The HDAC-containing multiprotein complex, Sin3 complex, is highly conserved from yeast to humans. In mammalian cells, the Sin3 complex consists of six core subunits, HDAC1/2, RbAp46, RbAp48, Sin3A/Sin3B, SAP18, and SAP30 () [12
]. Mammalian Sin3A and Sin3B (mSin3A and mSin3B) show high sequence similarity and an overlapping expression pattern, and both are essential in mouse development. However, these subtypes play distinct and nonoverlapping roles. mSin3A has a critical role in the early developmental preimplantation stage, since no mSin3A-null zygotes are found at E6.5 [15
]. mSin3A-depleted mouse embryonic fibroblasts (MEFs) exhibit the deregulation of genes involved in cell-cycle control, DNA replication, DNA repair, apoptosis, chromatin modification, and mitochondrial metabolism. Transcriptome analysis has revealed that mSin3A modulates the transcriptional network controlled by Myc-Mad, E2F, or p53. On the other hand, mSin3B has an essential function at the late-gestation stage, and its mutant is different from the mSin3A-/- embryo [16
]. In particular, erythrocyte and granulocyte differentiation and G0/G1 cell-cycle control are impaired in the mSin3B-/- embryo, and these phenotypes are due to the derepression of E2F target genes.
The Sin3 complexes. Schematic representation of the Sin3-containing HDAC complexes: (a) the Sin3-core and Sin3-RBP2 complexes in mammalian cells; (b) the Rpd3S complex in S. cerevisiae; (c) RLAF-N and LAF-A complexes in Drosophila. The empty dotted (more ...)
Transcriptional regulation is tightly coupled with dynamic change of histone acetylation in the promoter regions. However, the Sin3 complex functions not only at promoter regions but also at transcribed regions. In Saccharomyces cerevisiae
, Sin3 and a Class I HDAC, Rpd3, are involved with two functionally distinct complexes, Rpd3L and Rpd3S [17
]. Both complexes contain Sin3, Rpd3, and Ume1. In addition to these core subunits, Rpd3L contains at least six unique components: Rxt1, Rxt2, Dep1, Sds3, Pho23, and Sap30, whereas Rpd3S contains Rco1 and Eaf3 as unique components () [18
]. Rpd3L is localized primarily to promoter regions. In contrast, Rpd3S is localized to transcribed regions that are enriched in methylated H3K36 (H3K36me). H3K36me is a mark catalyzed by Set2 (KMT3) and is tightly coupled with the transcriptional elongation processes [19
]. Rpd3S recognizes this H3K36me mark via a combined action of chromodomain of Eaf3 and PHD (plant homeobox domain) of Rco1 [20
]. Deletion of Set2 or one of the Rpd3S-specific components results in spurious transcripts that emerge from incorrect transcription start sites in some transcriptionally active genes [17
]. Since the H4 acetylation levels within transcribed regions are increased in these mutant cells, Rpd3S is thought to repress unfavorable transcription by maintaining transcribed regions in a hypoacetylated state. In metazoans, however, the relationship between mSin3 complexes and the emergence of aberrant transcripts remains unclear.
One of the Rpd3S-specific components, Eaf3, is highly conserved from yeast to humans. The human Eaf3 homologue MRG15 was initially identified as a factor closely related to MORF4 (mortality factor on human chromosome 4), whose transient expression induces senescence in a subset of human tumor cell lines [23
]. MRG15 is a stable component of mSin3-HDAC complexes, and the MRG15-associated mSin3-HDAC complex also contains Pf1, a component that shares similarity with S. cerevisiae
], and a histone H3K4-specific demethylase, RBP2 (KDM5A) [26
] (). Of note, MRG15 is shared with at least two other complexes: the Tip60 (NuA4)-HAT complex [26
] and the BRCA2-containing DNA-damage-responsive complexes [29
]. Although we will not discuss these complexes further in this paper, the MRG15-associated complexes are implicated in DNA-damage responses [29
], in addition to transcriptional regulation, suggesting that histone acetylation dynamics is tightly coupled with histone eviction/deposition during DNA-repair processes.
