Ubiquitination of Histone H3 in Elongating Spermatids of Rat Testes*
- From the ‡Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0W3 Canada and the §Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Abstract
Because of the potential role of histone ubiquitination in altering chromatin structure, we characterized the levels of ubiquitination of specific histones in meiotic and postmeiotic germ cells in rat testes by two-dimensional gel electrophoresis. The levels of the major ubiquitinated histone forms, mono- and poly-ubiquitinated H2A, were highest in the pachytene spermatocyte stage, declined thereafter through the round spermatid stage, and reached their lowest levels in elongating spermatids. Three additional ubiquitinated histone species, besides H2A, were detected using anti-ubiquitin antibodies specifically in the fraction enriched in elongating spermatids. Based on their electrophoretic mobilities, they corresponded to uH3, uTH3, and uH2B. Polyubiquitinated forms of these proteins were also observed. The identity of these proteins was confirmed by immunoblotting with anti-H3 antisera and by differential extraction of the proteins from the nucleus with increasing salt concentrations. This is the first report of ubiquitination of H3in vivo. We speculate that its ubiquitination could loosen the nucleosome structure in preparation for histone removal, be a consequence of nucleosome relaxation or disruption caused by other means, or target H3 for degradation.
Footnotes
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↵* This work was supported by grants from the National Institutes of Health (HD-16843 and core grant CA-16672 to M. L. M) and the Medical Research Council of Canada (MT-9186 to J. R. D). The award of a Medical Research Council of Canada Senior Scientist Award (to J. R. D.) is gratefully acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed. Tel.: 1-713-792-3424; Fax: 1-713-794-4858; E-mail:meistrich{at}odin.mdacc.tmc.edu.
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↵1 I. Zalenskaya and M. L. Meistrich, in preparation.
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↵2 The abbreviations used are: uH2A, uH2B, uH3, mono-ubiquitinated forms of histones H2A, H2B, and H3, respectively; u2H2A, u2H2B, u2H3, diubiquitinated forms of H2A, H2B, and H3, respectively; AUT, acid-urea-Triton X-100; TCA, trichloroacetic acid.
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- Received February 2, 1998.
- Revision received March 9, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
