Over the last few years, a number of mammalian homologs of yeast RDB genes that exhibit a high degree of sequence and functional conservation have been identified, suggesting that RDB mechanisms might be conserved throughout eukaryotic evolution (21
). However, some remarkable functional differences between yeast and mammalian proteins (8
) and the identification of additional Y family DNA polymerases in mammalian species (for a review, see reference 19
) forbid simple extrapolation from yeast to mammals. Also, the identification of two mammalian RAD6
homologs provides a complicating factor. Previously, we demonstrated that the inactivation of the mHR6B
gene, one of the two mammalian homologs of yeast RAD6
, induces only a relatively mild phenotype in mice compared to that induced by a rad6
-null allele in yeast. Whereas yeast rad6
mutants display extreme sensitivity to all kinds of genotoxins, low growth rates, and impaired sporulation, the main phenotype of mHR6B−/−
mice is a severe derailment of spermatogenesis (55
). This observation raised the question whether the second homolog, mHR6A
, is responsible for the greater part of the somatic RAD6-like functions in mice or whether there is extensive functional redundancy between the two mammalian proteins.
RAD18 is the E3 that interacts with RAD6 in RDB in yeast. Both human RAD6 homologs can interact with human RAD18, and the overexpression of a dominant-negative human RAD18
gene results in increased UV sensitivity (68
). Analysis of mHR6A-deficient MEFs, however, failed to reveal any difference between their response to UV irradiation and that of wild-type cells. This outcome is comparable with results of UV survival assays of mHR6B-deficient MEFs, which also showed no increased UV sensitivity (55
). In addition, mHR6A-deficient mice appeared normal, apart from a slight reduction in body weight, and preliminary studies did not reveal an increased cancer predisposition up to 1 year of age.
Besides RAD18, UBR1 is another E3 capable of interacting with RAD6 (15
). The human and mouse homologs of UBR1
have been cloned, and their roles in the N-end rule pathway and interaction with mHR6A and mHR6B have been confirmed (36
). One of the observed phenotypes in Ubr1−/−
mice is a small but significant reduction in body weight (37
). Similar reductions in body weight were observed for mHR6A-deficient animals (present data) and for mHR6B-deficient animals (unpublished data). Ubr1−/−
mice are, however, normally fertile. This finding indicates that the infertility phenotype of both mHR6B−/−
male and mHR6A−/−
female mice is not determined by the interaction of mHR6A and/or mHR6B with Ubr1 and does not involve dysregulation of protein degradation via the N-end rule.
In the present breeding program, in addition to the absence of DKO mice, we found that mHR6A−/−/mHR6B+/−
females and mHR6A−/Y/mHR6B+/−
males were underrepresented and runted and often died around birth. The mHR6A−/Y/mHR6B+/−
males that survived were normally fertile. This finding indicates that the level of mHR6B protein obtained from one functional mHR6B
allele is sufficient to support spermatogenesis, even in the absence of mHR6A protein. On the one hand, the results obtained indicate that mHR6A and mHR6B have completely redundant activities both in somatic cell types and in germ line cells. On the other hand, the observed female and male infertility phenotypes in mHR6A- and mHR6B-deficient mice, respectively, indicate that germ line cells are subject to a dose-dependent effect which involves spermatogenesis- and oogenesis-specific differences in expression levels of mHR6A and mHR6B. However, the possibility that mHR6A and mHR6B have different functional properties at the molecular level cannot be completely excluded. Several observations that point in this direction have been described. Ubiquitin-protein ligases (E3s) can be found among proteins that contain either HECT or RING motifs, such as RAD18 and UBR1 (reviewed in reference 51
), but also among proteins with U-box domains. Hatakeyama et al. (23
) showed that the U-box motif-containing cyclophilin CYC4/hCyP-60 needs the E2 enzyme HR6B, but not HR6A, for polyubiquitination. Cyclophilins are thought to have chaperone functions, being involved in the proper folding of protein substrates in vivo. Although ubiquitously expressed, transcript levels of cyclophilins are elevated and differ in size in the testis (74
). This finding suggests the possibility that the spermatogenic derailment observed in mHR6B−/−
but not in mHR6A−/Y
males is not caused by the relatively low level of mHR6A but by the inability of mHR6A to interact with cyclophilin. Additional data on possible specificity come from the identification of mHR6A, and not mHR6B, as an interacting partner of Rfpl4 in a yeast two-hybrid screen (67
). Rfpl4 is present in maturing oocytes and remains expressed up to the eight-cell-stage embryo in the mouse. The protein shows the characteristics of a RING domain containing E3 ubiquitin ligase and can specifically interact with mHR6A. This protein could be useful in trying to identify the specificity-determining amino acid residues of mHR6A and mHR6B; mHR6A and mHR6B differ in only seven positions.
