During spermatid maturation, several chromatin rearrangements occur. Initially, the histones are replaced by transition proteins which in turn are finally replaced by protamines (34
). Histones are still degraded when genes encoding transition proteins are inactivated in the mouse, indicating that the loss of histones is not simply due to displacement by the transition proteins (56
). Ubiquitination of several histones is increased just prior to their degradation (29
), suggesting that ubiquitin-dependent proteolysis plays an important role in histone replacement. In this paper, we identify and characterize a new testis ubiquitin protein ligase, E3Histone
, which is an excellent candidate ligase for mediating the ubiquitination of histones. It ubiquitinates all core histones in vitro (Fig. ). With wild-type ubiquitin, E3Histone
produced much-higher-molecular-weight forms of histone than when MeUb was used (Fig. ), indicating that E3Histone
can form polyubiquitin chains on histones, thus conferring the potential for recognition and degradation by the proteasome. Its preferred interaction with UBC4 would also be consistent with such a role, as UBC4 isoforms are induced in early elongating spermatids (39
) when histones begin to be replaced. Indeed, the induction of UBC4 may be a mechanism by which histone ubiquitination and degradation are initiated and regulated.
Mass spectrometric analysis identified E3Histone as a HECT domain-containing ligase, previously named LASU1. This was consistent with our observations that E3Histone activity was abolished by the thiol reactive compound N-ethylmaleimide but not by the divalent cation chelators EDTA or TPEN (Fig. ). In addition, our EST sequence analysis corrected a frameshift-inducing sequencing error present in the previous sequence of LASU1, thus resulting in a much longer N terminus that includes what was hitherto a protein of unknown function. Remarkably, mass spectrometric coupled to bioinformatics analyses of this bovine enzyme was able to identify 88 peptides matching the human LASU1 or mouse unnamed protein sequence. This result suggests a significant degree of conservation of sequence among these species, which was confirmed by the observation of 97.7% identity and 98.5% similarity between mouse and human sequences present in the EST databases. All other proteins identified in the analyses had fewer than seven matching peptides, suggesting that LASU1 was the dominant protein in this enzyme and that these other proteins were likely contaminants. Indeed, they were not detected in MS analyses of protein bands in immunoprecipitates of E3Histone. Also, none of the other identified proteins appeared to be a potential ligase. The predicted molecular mass of 482 kDa was reasonably close to the estimated molecular mass of E3Histone of ~600 kDa, considering the limited precision of mass estimation by chromatography for such a large protein and the assumption of globular enzymes in these estimations. The enzyme bound to anion exchange columns, as would be expected for the predicted pI of 4.87. All of these observations together argue strongly that LASU1 is E3Histone. The latter findings also suggest that E3Histone functions as a monomeric protein, but the mass determinations are not precise enough to rule out the presence of an additional small subunit(s), particularly nonstoichiometrically in subsets of the enzyme molecules.
Interestingly, LASU1 was found to be expressed in many different tissues at both mRNA and protein levels and more highly expressed in the testis, brain, lung, and kidney (unpublished data). E3Histone
could therefore have other important roles in different tissues besides its function on chromatin condensation in elongated spermatids. It is possible that it may play a role in the ubiquitination of histones in other cells. However, in most cells, ubiquitination of histones appears to modify function rather than target for degradation. In eukaryotic cells, 10 to 15% of total histone H2A is ubiquitinated (51
), while 10% of histone H2B is ubiquitinated in yeast (40
) and 1.5% of cellular histone H2B is monoubiquitinated in higher eukaryotes (51
). The function of this ubiquitination remains largely unknown except for a few examples. Monoubiquitination of histone H2B in S. cerevisiae
leads to methylation of histone H3 on its lysine 4 and lysine 79 residues, which in turn causes telomeric gene silencing (6
). This ubiquitination is mediated by the RING domain-containing E3, Bre1, which relies on Rad6p as its cognate E2 (25
). Inactivation of a mouse homolog of Rad6p leads to defective spermatogenesis but does not appear to prevent histone degradation. Furthermore, monoubiquitination of histone H1 in drosophila by TAFII
250, another RING finger-containing protein, results in transcriptional activation (37
). These results taken together support a link between histone ubiquitination and gene transcription. In addition, two other ubiquitin protein ligases belonging to the RING domain E3 family, BRCA1/BARD1 (28
) and Np95 (11
), have been recently shown to promote ubiquitination of histone in vitro.
In this paper, we identify for the first time a HECT domain-containing E3 that ubiquitinates histones. Besides a role in mediating histone degradation, this ubiquitination could also be involved in transcriptional regulation. Indeed, previous studies with the rat homolog of LASU1 suggested that it may function as a DNA-binding transcriptional regulator (17
). The most similar other known E3s are S. cerevisiae
Tom1 and E3s closely related to Tom1. Tom1 mutants are pleiotropic and include defects in transcription that appear to be mediated through ADA coactivator proteins (43
) as well as defects in the export of mRNA (14
). Suppressors of tom1 mutants include STO1/G4p2/STM1, a basic protein partially homologous to histone H1 that can bind to G4 nucleic acids (16
). Thus, Tom1 appears to interact with basic nucleic acid binding proteins and in that way is similar to the demonstrated ability of LASU1 to interact with and ubiquitinate histones.
LASU1 is here demonstrated to be one of the largest E3s to be characterized to date. Like other large E3s, such as the 374-kDa Tom1 (50
) and 300-kDa EDD (35
), the large size likely permits it to interact with a variety of substrates and to exert a large number of functions which remain to be defined.