In this study, we identified novel BAP1-interacting proteins and showed that nearly all cellular BAP1 forms high-molecular-weight multiprotein complexes with several transcription factors and cofactors. The associated partners are likely to play critical roles in targeting BAP1 to potential substrates, thereby regulating its function. Based on the relative abundance of BAP1-associated proteins purified from HeLa or U2OS cells, and on data from other studies (31
), it appears that HCF-1, ASXL1 and/or ASXL2, OGT, and FOXK1 and/or FOXK2 might form a BAP1 core complex. This minimal complex may selectively associate with additional regulators or transcription factors to form specific functional complexes in a cell type- and/or promoter-dependent manner. Indeed, substoichiometric levels of several transcription factors copurified with BAP1. These factors are involved in a wide range of cellular processes, suggesting that BAP1 might exert a much broader role in regulating cell function than previously appreciated. Consistent with this notion, BAP1 depletion by RNAi induced profound changes in the expression of genes mediating and/or controlling numerous cellular pathways. Further studies will be needed to investigate how BAP1, via selective interactions with specific transcription factors and cofactors, regulates specific biological responses.
We provided strong evidence that BAP1 is a transcriptional coactivator, as follows. (i) BAP1 associates with transcriptionally active chromatin. (ii) BAP1 acts as an activator, in a DUB activity-dependent manner when targeted to a promoter by using the Gal4 system. (iii) Genomewide expression analysis reveals a considerable number of genes downregulated following BAP1 depletion. (iv) BAP1 directly occupies the cox7c
promoter, and depletion of BAP1 results in the downregulation of this gene. It is also possible that BAP1 possesses dual coactivator/corepressor functions, depending on its association with specific transcription factors and cofactors on the regulatory elements of target genes. In agreement with the latter hypothesis, some BAP1-interacting proteins, including HCF-1, YY1, OGT, and ASXL, are known to interact with both coactivators and corepressors (5
). In addition, a significant number of genes were upregulated following the depletion of BAP1. This suggests that BAP1 might exert a repressive effect on their promoters, although these genes could constitute indirect targets, i.e., their upregulation could result from secondary changes induced by BAP1 depletion.
Using YY1 as a model for sequence-specific transcription factors that interact with BAP1/HCF-1, we demonstrated that these three proteins form a ternary complex in vivo
that can associate with chromatin. Moreover, we found that BAP1 and HCF-1 are recruited by YY1 to coactivate cox7c
, a gene previously reported to depend on YY1 binding sites for transcriptional activation (50
). While depletion of BAP1 or HCF-1 reduces expression of cox7c
, in contrast, depletion of YY1 induces an increase in the expression of this gene. These results suggest that YY1 possesses a dual function as both a repressor and an activator of cox7c
, depending on its association with the HCF-1/BAP1 coactivator complex. A similar repression/activation mechanism by YY1 has been previously shown for the murine beta interferon promoter (37
). Consistent with this model, YY1 interacts with HCF-1 through the central region containing a GA/GK-rich domain, previously shown to be involved in interactions with HDACs (70
). This suggests that the association of YY1 with HCF-1/BAP1 is mutually exclusive with respect to its interaction with HDAC corepressive complexes. With respect to cox7c
expression, it is well-known that nucleus-encoded mitochondrial genes, including components of the cytochrome c
oxidase complex, are not constitutively expressed but rather are subject to tight regulation by several transcription factors and cofactors, depending on the state of cell growth, energy balance, and other tissue-specific needs (18
). Therefore, such genes are expected to oscillate between activation and repression states.
It is not clear at this time whether BAP1 regulates all YY1 target genes. It is possible that it might regulate only a subset of these targets, perhaps those on which YY1 acts as an activator only or on which YY1 might exert a dual activator/repressor function. Other transcription factors might dictate the specificity via interaction with YY1. Indeed, YY1 is well known to interact with numerous transcription factors, such as SP1, C-myc, and E2Fs (25
). HCF-1, via additional interactions, might also contribute to the selectivity of BAP1 recruitment to specific YY1 target genes. In this respect, it is not surprising that Gal4-BAP1 lacking HBM is only slightly impaired in transcription activation, suggesting that the interaction between HCF-1 and BAP1 might be involved mostly in the recruitment of the latter to specific promoters.