We previously demonstrated that MRG15 recruits RBP2 and controls the H3K4me levels on transcribed regions via the RBP2 activity [26
]. In addition, van Oevelen et al. reported that the majority of Sin3 target genes (58%) are bound by RBP2, which spreads over the region immediately downstream of the transcription start site on a subset of E2F target genes during differentiation [34
]. These results suggest that the Sin3-HDAC complex and RBP2 play a cooperative role in repressing target genes through deacetylation, demethylation, and, probably, the repositioning of nucleosomes. In S. cerevisiae
, no evidence has been reported for a physical interaction or functional link between the Sin3 complex and the KDM5 homologue Jhd2. In Drosophila
, RPD3, MRG15, and PF1, but not SIN3, were identified in an LID-(a KDM5 homologue) containing complex [35
]. Intriguingly, the deacetylation activity of RPD3 is inhibited by its interaction with any of these associated proteins, including LID, implying that the relationship between the HDAC and HDM activities is counteractive.
Recent reports have shown that LID associates with SIN3 and functions in Notch silencing [36
]. The Notch signaling pathway plays pleiotropic roles during embryonic development and is important for the regulation of cellular self-renewal [37
]. Moshkin et al., purified factors associated with histone chaperones (ASF1 and NAP1) from Drosophila
S2 cells or embryonic nuclear extract, and identified several peptides, including LID, SIN3A, PF1, RPD3, MRG15, and a BRCA2-binding protein, EMSY. Using a reciprocal purification approach, they further showed that NAP1 binds the RPD3 and LID-Associated Factors (RLAFs) consisting of RPD3, LID, SIN3A, PF1, EMSY, and MRG15, whereas ASF1 binds LAF, a similar complex lacking RPD3 (). Both of these complexes, RLAF-NAP1 (RLAF-N) and LAF-ASF1 (LAF-A), are required for the transcriptional repression of Notch-regulated genes. In agreement with the enzymatic activity of LID, the knockdown of ASF1, NAP1, or any of the LAF components results in an accumulation of H3K4me3 at the promoter and enhancer regions. Interestingly, H3K4me3 accumulation is not observed in RPD3 knockdown cells [36
]. This implies that LID recruitment to the promoter/enhancer regions of Notch target genes may not depend on the entire RPD3-containing RLAF complex. Considering that the RPD3-containing RLAF complex resembles Rpd3S in S. cerevisiae
in its subunit composition (Figures and ), it is possible that RLAF acts at the rest of transcribed regions, with or without the association with NAP1. This possibility will be tested by future studies.
RBP2/KDM5A was also found to be involved in the Notch pathway in a study using mammalian cells [39
]. RBP-J is a nuclear effector of Notch signaling. Upon ligand binding, RBP-J activates the expression of Notch target genes, and, in the absence of Notch signal, RBP-J switches off the target gene expression. Liefke et al. showed that RBP2/KDM5A associates with RBP-J in vivo
. RBP2/KDM5A is colocalized with RBP-J at the promoter region of Notch target genes and regulates their expression. Although the involvement of HDAC at the target promoter region was not described in this study, it is most likely that RBP2 works together with an HDAC complex, as in the case of the LAF-A complex in Drosophila
Another function of the Sin3 complex was revealed by a genome-wide study. The gene-expression profiles of Sin3-depleted Drosophila
cells were analyzed by high-density oligonucleotide array [40
]. This analysis revealed that the expression of ~3% of the Drosophila
genes is altered in Sin3-depleted cells. The affected genes are associated with diverse biological processes, including signal transduction, transcriptional regulation, and cell-cycle control. Interestingly, the list of affected genes also includes a substantial fraction of genes involved in cytosolic and mitochondrial energy-generating pathways. Furthermore, Sin3-deficient Drosophila
cells exhibit an increase in mitochondrial mass. In accordance with this observation, the genome-wide analysis of RBP2-binding sites using human breast cancer cell lines revealed that RBP2 is enriched at genes encoding proteins that localize to the mitochondrion, including mitochondrial ribosomal proteins [41
]. Moreover, the alteration of RBP2 leads to mitochondrial defects in human cells, as in Sin3-depleted Drosophila
Overall, the Sin3-HDAC complexes associated with KDM5 regulate the transcription of many genes, in processes such as Notch signaling and mitochondrial functions. Notably, the transcriptional regulation is achieved by dynamic changes in histone modifications not only at promoter regions, but also at transcribed regions. The Sin3-KDM5 complex appears to play an important role at both regions by exchanging its associated cofactors.