Chromatin structure and compaction can be altered by a combination of post-translational histone modifications and the specific positioning of nucleosomes. Histone acetyl-transferases (HATs) and deacetylases (HDACs), recruited by sequence-specific activators and repressors, respectively, antagonistically regulate the acetylation of lysines on amino-terminal histone tails and directly link histone modifications with gene expression (Grunstein, 1997
). Mammalian HDAC1 is a deacetylase that is highly homologous to yeast Rpd3 (Ekwall, 2005
). Studies in yeast and mammalian cells showed that HDAC1/Rpd3 is an enzymatic component of multi-protein complexes containing the Sin3 co-repressor protein. In mammalian cells, the Sin3 core complex consists of at least eight subunits (Alland et al., 2002
; Hassig et al., 1997
; Laherty et al., 1997
; Zhang et al., 1997
). However, there is considerable disagreement regarding a ‘holo-Sin3’ complex, most likely due to transient associations with, and heterogeneity of, sub-stoichiometric regulatory proteins, including Swi/Snf remodeling proteins, Retinoblastoma (RB) binding protein 2 (RBP2), and other proteins (Hayakawa et al., 2007
; Nagl et al., 2007
; Sif et al., 2001
). Interestingly, RBP2 was recently shown to be a demethylase specific for di- and tri-methylated lysine 4 of histone H3 (Christensen et al., 2007
; Klose et al., 2007
). Thus, the Sin3 complex provides a versatile platform for chromatin modifying and remodeling activities.
Sin3/Rpd3 co-repressor complexes are recruited to promoter regions via sequence-specific repressors such as Ume6 or Mad in yeast and mammalian cells, respectively, resulting in localized deacetylation of histones within promoter regions and transcriptional silencing (Ayer et al., 1995
; Kadosh and Struhl, 1997
; Schreiber-Agus et al., 1995
). Interestingly, in addition to its well-established role in promoter binding and gene repression, distinct Sin3/Rpd3 complexes have recently been identified in both budding and fission yeast and shown to deacetylate nucleosomes within the coding regions of active and repressed genes, preventing spurious forward and antisense transcription (Carrozza et al., 2005
; Keogh et al., 2005
; Li et al., 2007
; Nicolas et al., 2007
). However, the relationship between yeast and mammalian Sin3 complexes has not been completely resolved, and whether mammalian complex(es) exhibit regulatory activities (besides histone deacetylation) analogous to those in yeast is not known.
Expression of cell cycle genes is regulated by the reversible recruitment of the E2F/DP family of transcription factors and associated chromatin remodeling enzymes during discrete stages of the mammalian cell cycle (Blais and Dynlacht, 2007
; Frolov and Dyson, 2004
). In quiescent or early G1 cells, E2F4/DP and associated retinoblastoma tumor suppressor protein (pRb) family members p107 and p130 binds to the promoters of cell cycle regulated genes, in some instances recruiting the Sin3/HDAC complex and repressing transcription (Rayman et al., 2002
). As cells progress into S phase, E2F4 and Sin3 dissociate from genes, leading to increased histone H3 and H4 acetylation and gene expression (Balciunaite et al., 2005
; Rayman et al., 2002
; Takahashi et al., 2000
). This strongly suggests a model in which E2F4-pocket protein complexes periodically and reversibly recruit Sin3 to cell cycle regulated genes during cell cycle progression.
We have also postulated a role for E2F4 and HDACs in terminal differentiation of skeletal muscle cells, wherein cell cycle genes are de-acetylated during cell cycle arrest and are subsequently permanently silenced in differentiated myotubes through pRb-mediated methylation of histone H3 lysine 27 (Blais et al., 2007
). However, the molecular mechanisms by which genes are permanently repressed in a step-wise fashion during differentiation are not understood, and a general description of changes at the level of chromatin remodeling is completely lacking.
In order to gain insight into the molecular mechanisms of permanent gene repression in differentiated cells, we performed extensive genome-wide transcription factor binding analyses in which we coupled chromatin immunoprecipitation (ChIP) with promoter microarrays (ChIP-on-chip). Here, we performed large-scale binding analyses for Sin3A, Sin3B, RBP2 and E2F4, and in parallel, we analyzed histone H3 acetylation and methylation levels, nucleosome positioning, and gene expression in growing and differentiated skeletal muscle cells. Our data suggest that Sin3 and RBP2 play a concerted role in repressing expression of a subset of E2F4 target genes through the modification and repositioning of nucleosomes in differentiated cells.