In this study, we have shown that CABIN1 is a functional member of the HUCA histone chaperone complex. Our data confirm CABIN1 as the human ortholog of the yeast Hir3p protein.
To start, we have confirmed that CABIN1 physically interacts with other members of the HUCA complex in cells. The interaction with the complex is most likely direct, because we have successfully reconstituted a quaternary complex comprised of HIRA, UBN1, CABIN1, and ASF1a. While there are likely other proteins that interact with this complex in vivo
, based on previous complex purification studies (10
) and our own unpublished efforts in this regard, this quaternary complex appears to be a particularly stable core entity.
We have defined the region of CABIN1 that interacts with HIRA and vice versa
. Specifically, HIRA binds to the conserved N-terminal TPRs of CABIN1, and CABIN1 binds to the conserved C domain of HIRA. The latter result is particularly significant in light of a previous analysis that highlighted three conserved domains in HIRA: the N-terminal WD40 repeats, a central B domain, and a C-terminal C domain (22
). We now know that the N-terminal WD40 repeats bind to UBN1 (5
), the B domain binds to ASF1a (34
), and the C domain binds to CABIN1. By this view, HIRA forms a scaffold for the HUCA complex, acting as a binding platform to recruit UBN1, ASF1a, and CABIN1. This model is supported by our previous demonstration of heterodimeric complexes formed between purified recombinant HIRA and UBN1 (5
) and also HIRA and ASF1a (34
). While we have not yet achieved this for HIRA and CABIN1, we have shown that the C-terminal C domain of HIRA is required to recruit CABIN1 to the quaternary complex. While HIRA is the scaffold for the complex, UBN1, CABIN1, and ASF1a presumably have their own specialized functions. Indeed, ASF1a appears to be the primary histone binding subunit of this histone chaperone complex (8
). In sum, HUCA is a four-member quaternary complex comprised of HIRA, UBN1, CABIN1, and ASF1a, in which HIRA serves as a platform to bring the other members together.
Presumably, the other members of the complex, UBN1 and CABIN1, bring specific functionalities to the complex. In this regard, CABIN1 has been previously shown to facilitate transcriptional repression by transcription factors, including MEF2 and p53, by recruiting histone-modifying enzymes, mSin3, histone deacetylases (HDACs), and SUV39H1 (16
). These enzymes modify chromatin to achieve a more transcriptionally repressed state. Interestingly, CABIN1 is regulated by intracellular calcium, to link calcium signaling to control of gene expression (16
). Whether or not CABIN1 serves to recruit similar histone-modifying enzymes to the HUCA complex and whether HUCA is also regulated by calcium signaling remain to be established.
In addition to showing that CABIN1 is a member of some members of the HUCA complex, we have demonstrated that CABIN1 shares functional outputs with other members of the complex. Like other members of the complex, CABIN1 is involved in chromatin heterochromatinization in senescent human cells (5
). Two lines of evidence show this. First, CABIN1 is recruited to PML nuclear bodies, together with HIRA and UBN1, in senescent cells. Second, ectopic expression of CABIN1 induces a senescent-like state, as indicated by proliferation arrest, activation of the pRB tumor suppressor pathway, expression of SA β-Gal, and formation of SAHF. Previous studies from our lab have shown that recruitment of some members of the HUCA complex to PML bodies is linked to formation of SAHF (5
). While PML bodies and SAHF do not colocalize, it is thought that PML bodies serve as sites to modify or assemble the HUCA complex into higher-order complexes prior to its role in SAHF formation.
HUCA's role in formation of SAHF is likely to involve histone deposition and nucleosome assembly. Specifically, the HUCA complex is thought to preferentially deposit the histone variant H3.3 into chromatin in a DNA replication-independent manner (33
). Histone H3.3 is best viewed as a replacement variant histone, whose incorporation into chromatin outside DNA replication is associated with both transcription activation and repression (see the introduction). Here, we have underscored the link between HIRA and histone H3.3 deposition and similarly established a link between CABIN1 and histone H3.3 deposition. siRNA-mediated knockdown of HIRA and CABIN1 in HeLa cells modestly affects cellular gene expression (in terms of magnitude of the changes in RNA abundance) programs in both cases. The relatively subtle effect of HIRA and CABIN1 knockdown is in line with a previous study that failed to identify a marked effect on global gene expression after HIRA inactivation in mouse embryonic stem (ES) cells (12
). Presumably, the function of the HUCA complex at genes is redundant with other chromatin regulators. More important than the modest effect on gene expression, two results point to the shared function of HIRA and CABIN1. First, there is significant overlap of the genes regulated by HIRA and CABIN1. Second, genes that are downregulated by inactivation of HIRA and CABIN1 are both especially enriched in histone variant H3.3 at the TSS, gene body, and 3′ end of gene body, compared to genes that are upregulated or do not change. This supports the role of histone H3.3 and HUCA in gene activation. The presence of histone H3.3 at the gene's TSS is thought to promote gene expression, because nucleosomes containing histone H3.3 and variant H2AZ are more labile than canonical nucleosomes (20
). These labile nucleosomes are thought to facilitate access of transcription factors and chromatin remodeling events associated with transcription. Since HUCA is largely responsible for deposition of histone H3.3 at the TSS (12
), knockdown of HIRA or CABIN1 is likely to repress transcription at these genes by generating a more static, less transcriptionally permissive, and perhaps histone H3.1-containing, nucleosome structure at the TSS. Regardless of the precise mechanism, the similar link between both HIRA and CABIN1 and histone H3.3 supports the notion that CABIN1 is a functional member of the HUCA complex.