The most prominent effect of disrupting mHR6A is the observed complete failure of mHR6A-deficient females to produce offspring, resulting from developmental arrest after the first embryonic cell division. It is unlikely that the phenotype of the female mHR6A−/− mice is due to aberrant levels of gonadotropins, since vaginal smears for mHR6A-deficient females revealed regular 4- to 5-day cycles and histological examination of fixed and stained ovary sections showed the presence of all stages of follicular development. In addition, the animals mated readily, the number of fertilized oocytes was similar to that for mHR6A+/− littermates, and pseudopregnancy was induced. These data indicate a normal hypothalamic-hypophyseal-gonadal axis. Measurable deviations from normal serum gonadotropin levels would have resulted in dysregulated cycles.
The present results identify mHR6A
as one of the few mammalian genes that encode a factor essential for the very first steps in embryonic development. Thus far, only Mater
have been shown to encode proteins that have their major, if not exclusive, functions in early postfertilization development (12
The mechanism that is disrupted by the absence of mHR6A is still unclear but could be related to the regulation of transcription and/or chromatin rearrangements. Just recently, it has started to become clear how, and what kind of, core histone tail modifications play a role in chromatin remodeling, higher-order chromatin structure, and transcription (see reference 63
and references therein). These modifications include phosphorylation, acetylation, methylation, and ubiquitination. Robzyk et al. (54
) showed that the ubiquitination of histone H2B in yeast is dependent on a functional RAD6
gene. Mutation of lysine residue 123 of H2B, which is essential for ubiquitination, prevents the methylation of H3K4 and induces defects in sporulation and gene silencing. Other RAD6-dependent processes, such as RDB, appear unaffected (54
During fertilization, the chromatin in the paternal pronucleus is reorganized, with histones of maternal origin replacing protamines. From that point on, acetylated forms of histone H4 are detectable in the paternal pronucleus, associated with the onset of transcription (1
). The methylation of histone H3 at lysine residue 9 (H3K9), known to be involved in the formation of stable repressive heterochromatin (50
), was found in the maternal pronucleus but could hardly be detected in the paternal pronucleus (3
). In addition, H3K4 methylation, a hallmark of active chromatin (64
), was also observed predominantly in the maternal pronucleus rather than the paternal pronucleus. The antibody used in the present study recognizes the dimethylated form of H3K4. The presence of trimethylated H3K4 has been shown to be a prerequisite for active genes, and the role of dimethylated H3K4 in mammalian cells is not clear, but the presence of dimethylated H3K4 may correlate with a permissive state of chromatin in which genes are potentially active (59
). The observed H3K4 staining in the maternal pronucleus may mark regions that will become active shortly after the pronuclear stage. However, we did not observe a change in the histone H3 methylation pattern in the pronuclei in the mHR6A-deficient oocytes during fertilization.
cDNAs encode proteins that are able to induce H2B ubiquitination and subsequent methylation of H3K4 in rad6
), but there is a clear difference between ubiquitination of H2B in yeast and that in humans. In yeast, ubiquitinated H2B is most abundant and ubiquitinated H2A is undetectable, whereas in human somatic cells about 10% of H2A and only 1 to 1.5% of H2B is ubiquitinated (75
). In spermatogenesis, no change in histone ubiquitination patterns has been detected in mHR6B
knockout testis (5
), but this result does not exclude the possibility of a low, but functionally significant, rate of H2A and/or H2B ubiquitination by mHR6B. Similarly, the possibility that the ubiquitin-conjugating enzyme mHR6A takes part in chromatin remodeling during early embryonic development through low rates of ubiquitination of H2A and/or H2B, which then might affect the methylation of histone H3 in a manner which has not been detected in the present experiments, cannot be excluded. Finally, it is important to note that the ubiquitination of H2B by RAD6 in yeast appears to be unrelated to RDB mechanisms (54
) and that the role of mHR6A as a maternal factor for early embryonic development might involve a molecular mechanism of action outside the context of DNA repair.