Precisely how the assembled BAP1/HCF-1/YY1 complex acts to induce the activation of cox7c
or other target genes remains to be established. Nonetheless, the data suggest that the molecular mechanism involves ubiquitin signaling and the deubiquitination of specific substrates on target promoters. BAP1 might be continuously needed to prevent the degradation of HCF-1 (31
). Although the stability of the total cellular pool of HCF-1 is not significantly affected by BAP1 depletion, it is nonetheless possible that BAP1 stabilizes HCF-1 only on specific promoters following recruitment by YY1 or other transcription factors. Consistent with this, a BAP1 catalytically inactive mutant exerts a dominant negative effect on cox7c
expression. It is also plausible that HCF-1 association with BAP1 and YY1 targets the DUB activity to deubiquitinate histones, specific transcription factors, or components of the general transcription machinery. Consistent with this, the drosophila BAP1, Calypso, deubiquitinates monoubiquitinated H2A, a histone mark associated with gene repression (47
). However, Calypso does not possess HBM, and thus, the mammalian BAP1 appears to associate selectively with HCF-1 and numerous other proteins not found with Calypso. In addition, in contrast to Calypso, whose activity on ubiquitin-AMC is very low when not associated with ASX, the recombinant mammalian BAP1 appears to have the same activity as complexed BAP1. We note that although BAP1 partners do not affect its DUB activity on ubiquitin-AMC, this does not exclude the possibility of their effect in the context of a physiological substrate in vivo
Our results also shed light on the biological function of BAP1. This DUB was previously shown to be required for proper cell cycle progression, particularly the G1
/S transition (31
), Moreover, we observed similar effects in U2OS cells (data not shown), the cell type used here for global gene expression analysis. We also provided molecular insight linking BAP1 to the control of cell cycle genes, including subsets of E2F targets. In addition, HCF-1 is known to be required for a normal G1
/S transition and was recently shown to play a major role in regulating the expression of E2F target genes by promoting histone H3 K4 trimethylation (21
). Thus, BAP1 might play a pivotal role in regulating the G1
/S transition under normal and possibly stress conditions. Supporting this view, BAP1 is phosphorylated on an ATM/ATR consensus motif in response to DNA damage (33
), suggesting that these critical DNA damage-responsive checkpoint kinases might regulate BAP1 DUB activity and thus its function in controlling the expression of cell cycle genes.
BAP1 might also participate in transcriptional regulatory programs that coordinate cell growth with the cell cycle. For instance, in addition to cox7c
, the expression of several mitochondrial and general metabolism genes are shown here to be deregulated upon BAP1 knockdown. Interestingly, recent bioinformatics and genome-wide promoter occupancy studies indicated that YY1 binding sites are enriched in the promoter regions of nuclear genes that encode mitochondrial proteins (59
). Moreover, NRF1, a major regulator of mitochondrial respiration, copurifies with BAP1 (Fig. ), and both YY1 and NRF1 binding sites are frequently found in close proximity in a large number of promoters of genes encoding mitochondrial proteins (59
). Furthermore, HCF-1 has been found to interact with, and increase the transcriptional activity of, peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), a major transcriptional regulator of mitochondrial biogenesis (27
). Thus, BAP1 might play an important role in dynamically controlling transcriptional responses that coordinate mitochondrial function. Such responses, in turn, could constitute targets of stress signaling pathways (e.g., induced by DNA damage) that orchestrate adaptive metabolic responses.
In summary, our work indicates that BAP1 associates with several transcription factors and cofactors and is a gene-specific transcription regulator. As such, our findings establish a framework for further studies to (i) delineate the exact role of BAP1 in regulating the expression of genes involved in cell cycle progression and (ii) define how deregulation of BAP1 function contributes to tumorigenesis.