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.

BORIS（印记位点调节器的兄弟）是在羊膜动物早期进化过程中由于 CTCF 基因复制而出现的。 CTCF 和 BORIS 编码的蛋白质具有几乎相同的 DNA 结合域，可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 具有不同的功能，并且以相互排斥的方式行事。事实上，虽然 CTCF 普遍表达，但 BORIS 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，其在此背景下的功能尚未得到表征。为了解决这个问题，我们开发并利用了一组单克隆和多克隆抗体来绘制人类和小鼠基因组中的 CTCF 和 BORIS 结合位点。对几种表达 BORIS 的人类癌细胞系和小鼠生殖细胞进行 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，从而反映了其结合 BORIS 倾向的结合区域的潜在编码。我们发现这种编码很大程度上反映了这些区域与 CTCF 和 BORIS 异二聚体或 BORIS 或 CTCF 同二聚体共结合的能力。这些区域代表所谓的双“2xCTS”位点，能够将两个因子与至少两个相邻的 11ZF 识别基序结合，这些基序彼此紧密顺式靠近，并通过它们的协同蛋白质-蛋白质相互作用相互增强。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基因的缺失会导致大量基因的转录改变和K562细胞的异常分化，而BORIS的异位表达会导致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MAGE-A1基因表达调节中的相互作用。我们发现异位表达的 BORIS 可以激活 MCF-7 细胞和微转移 BCM1 癌细胞系中的内源性和甲基化报告基因 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 启动子抑制。 BORIS 和 Sp1 均与 TATA 结合蛋白 (hTBP) 相互作用，表明报告基因检测中 BORIS 和 Sp1 转录水平之间可能存在竞争作用机制。