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 H3 in 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.
Highlights
During spermiogenesis, which is the part of spermatogenesis involving the development of the spermatid, there are dramatic molecular and functional changes in chromatin structure
Displacement of the histones by the transition proteins occurs during the first phase of chromatin condensation, which begins at step 11
A pair of spots, which were prominent in pachytene spermatocytes (Fig. 1B), were observed at the position corresponding to uH2A
Summary
Prior to histone displacement and to identify possible targets for ubiquitination in elongating spermatids, we investigated the modifications of histones in three populations of cells from rat testes, including one enriched in elongating spermatids. We report that the histones showing elevated levels of ubiquitination in this cell type are variants of H3
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