions.

BackgroundCTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation.ResultsHere we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of the BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells.ConclusionsWe discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells

BORIS expression in ovarian cancer precursor cells alters the CTCF cistrome and enhances invasiveness through GALNT14

High-grade serous carcinoma (HGSC) is the most aggressive and predominant form of epithelial ovarian cancer and the leading cause of gynecological cancer death. We have previously shown that CTCFL (also known as BORIS, Brother of the Regulator of Imprinted Sites) is expressed in most ovarian cancers, and is associated with global and promoter-specific DNA hypomethylation, advanced tumor stage, and poor prognosis. To explore its role in HGSC, we expressed BORIS in human fallopian tube secretory epithelial cells (FTSEC), the presumptive cells of origin for HGSC. BORIS-expressing cells exhibited increased motility and invasion, and BORIS expression was associated with alterations in several cancer-associated gene expression networks, including fatty acid metabolism, TNF signaling, cell migration, and ECM-receptor interactions. Importantly, GALNT14, a glycosyltransferase gene implicated in cancer cell migration and invasion, was highly induced by BORIS, and GALNT14 knockdown significantly abrogated BORIS-induced cell motility and invasion. In addition, in silico analyses provided evidence for BORIS and GALNT14 co-expression in several cancers. Finally, ChIP-seq demonstrated that expression of BORIS was associated with de novo and enhanced binding of CTCF at hundreds of loci, many of which correlated with activation of transcription at target genes, including GALNT14. Taken together, our data indicate that BORIS may promote cell motility and invasion in HGSC via upregulation of GALNT14, and suggests BORIS as a potential therapeutic target in this malignancy

BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells

Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis

Global expression analysis of cancer/testis genes in uterine cancers reveals a high incidence of BORIS expression

Abstract Purpose: Cancer/testis (CT) genes predominantly expressed in the testis (germ cells) and generally not in other normal tissues are aberrantly expressed in human cancers. This highly restricted expression provides a unique opportunity to use these CTgenes for diagnostics, immunotherapeutic, or other targeted therapies. The purpose of this study was to identify those CT genes with the greatest incidence of expression in uterine cancers. Experimental Design: We queried the expression of known and putative CT gene transcripts (representing 79 gene loci) using whole genome gene expression arrays. Specifically, the global gene expressions of uterine cancers (n = 122) and normal uteri (n = 10) were determined using expression data from the Affymetrix HG-U133A and HG-U133B chips. Additionally, we also examined the brother of the regulator of imprinted sites (BORIS) transcript by reverse transcription-PCR and quantitative PCR because its transcript was not represented on the array. Results: Global microarray analysis detected many CT genes expressed in various uterine cancers; however, no individual CT gene was expressed in more than 25% of all cancers. The expression of the two most commonly expressed CT genes on the arrays, MAGEA9 (24 of 122 cancers and 0 of10 normal tissues) and Down syndrome critical region 8 (DSCR8)/MMA1 (16 if 122 cancers and 0 of 10 normal tissues), was confirmed by reverse transcription-PCR methods, validating the array screening approach. In contrast to the relatively low incidence of expression of the other CTgenes, BORIS expression was detected in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors. Conclusions: These data provide the first extensive survey of multiple CT genes in uterine cancers. Importantly, we detected a high frequency of BORIS expression in uterine cancers, suggesting its potential as an immunologic or diagnostic target for these cancers. Given the high incidence of BORIS expression and its possible regulatory role, an examination of BORIS function in the etiology of these cancers is warranted

Loss of Maternal CTCF Is Associated with Peri-Implantation Lethality of Ctcf Null Embryos

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development

BORIS, a paralogue of the transcription factor, CTCF, is aberrantly expressed in breast tumours

BORIS (for brother of the regulator of imprinted sites), a paralogue of the transcription factor, CTCF, is a novel member of the cancer-testis antigen family. The aims of the present study were as follows: (1) to investigate BORIS expression in breast cells and tumours using immunohistochemical staining, western and real-time RT–PCR analyses and (2) assess potential correlation between BORIS levels in tumours with clinical/pathological parameters. BORIS was detected in all 18 inspected breast cell lines, but not in a primary normal breast cell culture. In 70.7% (41 of 58 cases) BORIS was observed in breast tumours. High levels of BORIS correlated with high levels of progesterone receptor (PR) and oestrogen receptor (ER). The link between BORIS and PR/ER was further confirmed by the ability of BORIS to activate the promoters of the PR and ER genes in the reporter assays. Detection of BORIS in a high proportion of breast cancer patients implies potential practical applications of BORIS as a molecular biomarker of breast cancer. This may be important for diagnosis of the condition and for the therapeutic use of BORIS. The ability of BORIS to activate promoters of the RP and ER genes points towards possible involvement of BORIS in the establishment, progression and maintenance of breast tumours

The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer

BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions.Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates.The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation

Histone Modifications at Human Enhancers Reflect Global Cell-Type-Specific Gene Expression

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene1, 2, 3, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome4, 5, 6. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression