title: Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification.
abstract: RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called "N-rule" protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a preferential localisation of HHR6 in euchromatin areas, suggesting that the protein is associated with transcriptionally active regions. Our findings support the idea that both HHR6 genes have overlapping, constitutive functions related to chromatin conformation and that they have a specific role in spermatogenesis, involving Ub-mediated histone degradation.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
authors.forename: M H
authors.initials: MH
authors.lastname: Koken
authors.forename: J W
authors.initials: JW
authors.lastname: Hoogerbrugge
authors.forename: I
authors.initials: I
authors.lastname: Jasper-Dekker
authors.forename: J
authors.initials: J
authors.lastname: de Wit
authors.forename: R
authors.initials: R
authors.lastname: Willemsen
authors.forename: H P
authors.initials: HP
authors.lastname: Roest
authors.forename: J A
authors.initials: JA
authors.lastname: Grootegoed
authors.forename: J H
authors.initials: JH
authors.lastname: Hoeijmakers
mesh_terms: D000818:Animals; D002451:Cell Compartmentation; D002843:Chromatin / Q000378:metabolism*; D004260:DNA Repair; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018869:Heat-Shock Response; D006657:Histones / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007985:Leydig Cells / Q000737:chemistry; D008025:Ligases / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D051379:Mice; D008938:Mitosis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins; D013091:Spermatogenesis / Q000502:physiology*; D013737:Testis / Q000033:anatomy & histology / Q000502:physiology; D014018:Tissue Distribution; D044763:Ubiquitin-Conjugating Enzymes; D014466:Ultraviolet Rays
chemical_list: D002843:Chromatin; D006657:Histones; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C471606:RAD6 protein, S cerevisiae; C471545:UBE2A protein, human; C471546:UBE2B protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases