title: Identification of an ubinuclein 1 region required for stability and function of the human HIRA/UBN1/CABIN1/ASF1a histone H3.3 chaperone complex.
abstract: The mammalian HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex deposits the histone H3 variant H3.3 into chromatin and is linked to gene activation, repression, and chromatin assembly in diverse cell contexts. We recently reported that a short N-terminal fragment of UBN1 containing amino acids 1-175 is necessary and sufficient for interaction with the WD repeats of HIRA and attributed this interaction to a region from residues 120-175 that is highly conserved with the yeast ortholog Hpc2 and so termed the HRD for Hpc2-related domain. In this report, through a more comprehensive and refined biochemical and mutational analysis, we identify a smaller and more moderately conserved region within residues 41-77 of UBN1, which we term the NHRD, that is essential for interaction with the HIRA WD repeats; we further demonstrate that the HRD is dispensable for this interaction. We employ analytical ultracentrifugation studies to demonstrate that the NHRD of UBN1 and the WD repeats of HIRA form a tight 1:1 complex with a dissociation constant in the nanomolar range. Mutagenesis experiments identify several key residues in the NHRD that are required for interaction with the HIRA WD repeat domain, stability of the HUCA complex in vitro and in vivo, and changes in chromatin organization in primary human cells. Together, these studies implicate the NHRD domain of UBN1 as being an essential region for HIRA interaction and chromatin organization by the HUCA complex.
authors.affiliation: The Wistar Institute, Philadelphia, Pennsylvania 19104, United States.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tang
authors.forename: Aastha
authors.initials: A
authors.lastname: Puri
authors.forename: M Daniel
authors.initials: MD
authors.lastname: Ricketts
authors.forename: Taranjit Singh
authors.initials: TS
authors.lastname: Rai
authors.forename: Jason
authors.initials: J
authors.lastname: Hoffmann
authors.forename: Elise
authors.initials: E
authors.lastname: Hoi
authors.forename: Peter D
authors.initials: PD
authors.lastname: Adams
authors.forename: David C
authors.initials: DC
authors.lastname: Schultz
authors.forename: Ronen
authors.initials: R
authors.lastname: Marmorstein
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D056488:Histone Chaperones / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D012995:Solubility; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C491641:ASF1A protein, human; D048868:Adaptor Proteins, Signal Transducing; C491861:CABIN1 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; C086057:HIRA protein, human; D056488:Histone Chaperones; D006657:Histones; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D014157:Transcription Factors; C406814:UBN1 protein, human