Mendiratta Shweta, Bhatia Shipra, Jain Shruti, Kaur Taniya, Brahmachari Vani
Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India.
PLoS One. 2016 Jul 18;11(7):e0159370. doi: 10.1371/journal.pone.0159370. eCollection 2016.
The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity in vitro. Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes.
INO80亚家族中存在高度保守的DNA结合结构域，这预示着INO80可直接与DNA相互作用，并且我们在体外证实了其DNA结合活性。在此，我们报告通过SELEX方法鉴定出的DBINO结构域识别的共有基序，并展示INO80与该共有基序的特异性相互作用。我们发现INO80通过其结合基序显著下调报告基因的表达，且这种抑制作用依赖于细胞中INO80的存在，而非YY1的存在。如果共有基序内的特定残基发生改变，这种相互作用就会消失。我们通过计算机分析在人类基因组中鉴定出大量INO80的潜在靶位点，这些位点可分为三类：包含INO80和YY1识别序列的位点、仅包含YY1识别序列的位点以及仅包含INO80识别序列的位点。我们证实了INO80与HEK细胞中一组代表性位点的结合以及结合基序周围相关的抑制性组蛋白修饰。鉴于INO80在果蝇同源异型基因调控中作为三胸节和多梳蛋白增强子（ETP）的作用，它既能改变多梳等抑制性复合物的作用，也能改变三胸节等激活复合物的作用，INO80是否能作为染色质修饰复合物的招募因子还有待观察。
