Gopalakrishnan Suhasni, Sullivan Beth A, Trazzi Stefania, Della Valle Giuliano, Robertson Keith D
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA.
Hum Mol Genet. 2009 Sep 1;18(17):3178-93. doi: 10.1093/hmg/ddp256. Epub 2009 May 29.
DNA methylation is an epigenetically imposed mark of transcriptional repression that is essential for maintenance of chromatin structure and genomic stability. Genome-wide methylation patterns are mediated by the combined action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B. Compelling links exist between DNMT3B and chromosome stability as emphasized by the mitotic defects that are a hallmark of ICF syndrome, a disease arising from germline mutations in DNMT3B. Centromeric and pericentromeric regions are essential for chromosome condensation and the fidelity of segregation. Centromere regions contain distinct epigenetic marks, including dense DNA hypermethylation, yet the mechanisms by which DNA methylation is targeted to these regions remains largely unknown. In the present study, we used a yeast two-hybrid screen and identified a novel interaction between DNMT3B and constitutive centromere protein CENP-C. CENP-C is itself essential for mitosis. We confirm this interaction in mammalian cells and map the domains responsible. Using siRNA knock downs, bisulfite genomic sequencing and ChIP, we demonstrate for the first time that CENP-C recruits DNA methylation and DNMT3B to both centromeric and pericentromeric satellite repeats and that CENP-C and DNMT3B regulate the histone code in these regions, including marks characteristic of centromeric chromatin. Finally, we demonstrate that loss of CENP-C or DNMT3B leads to elevated chromosome misalignment and segregation defects during mitosis and increased transcription of centromeric repeats. Taken together, our data reveal a novel mechanism by which DNA methylation is targeted to discrete regions of the genome and contributes to chromosomal stability.
DNA甲基化是一种表观遗传施加的转录抑制标记，对维持染色质结构和基因组稳定性至关重要。全基因组甲基化模式由三种DNA甲基转移酶（DNMT1、DNMT3A和DNMT3B）的联合作用介导。DNMT3B与染色体稳定性之间存在令人信服的联系，这一点在ICF综合征（一种由DNMT3B种系突变引起的疾病）的有丝分裂缺陷中得到了强调，而有丝分裂缺陷是ICF综合征的一个标志。着丝粒和着丝粒周围区域对于染色体凝聚和分离的保真度至关重要。着丝粒区域包含独特的表观遗传标记，包括密集的DNA高甲基化，但DNA甲基化靶向这些区域的机制在很大程度上仍然未知。在本研究中，我们使用酵母双杂交筛选，鉴定出DNMT3B与组成型着丝粒蛋白CENP-C之间的一种新的相互作用。CENP-C本身对于有丝分裂是必不可少的。我们在哺乳动物细胞中证实了这种相互作用，并确定了负责的结构域。使用siRNA敲低、亚硫酸氢盐基因组测序和染色质免疫沉淀，我们首次证明CENP-C将DNA甲基化和DNMT3B募集到着丝粒和着丝粒周围的卫星重复序列，并且CENP-C和DNMT3B调节这些区域的组蛋白密码，包括着丝粒染色质的特征性标记。最后，我们证明CENP-C或DNMT3B的缺失会导致有丝分裂期间染色体错配和分离缺陷增加，以及着丝粒重复序列的转录增加。综上所述，我们的数据揭示了一种新的机制，通过该机制DNA甲基化靶向基因组的离散区域并有助于染色体稳定性。
