In the present study, we characterized MTA3 in the mouse ovary and examined its function in primary mouse granulosa cells. MTA3 not only interacts with NuRD complex proteins, as expected, but also coprecipitates with the major cohesin complex subunit RAD21. MTA3 protein knockdown in primary granulosa cells results in slowing of cell proliferation associated with reductions in cyclin B1 and cyclin B2 expression and a delay in the G2/M cell cycle transition. These findings demonstrate that MTA3 is a NuRD complex constituent that can interact with the cohesin complex in the ovary and suggest that the MTA3-NuRD complex functions with the cohesin complex to support cell cycle progression and growth of the granulosa cell compartment during follicle development in vivo.
Based on coprecipitation with CHD4 and HDAC1, at least some of the NuRD complex in the ovary contains MTA3. Although this does not exclude the possibility that MTA3 could act in association with other complexes or alone, it is likely that the slowing of cell proliferation is caused by alterations in NuRD complex function that result from a lack of MTA3. Indeed, knockdown of MBD3, a subunit required for NuRD integrity and stability, delays the G2/M transition and is associated with downregulation of cyclin B1 in several cancer cell lines [32
]. Similar delays in the G2/M transition associated with reduced cyclin B1 expression are observed following chemical and functional inhibition of HDAC1 [33
]. These observations suggest that MTA3 is required to support effective chromatin-remodeling activity of the NuRD complex in granulosa cells.
Cells duplicate their DNA content during S phase, and toward the end of this phase, newly synthesized sister chromatids are held together by the cohesin complex, particularly in pericentromeric regions, to facilitate faithful chromosome segregation during M phase [34
]. In human cell lines, association of the cohesin complex with chromatin is facilitated by both ISWI and NuRD chromatin-remodeling complexes through their SMARCA5 and CHD4 subunits, respectively, which interact directly with the core cohesin component RAD21 [18
]. Interestingly, NuRD associates with pericentromeric heterochromatin during late S phase in rapidly proliferating lymphoid cell lines and in primary germinal center B cells, suggesting that it facilitates cohesin loading in lymphoid cells [35
]. In the present study, we show that, similar to the ISWI complex subunit SMARCA5 [36
], MTA3 expression is significantly lower in terminally differentiated nondividing corpus luteum cells than in ovarian granulosa cells. These findings, combined with the observation that MTA3 depletion slows granulosa cell proliferation by affecting the G2/M transition, suggest that both the ISWI and NuRD complexes function to facilitate cohesin loading and sister chromatid stabilization during mitosis in granulosa cells.
The NuRD components MTA1, MTA2, and CHD4 facilitate DNA double-strand break repair [17
], and this effect could be mediated by modulation of cohesin association with repair foci. It is unlikely that MTA3 depletion in granulosa cells resulted in slowing of cell proliferation due to accumulation of DNA double-strand breaks. This statement is based on our findings that MTA3 depletion did not affect Cdkn1a
expression, TRP53 protein levels, or γH2AFX phosphorylation. Instead, slowing of the transition through the G2/M checkpoint due to inhibition of cyclin B expression or slower cohesin complex loading in the absence of MTA3 is a more likely explanation.
The concerted activity of several kinases is required for mitotic entry to occur with normal timing. One such kinase, aurora B, phosphorylates H3Ser10 in pericentromeric chromatin regions beginning in late G2 phase [37
]. This phosphorylation mark spreads along the chromosomes as mitosis proceeds and is complete in prophase; thus, it can serve as a mark of G2/M progression [38
]. In the rat, H3Ser10 phosphorylation is most prominent during proestrus in granulosa cells of growing follicles [39
], confirming an association of this mark with estrogen-mediated proliferation of primary granulosa cells. In mouse primary granulosa cell cultures, we found that MTA3 depletion caused more than a two-fold decrease in cells positive for H3Ser10 phosphorylation; however, less than 1% of cells in either treatment group exhibited this mark. The low incidence of H3Ser10 phosphorylation in both groups is consistent with the relatively slow cell proliferation we observed in these primary cells cultured in the absence of mitogenic stimuli like follicle-stimulating hormone or estradiol.
Later stages of chromosome condensation rely on the master mitotic complex cyclin-B1-CDK1, which triggers mitotic entry by initiating nuclear envelope breakdown [40
]. At this point, cells become committed to mitosis and proceed into prometaphase, even if stressed [41
]. Expression of type B cyclins in normally proliferating animal cells begins to rise in late S phase, reaches a maximum in the middle of G2/M phase, and drops immediately upon completion of mitosis [42
]. Transcription of cyclin B1 and cyclin B2 is regulated by transcriptional activators and repressors that bind promoter elements common to many cell cycle genes [43
]. We found that expression of cyclin B1 and cyclin B2 is significantly reduced following MTA3 knockdown. One explanation for this finding is that MTA3 serves directly as a transcriptional activator or coactivator. There are precedents for MTA proteins serving in this role because MTA1 stimulates PAX5 transcription in B cells by a direct interaction with its promoter and interacts with the transactivator HBx to promote nitric oxide synthase 2 transcription in hepatocellular carcinoma cells [44
]. A direct role for MTA3 in regulating cyclin B expression via promoter interactions is possible even though there is no evidence published to date that MTA3 serves as a transcriptional activator. However, it was recently shown that the cohesin complex can function to modulate chromatin architecture in ways that stabilize enhancer-promoter interactions important for gene transcription [19
]. This observation raises the possibility that MTA3 and/or the MTA3-NuRD complex supports cohesin-chromatin interactions required to promote sufficient cyclin B expression to transit the G2/M checkpoint.
Taken together, our studies indicate that MTA3 is a NuRD complex subunit in granulosa cells and support the idea that the MTA3-NuRD complex functions to support chromatin remodeling required for cohesin loading and G2/M progression. These findings highlight the critical role of the NuRD chromatin-remodeling complex in cell cycle control, in addition to its well-documented functions in modulating epigenetic marks that control gene transcription and in DNA double-strand break repair.