Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

BORIS（Brother Of The Regulator of Imprinting Sites）是在黑猩猩的早期进化过程中出现的，是CTCF基因复制的结果。CTCF和BORIS编码的蛋白质在体内和体外具有几乎相同的DNA结合结构域，识别相同的DNA序列。长期以来，人们一直认为CTCF和BORIS具有不同的功能，并以相互排斥的方式发挥作用。事实上，虽然CTCF广泛表达，但BORIS表达严格限于正常发育中的生殖细胞。然而，BORIS在广泛的癌症中异常表达，其在该背景下的功能尚未被表征。为了解决这个问题，我们已经开发并利用了一组单克隆和多克隆抗体来定位人类和小鼠基因组中的CTCF和BORIS结合位点。几种表达BORIS的人癌细胞系和小鼠生殖细胞的ChIP-seq分析确定，BORIS结合模式在独立来源的细胞系中是相似的，概括了生殖细胞中的BORIS结合，因此反映了结合区域结合BORIS的倾向的潜在编码。我们发现，这种编码在很大程度上反映了这些区域被CTCF和BORIS异二聚体或被BORIS或CTCF同源二聚体共结合的能力。这些区域代表了所谓的双“2xCTS”位点，其能够将两种因子结合到至少两个相邻的11 ZF识别的基序上，所述基序彼此接近地顺式接近，并且通过它们的合作蛋白质-蛋白质相互作用而相互增强。我们发现CTCF和BORIS在2xCTSes上的合作对于癌症和生殖细胞的转录程序至关重要。BORIS基因缺失导致K562细胞大量基因转录改变和异常分化，而BORIS的异位表达导致MCF 7细胞转录的干细胞特异性改变。在接下来的研究中，我们分析了BORIS和转录因子Ets-1和Sp1在MAGE-A1基因表达调控中的相互作用。我们发现，异位表达的BORIS可以激活和去甲基化的内源性和甲基化的报告基因MAGE-A1启动子在MCF-7细胞和微转移癌细胞系的BCM 1。Ets-1的过表达不能进一步上调BORIS介导的启动子活性。令人惊讶的是，在共转染实验中，我们观察到Sp1部分抑制BORIS介导的刺激，而添加Ets-1表达质粒废除了Sp1介导的MAGE-A1启动子的抑制。BORIS和Sp1都与TATA结合蛋白（hTBP）相互作用，这表明在报告分析中BORIS和Sp1转录水平之间存在竞争性作用机制的可能性。

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.