Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

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BORIS (Brother Of the Regulator of Imprinting Sites) emerged during early evolution of amniotes as the result of CTCF gene duplication. CTCF and BORIS encode proteins that share an almost identical DNA binding domain recognizing the same DNA sequences in vivo and in vitro. It has long been thought that CTCF and BORIS possess distinct functions and act in a mutually exclusive manner. Indeed, while CTCF is ubiquitously expressed, BORIS expression is strictly restricted to germ cells in normal development. However, BORIS is aberrantly expressed in a wide range of cancers, and its function in that context has not been characterized. To address this problem, we have developed and utilized a set of monoclonal and polyclonal antibodies to map CTCF and BORIS binding sites in both human and mouse genomes. ChIP-seq analysis of several BORIS-expressing human cancer cell lines and mouse germ cells established that the pattern of BORIS binding is similar across cell lines of independent origin, recapitulates BORIS binding in germ cells, and thus reflects an underlying encoding of the binding regions for their propensity to bind BORIS. We uncovered that this encoding largely reflects the ability of these regions to be co-bound by CTCF and BORIS heterodimers or by BORIS or CTCF homodimers. Those regions represent the so-called dual "2xCTS" sites capable of binding both factors to at least two adjacent 11ZF-recognized motifs placed in close cis proximity near each other, and mutually reinforced by their cooperative protein-protein interactions. We found that the cooperation of CTCF and BORIS at 2xCTSes is critical for the transcriptional program of cancer and germ cells. Depletion of BORIS gene leads to altered transcription of a large number of genes and aberrant differentiation of K562 cells, while the ectopic expression of BORIS leads to stem cell specific changes in transcription of MCF7 cells. In the next study, we analyzed the interplay of BORIS and the transcription factors Ets-1 and Sp1 in the regulation of MAGE-A1 gene expression. We found that ectopically expressed BORIS could activate and demethylate both the endogenous and methylated reporter MAGE-A1 promoter in MCF-7 cells and in the micrometastatic BCM1 cancer cell lines. Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS. Surprisingly, in co-transfection experiments we observed that Sp1 partly repressed the BORIS-mediated stimulation, while addition of an Ets-1 expression plasmid abrogated the Sp1 mediated repression of the MAGE-A1 promoter. Both BORIS and Sp1 interacted with the TATA binding protein (hTBP) suggesting the possibility of a competitive mechanism of action between BORIS and Sp1 transcript levels in reporter assays.