Abstract
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.
Highlights
The SNF2 family of chromatin remodelers is known to facilitate different regulatory functions involving chromatin
We identified the DNA binding motif of INO80 using Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach using GST-DBINO immobilized on glutathione sepharose (S2 Fig)
The 11 mer consensus motif was identified as the hINO80 binding DNA sequence motif and used in further experiments to study its interaction with the INO80 protein
Summary
The SNF2 family of chromatin remodelers is known to facilitate different regulatory functions involving chromatin. The INO80, a highly conserved member of the SNF2 family of DNA dependent ATPase shows functional diversity and is implicated in transcription, replication, cell division and DNA repair [1]. The alteration of chromatin structure to facilitate the recruitment of various interacting complexes for gene expression regulation is brought about by chromatin remodeling and histone modifications to maintain the status of gene expression [2]. One of the mechanisms by which chromatin structure can be altered involves the disruption, mobilization and stabilization of the histone octamer by multiprotein complexes, leading to either repression or activation of transcription [3]. The chromatin remodeling complexes hydrolyze ATP through a subunit that belongs to the superfamily of SNF2-type ATPases which can be PLOS ONE | DOI:10.1371/journal.pone.0159370. The chromatin remodeling complexes hydrolyze ATP through a subunit that belongs to the superfamily of SNF2-type ATPases which can be PLOS ONE | DOI:10.1371/journal.pone.0159370 July 18, 2016
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