title: Nuclear protein LEDGF/p75 recognizes supercoiled DNA by a novel DNA-binding domain.
abstract: Lens epithelium-derived growth factor (LEDGF) or p75 is a co-activator of general transcription and also involved in insertion of human immunodeficiency virus type I (HIV-1) cDNA into host cell genome, which occurs preferentially to active transcription units. These phenomena may share an underlying molecular mechanism in common. We report here that LEDGF/p75 binds negatively supercoiled DNA selectively over unconstrained DNA. We identified a novel DNA-binding domain in the protein and termed it 'supercoiled DNA-recognition domain' (SRD). Recombinant protein fragments containing SRD showed a preferential binding to supercoiled DNA in vitro. SRD harbors a characteristic cluster of lysine and glutamic/aspartic acid residues. A polypeptide mimicking the cluster (K(9)E(9)K(9)) also showed this specificity, suggesting that the cluster is an essential element for the supercoil recognition. eGFP-tagged LEDGF/p75 expressed in the nucleus distributed partially in transcriptionally active regions that were identified by immunostaining of methylated histone H3 (H3K4me3) or incorporation of Br-UTP. This pattern of localization was observed with SRD alone but abolished if the protein lacked SRD. Thus, these results imply that LEDGF/p75 guides its binding partners, including HIV-1 integrase, to the active transcription site through recognition of negative supercoils generated around it.
authors.affiliation: Department of Neurogenomics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8558, Japan.
authors.forename: Kimiko M
authors.initials: KM
authors.lastname: Tsutsui
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Sano
authors.forename: Osamu
authors.initials: O
authors.lastname: Hosoya
authors.forename: Tadashi
authors.initials: T
authors.lastname: Miyamoto
authors.forename: Ken
authors.initials: K
authors.lastname: Tsutsui
mesh_terms: D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D004278:DNA, Superhelical / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
chemical_list: D004278:DNA, Superhelical; D036341:Intercellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C119380:lens epithelium-derived growth factor