There are two known histone acetyltransferase complexes that modify histone H4 at K16; the MSL (male-specific silencing) complex in humans and flies, and the SAS (something about silencing) complex in yeast cells (1
). The MSL complex specifically acetylates H4K16 in Drosophila
and humans. In human cells, knockdown of hMOF results in the loss of H4K16ac (12
). H4K16 acetylation has been shown to destabilize nucleosomes and correlates with regions of chromatin decondensation (39
). Here, we report the first extensive study showing that MOF is required for proliferation during embryogenesis and in oncogenesis. However, the exact role of MOF in these processes remains to be determined. An intriguing possibility is that MOF acetylation of histone H4 at K16 may have a regulatory role during transcription (1
), which is poorly understood in mammals, but well documented in yeast and Drosophila
). The significance of H4K16 acetylation lies in the fact that in budding yeast, most of the genome exists in a decondensed state with more than three fourths of histone H4 acetylated at lysine 16 (28
). In Drosophila
, the transcriptionally enhanced X chromosome of male flies, a site of ubiquitous H4K16ac, is also decondensed (1
). In human cell lines, decreased levels of hMOF correlate with loss of H4K16ac and is associated with defective DNA repair (12
). In this study, we found that the loss of H4K16ac correlated with the progressive depletion of maternal MOF during embryogenesis in mMof
null embryos. In wild-type mMof embryos, the rapidly proliferating ICM cells have convincingly higher levels of MOF protein than trophectoderm cells, which is consistent with previous results showing higher levels of acetylated H4K16 in ES cells than in trophectoderm cells. Thus, increased acetylation of H4K16 in ES cells is indicative of the significance of this chromatin modification for proliferation during development. Complete ablation of MOF and loss of H4K16ac results in a marked delay in developmental progression, sluggish hatching and implantation, proliferation arrest, and death.
The loss of cell proliferation could be due to cell cycle arrest specifically induced by p53 and by ATM as suggested by a previous report of G2
/M arrest in hMOF knockdown cells (46
). In addition, hMOF has been reported to acetylate p53 at lysine 120, and this acetylation may help distinguish between the cell cycle arrest and apoptotic functions of p53 (45
). If the mMof depletion-dependent cell cycle arrest were the cause of embryonic lethality or cellular death, then depletion of p53 or ATM should have rescued or at least partially ameliorated the observed embryonic lethality. However, our results do not support this hypothesis.
Inactivation of the mMof gene in ES cells caused depletion of Mof protein accompanied with loss of acetylated H4K16 and resulted in reduced proliferation and ultimately cell death. Taken together with the lack of success in deriving homozygous Mof mutant ES cells, these experiments strongly suggest that Mof function is indispensable for cellular proliferation and/or viability. These results also raised the question of whether tumor cells can survive in the absence of MOF and the associated loss of H4K16 acetylation.
Multiple assays were utilized previously to quantitate H4K16 monoacetylation at the global level and genome region/locus-specific level, and the data suggested that loss of monoacetylation at H4K16 occurs in tumors and tumor-derived cell lines (8
). Our results, however, demonstrate that tumors and tumor-derived cell lines have similar or higher levels of hMOF or H4K16ac compared to those of normal control cells (Fig. and and data not shown). Similar results were found in multiple different transformed cell lines (Fig. and data not shown). Moreover, we found that primary cells have lower levels of hMOF and H4K16ac than the corresponding isogenic hTERT immortalized cells do (Fig. ). These in vitro results were also supported by the in vivo hMOF expression analysis of 300 different tumors, which demonstrated no loss of hMOF expression. In addition, tumor cells with higher levels of hMOF had faster initial growth and immediate regrowth (recurrence) of tumors after irradiation exposure (Fig. ), suggesting that hMOF promotes cellular proliferation. Therefore, after examination of a large number of tumor-derived cell lines and tumor samples, we propose that hMOF and H4K16ac may be required for the process of transformation, either in vitro or in vivo.
Intriguingly, MOF overexpression was associated with both increased K16H4 acetylation and increased cell proliferation (12
; also the present study), while cells treated with SIRT1 inhibitors had increased K16H4 acetylation levels but a reduction in cell proliferation (29
). Thus, although MOF seems to be closely associated with cell proliferation, the relationship between the H4K16 acetylation status and cell proliferation requires further study.
The loss of MOF and H4K16ac correlates with increased genomic instability (Tables and and Fig. ) (12
), which is considered an important step in cancer development because it can greatly accelerate the genetic changes leading to tumor cell progression (14
). However, induction of genomic instability and increased radiosensitivity caused by depletion of MOF result in decreased oncogenic transformation, whereas overexpression of MOF resulted in increased oncogenic transformation (Fig. ); this suggests that the loss of MOF is probably not a marker of oncogenesis.
Collectively, our data suggest that the requirement for MOF during tumor cell proliferation is similar to that during embryogenesis prior to tissue differentiation. The role of MOF in cellular proliferation is supported by the following. (i) There is a strong correlation observed between the ectopic expression of the catalytic unit of telomerase (hTERT) and the levels of hMOF and H4K16ac seen in isogenic cell systems (Fig. ). (ii) Depletion of MOF results in cellular lethality. (iii) Overexpression of MOF results in enhanced oncogenic transformation and faster relapse of tumor growth after radiation treatment (Fig. and ). Thus, the results suggest that expression of hMOF and acetylation of H4K16 are linked with proliferation status and is supported by the fact that all the tumor-derived cell lines and immortalized cell lines examined have both hMOF and acetylated H4K16. These novel observations presented here provide a foundation for future molecular studies on the significance of epigenetic modifications in varied contexts of embryonic development, tumorigenesis, and DNA metabolism.