TY - JOUR
T1 - Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification
AU - Koken, Marcel H.M.
AU - Hoogerbrugge, Jos W.
AU - Jaspers-Dekker, Iris
AU - De Wit, Jan
AU - Willemsen, Rob
AU - Roest, Henk P.
AU - Grootegoed, J. Anton
AU - Hoeijmakers, Jan H.J.
N1 - Funding Information:
We thank Drs. L. and S. Prakash for the generous gift of the RAD6 polyclonal antiserum, Drs. F. J. Benham for the GAPDH probe, P. Herrlich for the probes for collagenase and metallotheio-nin, T. Butt for ubiquitin probes, I. Laird for the human elongation factor 1α gene, and D. Meijer for the mouse protamine 1 probe. We are very thankful for the help of the following colleagues: Dr. Theo Luider for help with the 2-dimensional gel electrophoresis, Ton Verkerk for FACS analysis, Dr. Claude Backendorf and Li Bin Ma for some of RNA samples of UV-irradiated cells, Dr. Andre Hooge-veen for preparation of synthetic oligopeptides, Wim Vermeulen for microinjection experiments, Maarten Fornerod for the protein samples of cell cycle synchronisation experiments, Drs. Carel Meij-ers, Axel Themmen, and Leen Blok for helpful discussions, and Jan van Klaveren for helpful discussions and excellent technical assistance. Dr. D. Bootsma is acknowledged for critical reading of the manuscript and continuous support. We are grateful to Mirko Kuit and Tom de Vries Lentsch for photography, and Sjozef van Baal for computer assistance. This work was supported by the Dutch Cancer Society (Project IKR 88-2, 90-20 and 92-118) and the European Community (Contract B16-141-NL).
PY - 1996/1/10
Y1 - 1996/1/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0030021770&partnerID=8YFLogxK
U2 - 10.1006/dbio.1996.0011
DO - 10.1006/dbio.1996.0011
M3 - Article
C2 - 8575614
AN - SCOPUS:0030021770
SN - 0012-1606
VL - 173
SP - 119
EP - 132
JO - Developmental Biology
JF - Developmental Biology
IS - 1
ER -