1.13.1 Histone modification and acetylation
Eukaryotic chromatin consists of many fundamental unit nucleosomes and genomic DNA. Each nucleosome consists of a protein octamer of two molecules each of histone H2A, H2B, H3 and H4, wrapped by 147 bp of genomic DNA. The amino terminal tails of the histones extend from the core structure and are subject to post-translational modifications (PTMs) such as acetylation, phosphorylation, methylation, ubiquitylation and Sumoylation (Li et al, 2007). These modifications can influence nucleosome stability packing, and facilitate recruiting other TFs; modification of a histone at one amino acid position can influence the type of modification at other positions. Remarkable progress has been made during the past few years in the characterization of histone modifications on a genome-wide scale. The general picture emerging is that promoter regions of active genes have reduced nucleosome occupancy and elevated histone acetylation (Barrera & Ren, 2006; Kim et al, 2005; Wang et al, 2009). The modifications marking either active or inactive genes are highly specific. High levels of histone H3K9 acetylation and H3K4 methylation are detected in promoter regions of active genes (Barski et al, 2007; Bernstein et al, 2005; Kim et al, 2005; Roh et al, 2007), whereas elevated levels of H3K27 methylation correlates with gene repression (Boyer et al, 2006; Lee et al, 2006; Roh et al, 2006).
1.13.2 Histone methylation
Significant progress has also been made in characterizing global levels of histone methylation modifications in mammals. High levels of H3K4me1, H3K4me2, and H3K4me3 are detected surrounding TSSs. In addition to the promoter regions, these
modifications are also detected in intergenic regions. The H3 K4/9acetylation and H3K4 methylation signals outside of promoter regions have been correlated with functional enhancers in various cell types (Heintzman et al, 2007; Roh et al, 2007). The mono methylations of H3K27, H3K9, H4K20, H3K79 and H2BK5 are all linked to gene activation, whereas trimethylations of H3K27, H3K9, and H3K79 are linked to gene repression. Although H3K9 methylation has been implicated in heterochromatin formation and gene silencing, a large-scale analysis suggested that H3K9me3 is enriched in many active promoters. In a recent genome-wide ChIP-seq analysis, a significant dip in the signal was observed between -200 to +50 for H3K4me3, which correlated with the nucleosome loss in active genes. A series of peaks of H4K4me3 signals at +50, +210, and +360 were detected, suggesting similar nucleosome positioning relative to TSS in active genes (Barski et al, 2007)
The levels of H3K4me1 and H3K4me2 positively correlated with transcriptional levels. However, methylation of H3K27 correlated with gene repression (Boyer et al, 2006; Lee et al, 2006; Roh et al, 2006). Indeed, H3K27me3 levels were higher at silent promoters than at active promoters (Barski et al, 2007). Also, high levels of H3K4me1 combined with low levels of H3K4me3 as a signature for predicting enhancers was also observed (Heintzman et al, 2007). In summary, active genes are characterized by high levels of H3K4me1, H3K4me2, H3K4me3 and H3K9me. In contrast, inactive genes are characterized by low or negligible levels of H3K4 methylation at promoter regions, high levels of H3K27me3 and H3K79me3 in promoter and gene-body regions.
1.13.3 RNA Pol II
The synthesis of eukaryotic mRNA is carried out by RNA pol II. During this process, pol II associates transiently with many different factors, including the general transcription factors TFIIB, TFIID, TFIIE, TFIIF and TFIIH, coactivators and elongation factors. Pol II is sufficient to catalyse DNA-directed RNA synthesis, but it is unable to recognize promoter DNA on its own. Thus, general transcription factors TFIIB, -D, -E, -F, and -H, which mediate promoter recognition and opening, are required for transcription initiation.
Although the general transcription factors and pol II are sufficient for accurate transcription initiation in vitro, these components alone fail to respond to activator proteins bound to
enhancer or upstream activation sequences. Mediator, an enormous complex composed of many subunits, is required for transcription from most pol II promoters. It appears to function as a ‘control panel’ that integrates regulatory signals from enhancer-bound activators, and transduces this information to pol II and the general transcription factors (Sikorski & Buratowski, 2009). The composition of Mediator complexes is different, depending upon the specific activator, suggesting that Mediator is a dynamic complex that allows for mixing-and-matching of sub complexes in response to different activator or repressor requirements.
1.13.4 CBP
p300 and CBP were originally identified as proteins that bound to the adenoviral E1A and the cAMP-response-element binding protein (CREB), respectively (Chrivia et al, 1993; Eckner et al, 1994a). The human CBP gene is located in chromosomal region 16p13.3.
Interestingly, this region shows extensive homology to a region on chromosome 22 where the p300 gene resides (Eckner et al, 1994b). Thus CBP and its paralog p300 are functionally closely related, both versatile transcriptional co-activators that can influence many physiological processes including proliferation, differentiation and apoptosis (Giordano & Avantaggiati, 1999; Goodman & Smolik, 2000).
CBP and p300 are thought to regulate gene expression acting as adaptor molecules, interacting both with a wide variety of TFs and with components of the basal transcriptional machinery, including TBP, TFIIB, TFIIE and TFIIF (Figure 1.9) (Goodman & Smolik, 2000; Wang et al, 2013). Therefore, p300/CBP is thought provide such a bridge to the
transcriptional machinery. Study of the IFNβ enhancer has shown that the surface of
p300/CBP provides a scaffold for different components of the transcription apparatus (Kim
et al, 1998; Munshi et al, 1998). ATF2/JUN, NF-κB p50/p65 and IRF3/IRF7 can be bound to CBP/p300 to form the IFNβ enhancesome (Munshi et al, 1998).
Figure 1.9 CBP and gene transcription
Another important function of CBP is to acetylate multiple sites in the core histone tails through its HAT activity (Figure 1.9). Acetylation of lysine residues of histone tails helps transcription factors access the DNA in chromatin (Vettese-Dadey et al, 1996), may also weaken internucleosomal interactions and de-stabilise higher-order chromatin structure (Garcia-Ramirez et al, 1995; Luger et al, 1997; Tse et al, 1998), and may therefore facilitate the processivity of pol II through nucleosome arrays (Nightingale et al, 1998). Besides acetylating all four histones, CBP/p300 HATs have also been shown to modify other proteins (Figure 1.9) (Wang et al, 2013). Examples include tumour suppressor protein p53, acetylation of which results in an enhancement of its DNA binding activity (Gu & Roeder,
1997), NF-κB (RelA/p65) (Huang et al, 2010; Kiernan et al, 2003), STAT1(Zhuang, 2013)
and the basal transcription factors TFIIE and TFIIB (Chen et al, 2001).
As discussed (section 1.13.1), specific histone acetylation is closely correlated with transcriptional activity in eukaryotic cells. To date, four mainly multi-gene families of nuclear proteins have been described that possess HAT activity including GCN5 and P/CAF, p300 and CBP (Bedford et al, 2010). GCN5 preferentially acetylates H3 and H4 histones (Kuo et al, 1996) whereas CBP can acetylate all four histones (Bannister & Kouzarides, 1996). CBP and p300 have unique functions that cannot be substituted for by other HATs. For instance, the acetylation of histone H3 lysine 18 is completely dependent on CBP/p300