Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

Boris(印迹位点调节基因的兄弟)出现在羊膜动物的早期进化过程中，这是CTCF基因复制的结果。CTCF和Boris编码的蛋白质具有几乎相同的DNA结合域，在体内和体外识别相同的DNA序列。长期以来，人们一直认为CTCF和Boris拥有截然不同的职能，并以相互排斥的方式行事。事实上，虽然CTCF普遍表达，但Boris的表达严格限制在正常发育的生殖细胞。然而，Boris在广泛的癌症中异常表达，其在这一背景下的功能尚未被描述。为了解决这个问题，我们已经开发和利用了一组单抗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。对几个表达Boris的人类癌细胞和小鼠生殖细胞的芯片序列分析证实，在独立起源的细胞系中，Boris结合的模式相似，概括了生殖细胞中的Boris结合，从而反映了它们与Boris结合倾向的结合区域的潜在编码。我们发现，这种编码在很大程度上反映了这些区域与CTCF和Boris异源二聚体或Boris或CTCF同源二聚体共同结合的能力。这些区域代表所谓的双“2xCTS”位点，能够将这两个因子与至少两个相邻的11ZF识别的基序结合在一起，这些基序彼此靠近，并通过它们的合作蛋白质-蛋白质相互作用而相互加强。我们发现，CTCF和Boris在2xCTSes上的合作对癌细胞和生殖细胞的转录程序至关重要。Boris基因缺失导致大量基因转录改变，导致K562细胞分化异常，而Boris基因的异位表达导致MCF7细胞的干细胞特异性转录改变。在接下来的研究中，我们分析了Boris与转录因子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MCF-7细胞和微转移BCM1癌细胞中内源性和甲基化报告MAGE-A1启动子并使其去甲基化。过表达Ets-1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部分抑制了Boris介导的刺激，而添加Ets-1表达载体则取消了Sp1介导的对MAGE-A1启动子的抑制。在报告分析中，Boris和Sp1都与TATA结合蛋白(HTBP)相互作用，这表明Boris和Sp1转录水平之间可能存在竞争作用机制。

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.