Histone modifications and transcription

The nucleosomes are subjected to a variety of post-translational modifications which play an important role in the regulation of transcription through chromatin. Particularly, the N-terminal histone tails are targeted by multiple enzymes that covalently modify specific residues. These modifications include acetylation, methylation, ubiquitination, ADP-ribosylation, sumoylation and phosphorylation (reviewed by Shilatifard, 2006 ; Li et al., 2007). Genes that are transcriptionally active have been associated with specific modifications such as acetylation of histone H3 and H4 (H3ac, H4ac), di- and trimethylation of H3 at lysine 4 (H3K4me2/3), lysine 36 (H3K36me2/3) and lysine 79 (H3K79me) or phosphorylation of H3 at serine 10 (H3S10p). Most of these modifications are deposited sequentially at the histone tails during transcription, and are distributed in a distinct pattern throughout the transcription unit (Fig. 5B). These modifications are notably involved in the maintenance of an open chromatin structure and modulate specific elongation events by promoting the recruitment of multiple transcription factors (Fig. 6).

Histone H3 and H4 acetylation occurs at several lysine residues and is carried out by multiple histone acetyltransferase (HATase) complexes (reviewed by Brown et al., 2000).

Histone acetylation is an important prerequisite for productive elongation, and H4K16ac was notably shown to inhibit higher-order chromatin structure (Shogren-Knaak et al., 2006).

Histone acetylation occurs at active promoter prior to PIC assembly, and both HATase enzymes and chromatin remodelers such as SAGA (Spt-Ada-Gcn5-acetyltransferase complex) and SWI/SNF are concomitantly recruited to increase DNA accessibility for PIC formation in yeast (Hassan et al., 2001 ; Bhaumik and Green, 2001 ; Neely et al., 2002).

Recent genome-wide analysis of HATases binding and histone acetylation in yeast showed that H3ac and H4ac peaks sharply at the promoter region of active genes, and that both Gcn5 and Esa1 HATases are generally recruited to promoters (Robert et al., 2004 ; Pokholok et al., 2005). Some HATase complexes including Elongator in yeast and human cells (Winkler et al., 2002 ; Kim et al., 2002 ; Gilbert et al., 2004), as well as TAC1 (Trithorax acetylation complex 1) in Drosophila, which display both HATase and HMTase (histone methyltransferase) activities (Petruk et al., 2001, 2004), remain associated with the elongation complex during transcription. In Drosophila, histone acetylation can be detected across relatively large chromatin domains, which may be used for a long-term maintenance and modulation of patterns of gene expression (Turner, 2000). Particularly, high levels of H4K16ac along the body of dosage-compensated genes on the male X chromosome are

required to increase the rate of transcription relative to the female levels (Smith et al., 2001 ; Gilfillan et al., 2006 ; Bell et al., 2008).

The early elongation phase coincides with methylation of H3K4. In yeast, H3K4 is methylated by the Set1 HMTase subunit of the COMPASS (complex proteins associated with Set1) complex (Miller et al., 2001 ; Briggs et al., 2001 ; Roguev et al., 2001). Set1 is responsible for H3K4me3 around the transcription initiation site of active promoter and H3K4me2 across the coding sequence (Pokholok et al., 2005). Importantly, Set1 is recruited at the promoter region through its interaction with the RNAPII CTD that has previously been phosphorylated at Ser-5 by TFIIH (Krogan et al., 2003a ; Ng et al., 2003b). Functional homologues of Set1 have been identified in higher eukaryotes including the Trx (Trithorax, Petruk et al., 2004 ; Smith et al., 2004) and Ash1 (Absent, small, and homeotic complex 1) (Beisel et al., 2002 ; Byrd and Shearn, 2003) proteins in Drosophila, as well as the SET1 and the MLL (mixed lineage leukaemia) protein family in human (Milne et al., 2002 ; Nakamura et al., 2002). The recruitment of Set1 to the phosphorylated CTD is assisted by the PAF (RNA polymerase-associated factor 1) complex. PAF is an evolutionarily conserved elongation factor from yeast to human involved at multiple stages of the transcription cycle (reviewed by Rosonina and Manley, 2005). PAF function is notably required for the local H3K4me3 distribution at the promoter region and the wide-spread H3K4me2 pattern deposited by Set1 in yeast. Similarly, human MLL HMTases require their association with the WRD5 protein for efficient H3K4me3 activity (Wysocka et al., 2005). In Drosophila, the Ash2 protein was shown to specifically affect H3K4me3 (Beltran et al., 2007). Although Ash2 does not possess intrinsic HMTase activity, its association with Ash1 or a currently unknown HMTase is required for subsequent H3K4me3 at the promoter. The functional relevance of the H3K4 methylation pattern has not been yet fully elucidated. Disruption of PAF activity in yeast affects mRNA processing but not the rate of transcription elongation, suggesting that Set1-mediated H3K4 methylation is rather involved in signalling mechanisms than in transcription (Mueller et al., 2004 ; Mason and Struhl, 2005). This hypothesis is notably supported by the observation that several chromatin remodelers such as NURF (nucleosome remodeling factor) (Santos-Rosa et al., 2003) and Chd1 (Pray-Grant et al., 2005) selectively interact with H3K4 methylated nucleosomes.

Subsequently to RNAPII CTD phosphorylation at Ser-5, the P-TEFb kinase is recruited to the TEC to trigger productive elongation. As RNAPII travels towards the 3’ end of the coding sequence, nucleosomes became trimethylated at H3K36 (Pokholok et al., 2005).

In yeast, H3K36 methylation is mediated by the Set2 HMTase, which also associates with the

phosphorylated CTD (Li et al., 2002, 2003 ; Krogan et al., 2003b ; Schaft et al., 2003 ; Xiao et al., 2003). P-TEFb depletion in yeast results in the loss of Set2 recruitment and H3K36 methylation across the coding region of genes, demonstrating that Set2 preferentially interacts with the Ser-2-phosphorylated CTD (Krogan et al., 2003b ; Xiao et al., 2003). Similarly to Set1, the recruitment of Set2 to the CTD and its HMTase activity is dependent on the PAF complex (Krogan et al., 2003b). H3K36me3 is recognized by the yeast histone deacetylase Rpd3, which generates an hypoacetylated environment within the coding sequence (Carrozza et al., 2005 ; Joshi and Struhl, 2005 ; Keogh et al., 2005). This process is thought to prevent inappropriate binding of transcription factors within the coding sequence and the generation of cryptic transcripts initiated from internal promoter-like sequences (Carrozza et al., 2005 ; Joshi and Struhl, 2005).

As mention above, PAF complex is involved in the recruitment of both Set1 and Set2 HMTases onto the phosphorylated CTD. PAF function is also required for H2B ubiquitination, which is a prerequisite for H3K4me2/3 at active genes (Ng et al., 2003a ; Wood et al., 2003 ; Dehe et al., 2005 ; Shahbazian et al., 2005). At the promoter region, PAF is recruited to the polymerase through interaction with the Ser-5-phosphorylated CTD and DSIF (Qiu et al., 2006), and is thought to play a pivotal role in regulating the binding of various CTD-associated chromatin regulators. These factors include notably Chd1 (Simic et al., 2003), Spt6 and FACT (Adelman et al., 2006 ; Pavri et al., 2006).

Several studies have reported a role for H3S10 phosphorylation in the regulation of early transcriptional events. H3S10 phosphorylation can be mediated by Snf1, the AMP-activated protein kinase (AMPK) homologue in yeast, which acts in concert with the Gcn5 HATase to activate expression of many stress-induced genes (Lo et al., 2001). Inhibition of H3S10 phosphorylation at such promoter eventually prevents the recruitment of TBP suggesting a role for this modification during PIC assembly (Lo et al., 2005). Snf1 physically interacts with P-TEFb (Ctdk1) (Van Driessche et al., 2005) and Mediator (Kuchin et al., 2000), and was recently shown to function in early elongation to alleviate transcriptional pausing (Tachibana et al., 2007). However the role of H3S10p in transcription remains elusive, and Snf1 can stimulate transcription independently of its H3S10-kinase activity. In mammalian cells, H3S10p also occurs on nucleosomes at the promoter and/or within the body of genes following induced activation, and is mediated by the mitogen- and stress-activated protein kinase (MSK)1 and MSK2 (reviewed by Davie, 2003 ; Soloaga et al., 2003).

A recent study also showed that human PIM1 kinase associates with the oncogene c-Myc and contributes to the activation of its target genes by phosphorylation of H3S10 at the

c-Myc-binding sites (Zippo et al., 2007). The function of H3S10p in Drosophila is more controversial. H3S10p is detectable at most euchromatic regions of polytene chromosomes, and the dynamic relocation of this modification at heat-shock genes along with the transcription machinery during the heat-shock response suggested a role for H3S10p in transcription activation (Nowak and Corces, 2000 ; Nowak et al., 2003 ; Ivaldi et al., 2007).

Ivaldi et al. (2007) have recently proposed that H3S10p mediated by JIL-1, the major Drosophila H3S10-kinase, is a hallmark for early transcription elongation and would be required for expression of the majority, if not all, genes in this species. The authors further proposed a model in which JIL-1 activity is required in the release of RNAPII from promoter-proximal pausing and that H3S10p is a prerequisite for P-TEFb recruitment. However, these data were not confirmed by others, and the group of Johansen claims that JIL-1-mediated H3S10 phosphorylation is actually required in the maintenance of euchromatin structure by counteracting the spreading of heterochromatin proteins (Zhang et al., 2006 ; Deng et al., 2008 ; Cai et al., 2008). It is conceivable that H3S10p is involved in both maintenance of chromatin architecture and transcription regulation, and further works in Drosophila are thus necessary to clarify the role of H3S10p in the transcriptional activation of certain stress-inducible genes such as the heat-shock genes.

2. P-TEFb : a key factor in the regulation of cellular processes and HIV-1