During spermatogenesis histones must be degraded in late round and early elongating spermatids to permit chromatin condensation. Ubiquitin conjugation is activated and histones are ubiquitinated at this stage, suggesting that histone degradation may be mediated by ubiquitination. The activation of ubiquitin conjugation during spermatogenesis is dependent on the ubiquitin conjugating enzyme (E2) UBC4. We therefore studied whether histones are ubiquitinated by a UBC4 dependent ubiquitin protein ligase (E3) during spermatogenesis. E3Histone was identified by a biochemical screen and purified to near homogeneity. Mass spectrometry identified E3Histone as LASU1, a 482 kDa HECT domain protein and E3Histone conjugates ubiquitin to all core histories in vitro. UBC4-1 and UBC4-testis were the preferred E2s for E3Histone-dependent ubiquitination of histones. E3Histone was the major UBC4-1 dependent histone ubiquitinating E3 in testis. Anti-LASU1 antibody immunodepleted E3 Histone activity. Immunohistochemistry showed that E3Histone /LASU1 was predominantly expressed in nuclei from spermatogonia to mid-pachytene cells, but not detectable in spermatids. Histones are also ubiquitinated in spermatocytes. E3Histone/LASU1 was widely expressed in different mouse tissues. It was mainly expressed in the cytoplasm in most tissues, except in neurons of the brain and in early germ cells of the testis where it was expressed in the nucleus. In most tissues, E3Histone/LASU1 was expressed in epithelia. The wide expression of E3Histone/LASU1 suggests the existence of substrates of this E3 other than histones. Indeed, our assays showed that in vitro purified E3Histone stimulates polyubiquitination of Mcl-1, a BH3 region containing antiapoptotic protein. E3Histone may therefore regulate cell apoptosis by mediating degradation of Mcl-1. Since E3Histone/LASU1 was found previously to affect gene transcription and histone monoubiquitination is known to regulate gene transcription, we also evaluated the role of E3 Histone/LASU1 in histone ubiquitination in somatic cells. Depletion of E3Histone/LASU1 protein by siRNA did not affect the levels of free or ubiquitinated histories. In summary, E3Histone /LASU1 is a novel multi-functional protein that may mediate histone ubiquitination during meiosis and may be involved in apoptosis by triggering Mcl-1 degradation. Its wide expression and large non-catalytic region indicate that there are likely many other substrates of E3Histone/LASU1
