Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

BORIS 对正常和病理 CTCF 功能的表观遗传调控

• 批准号: 9354824
• 负责人: Victor Lobanenkov
• 金额: $ 60.77万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9354824
• 关键词: Address; Affect; Area; Binding; Binding Sites; Biological Assay; Brothers; Cancer cell line; Cell Line; Cells; ChIP-seq; Chromatin; DNA Binding; DNA Binding Domain; DNA Sequence; Derivation procedure; Development; Diagnostic; Disease; ES Cell Line; Ectopic Expression; Epigenetic Process; Evolution; Gametogenesis; Gene Duplication; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Germ Cells; Goals; Human; Human Genome; Immunological Diagnosis; In Vitro; K562 Cells; Knock-out; MCF7 cell; Maintenance; Malignant Neoplasms; Maps; Mediating; Meiosis; Mitotic; Molecular; Monoclonal Antibodies; Mus; Pathologic; Pattern; Plasmids; Play; Proteins; Protocols documentation; Rattus; Regulation; Regulatory Element; Reporter; Repression; Role; Site; Stem cells; Structure; TATA-Box Binding Protein; Technology; Transcript; Transcriptional Activation; Transfection; Translational Research; Vaccination; base; c-ets1 transcription factor; cancer cell; cancer immunotherapy; cancer testis antigen; cancer therapy; clinical application; embryonic stem cell; epigenetic regulation; genome-wide; imprint; improved; in vivo; malignant breast neoplasm; melanoma-associated antigen-A1; mouse genome; neuronal cell body; novel; overexpression; paralogous gene; pluripotency; polyclonal antibody; programs; promoter; protein protein interaction; research study; treatment site; zinc finger nuclease

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.

作为CTCF基因复制的结果，BORIS（印迹位点调控的兄弟）在羊膜早期进化中出现。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，并以互斥的方式发挥作用。事实上，虽然CTCF是普遍表达的，但BORIS的表达严格限于正常发育的生殖细胞。然而，BORIS在多种癌症中异常表达，其在这种情况下的功能尚未被表征。为了解决这一问题，我们开发并利用了一套单克隆和多克隆抗体来定位人和小鼠基因组中的CTCF和BORIS结合位点。通过对几种表达BORIS的人类癌细胞系和小鼠生殖细胞的ChIP-seq分析发现，BORIS的结合模式在不同来源的细胞系之间是相似的，从而概括了BORIS在生殖细胞中的结合，从而反映了它们倾向于结合BORIS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同源二聚体共结合的能力。这些区域代表了所谓的双重“2xCTS”位点，能够将两个因子结合到至少两个相邻的11zf识别的基序上，这些基序彼此紧密地顺式靠近，并通过它们的合作蛋白-蛋白相互作用相互加强。我们发现CTCF和BORIS在2xCTSes的合作对癌症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细胞发生异常分化，而BORIS基因的异位表达导致MCF7细胞的干细胞特异性转录变化。在接下来的研究中，我们分析了BORIS与转录因子Ets-1和Sp1在调节MAGE-A1基因表达中的相互作用。我们发现异位表达的BORIS可以激活MCF-7细胞和微转移BCM1癌细胞系中内源性和甲基化的报告基因MAGE-A1启动子并使其去甲基化。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部分抑制了boris介导的刺激，而添加Ets-1表达质粒则消除了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BORIS和Sp1转录本水平之间可能存在竞争机制。