We demonstrate that Bre1 (human BRE1A/B (RNF20/40) and mouse Bre1a/b (Rnf20/40)) acts as an important suppressor of chromosomal instability (CIN). This finding complements the previously suggested mechanism for Bre1 tumor suppression through transcriptional regulation of select oncogenes and tumor suppressor genes (14
). The types of chromosomal aberrations we observed after knockdown of Bre1 indicated that a defect in homologous recombination (HR) contributes to CIN in the Bre1-deficient cells (40
). This conclusion is consistent with our previous observation that reduced monoubiquitination of H2B in Saccharomyces bre1null
mutants and in mouse cells leads to defective recombinational repair of double-strand breaks (DSBs) (10
). We show that R-loops, the RNA-DNA hybrid structures usually formed behind elongating RNA polymerase II when mRNA processing is disturbed (30
), constitute a significant source of DSBs in Bre1-deficient cells. Overall, our data support a model in which reduction in Bre1-dependent ubiquitination of histone H2B increases genomic instability through increased generation of DSBs resulting from a defect in correct processing of canonical histone mRNA and through inhibition of HR needed for DSB resolution ().
A model depicting sources of genomic instability in Bre1-deficient cells
It should be noted that while we interpret the observed Bre1 knockdown phenotype as arising from an impact on the well-known role of Bre1 in H2B ubiquitination, it is formally possible that additional targets for the Bre1 ubiquitin ligase exist, which could contribute to the knockdown phenotype. Testing this with an H2B K120 substitution mutant is not straightforward in mammals, because their genomes contain at least 17 H2B genes (43
). However studies using overexpressed ectopic H2BK120R mutant gene in cells with wild-type chromosomal copies of H2B have shown that effects of overexpression of H2BK120R mimic those of loss of BRE1, including the effect on the formation of γH2AX foci, which we used in our study to detect DSBs (11
). Also, in Saccharomyces
, where chromosomal copies of the H2B gene number only two and can be deleted, H2BK123R mutants do mimic the bre1
deletion phenotype (reviewed in (9
)), implying that the phenotypes are likely to be conferred through effects on the H2B target. Questions remain, however, as to whether the phenotypes we observed after loss of Bre1 are mediated via the well-known impact of H2B ubiquitination on methylation of histone H3, and specifically whether H3K4me3 and or H3K79me2 are involved. Detailed investigation of the role that H3K4 methylation plays in different Bre1 phenotypes is hampered by the fact that there are many SET1 homologues in mammals, some of which may not be uH2B-dependent (reviewed in (9
)). Also, knocking down the H3K79 methyltransferase Dot1 would completely eliminate methylation of H3K79, while loss of Bre1-mediated H2B ubiquitination only eliminates the higher states of methylation of H3K79, so additional phenotypes may be conferred by the Dot1 knockdown beyond those mediated by the impact of uH2B on H3K79. Although the effects of Dot1 knockdown in mice (44
) paralleled the impaired cell growth, increased ploidy and centromeric abnormalities we observed after Bre1 knockdown, specific clarification of these issues requires further study.
A crucial unresolved question in cancer biology is whether CIN represents an early event and is therefore a driving force of carcinogenesis. Our results support models in which CIN drives tumorigenesis rather than being its consequence. We provide a comprehensive demonstration of a stepwise accumulation of chromosomal changes that start with downregulation of Bre1. Consistent with the CIN model, we also show that BRE1A/B deficiency accompanies early steps of testicular cancer development.
Correct processing of replication-associated histone mRNA is particularly relevant in connection to testicular carcinogenesis. In the testis a massive synthesis of histone variants accompanies dramatic reorganization of the genome, during which the majority of the histones are replaced by transition proteins and protamines. Lack of Bre1 leading to abnormal presence of polyadenylated histone mRNAs, which are not rapidly degraded at the end of S-phase, could interfere with proper incorporation of the variant histones into chromatin, and lead to testicular dysgenesis. We demonstrated that low levels of BRE1A and BRE1B, and low H3K79me2 are found in intratubular cell neoplasia (CIS), as well as in seminomas. Seminomas show chromosomal changes similar to those found in CIS and therefore are considered a default pathway from the CIS precursor lesion to invasive testicular germ cell tumors (TGCT) (39
). Like all TGCTs, seminomas are characterized by high levels of CIN and aneuploidy, and a gain of chromosome 12p (46
). Gain of 12p is not present in CIS, and so it is believed that overexpression of gene(s) on 12p is pertinent to invasive growth. Thus, we can speculate that genomic instability initiated by the abnormal downregulation of BRE1A/BRE1B function may facilitate gain of 12p and thus constitute one possible route to seminoma. Seminomas recapitulate the undifferentiated and pluripotent primordial germ cell (PGC) phenotype, and are thought to arise when a block in maturation of PGCs prevents them from forming spermatogonia. Downregulation of BRE1A/BRE1B may be associated with such a maturation block (39
), and thus may lead to infertility. In fact, men from families with fertility problems are known to have an elevated risk of testicular cancers, especially seminoma (47
). Hence, deficiency in BRE1A/B may be among etiological factors in common for both infertility and testicular cancer. In addition, mutation of BRE1 (RNF20) has been found among other CIN genes mutated in colorectal cancers (50
) suggestive of a more general role of Bre1 in CIN.
In conclusion, we propose that the mammalian homologs of the yeast BRE1 gene serve as tumor suppressors by preventing replication stress and chromosomal instability that arise from DSBs associated with incorrect processing of replication-associated histone mRNAs and inefficient HR. In addition to clarifying basic cellular mechanisms, the identification of Bre1 as a CIN gene may have specific relevance for estimation of risk and diagnosis of testicular cancer.