Despite considerable effort, our understanding of the mechanistic contribution of histone modifications during active transcription remains poor. Here, we provide biochemical and functional evidence that CHD1 functions to modulate the efficiency of pre-mRNA splicing in part through physical bridging of spliceosomal components to H3K4me3. Of the many steps involved in pre-mRNA processing, the recruitment of factors required for pre-mRNA splicing to nascent transcripts is perhaps the most poorly understood. In crude extracts, H3K4me3 has the ability to enrich components of the spliceosome, notably U2 snRNP and associated factors. Moreover, CHD1 can physically associate with multiple subunits of U2 snRNP in addition to the SF3a sub-complex. Considering that conventional chromatography identified a stable complex between CHD1 and SF3a, SF3a is likely required for U2 snRNP binding to H3K4me3. Currently, we are unaware what triggers the association of additional U2 snRNP components, besides the SF3a sub-complex, to CHD1. Perhaps CHD1 binding to the H3K4me3 mark itself facilitates the nucleation of larger spliceosomal complexes to the 5′ end of active genes. Overall, the observed effect on U2 snRNP chromatin association and the efficiency of pre-mRNA splicing is most dramatic when reducing the level of CHD1 protein, as compared to H3K4me3 levels. This could simply be due to the remaining H3K4me2 that is present in the absence of H3K4me3, as CHD1 binds both H3K4me2 and H3K4me3. CHD1 was also observed to affect the amount of H3K4me3 on active genes as well, perhaps indicating a combinatorial action of reducing both CHD1 and H3K4me3 levels at the same time. We are currently exploring these differing scenarios.
It is notable that the U1 snRNP and SR proteins, which act very early in spliceosome assembly, were largely absent from the H3K4me3 affinity and anti-CHD1 co-immunoprecipitation fractions. Thus alternative mechanisms for recruitment of spliceosomal components must exist. This is especially true given that pre-mRNA splicing in vivo fully recovers with time, even under reduced levels of CHD1 and H3K4me3 on active genes. As such, U1 snRNP and SR proteins associate, albeit perhaps indirectly, with the carboxy terminal domain of the largest subunit of RNAP II (for review see (Hirose and Manley, 2000
)), indicating that splicing factors can be recruited in multiple fashions. It is also possible that the restored ratio of un-spliced to total transcripts observed in is a consequence of post-transcriptional splicing, or alternatively, via degradation of un-spliced pre-mRNAs by the mRNA surveillance pathway.
Mass spectrometry and western blot analyses of the H3K4me3 affinity purified material identified numerous proteins implicated in transcription post-initiation events, in addition to pre-mRNA splicing. Among the mixture of complexes affinity purified through H3K4me3, we identified a large protein complex consisting of CHD1, SNF2h, and the PAF complex. CHD1 was also demonstrated to bridge the FACT heterodimer to H3K4me3. The interactions between CHD1, SNF2h, PAF, and FACT described above are also supported by numerous previous findings, primarily in yeast (Alen et al., 2002
; Gavin et al., 2002
; Kelley et al., 1999
; Krogan et al., 2002
; Simic et al., 2003
; Tsukiyama et al., 1999
). Consistent with our results, human SNF2h precisely overlaps with H3K4me2/3 at active genes in vivo, as determined by quantitative chromatin immunoprecipitation analyses (Kouskouti and Talianidis, 2005
). Knockdown of CHD1 by siRNA treatment resulted in the altered association of SNF2h, FACT and PAF on chromatin as determined by sub-cellular fractionation and ChIP (Supplementary Figure 3B and 3D
). These collective results suggest that H3K4me3 may serve to position FACT, PAF, and SNF2h via their interactions with CHD1 near the 5′ region of active genes.
Of particular interest is FACT enrichment on H3K4me3. The functional role of FACT in transcript elongation on chromatin templates is well established (Sims et al., 2004
). However, how FACT is properly placed near the first nucleosome after transcription initiation remained unclear. Our findings provide a possible mechanism for FACT recruitment and/or placement upon gene activation in human cells. The localization of H3K4me3 near the transcription start site is consistent with the required positioning of FACT to allow passage of the first nucleosomal barrier. Furthermore, ChIP studies in human and Drosophila cells identified the H3K4 tri-methyl mark co-localizing with FACT on active genes (Kouskouti and Talianidis, 2005
; Saunders et al., 2003
). Consistent with our findings, reduction of H3K4me3 on active genes via knock down of PAF subunits results in reduced recruitment of both FACT and the chromatin modulator Spt6, although RNAPII levels remained unaffected (Adelman et al., 2006
). Interestingly, Spt6 was also identified by mass spectrometry in the H3K4me3 eluted material (Supplementary Table 1
), although its relationship to CHD1 remains unknown at present.
In a fully reconstituted transcription system, the generation of H3K4me3 was observed to be coupled to transcription, but also dependent on H2B monoubiquitination, as previously described in yeast (Dover et al., 2002
; Pavri et al., 2006
; Sun and Allis, 2002
). These results suggest that H3K4me3 must occur subsequent to transcription initiation and after FACT and the H2B monoubiquitination machinery has located the first nucleosome during the initial round of transcription. Thus, H3K4me3 may function to expedite FACT/PAF localization during subsequent rounds of transcription, similar to re-initiation, a process that is much faster than initial PIC formation (Orphanides and Reinberg, 2000
). Given that H2B monoubiquitination is reversed during transcription (Henry et al., 2003
), the more stable H3K4me3 mark would serve as a permanent recruitment platform for facilitating RNAPII passage through nucleosomes.
An interesting question is whether the function of histone methylation (and perhaps other histone modifications) during active transcription is to facilitate the timing, but not the absolute levels of gene expression. In this context, histone methylation on active genes would be critical during embryonic development of multi-cellular organisms as well as on genes that respond to stimuli. Perhaps this is why histone methyl “writers” and “readers” are so instrumental during mammalian development (Margueron et al., 2005
). Moreover, tightly regulated gene regulatory networks may be particularly sensitive to the altered timing of gene expression, which may explain why histone methylation appears to play a prominent role in pleuripotency and oncogenesis.
H3K4me3, a histone tail modification associated with transcription activity, has now been shown to facilitate pre-mRNA maturation (). This adds credence to the general belief that modifications within the histone tails are not passive, but function to recruit factors that regulate different steps of gene expression, some to maintain genes in a silent state, and others, like H3K4me3, to facilitate the production of a mature mRNA.
Figure 7 Model of H3K4me3 function during transcription. Upon transcriptional activation, H3K4me2 is converted to H3K4me3 by an ASH2 containing complex, which places CHD1 near the transcriptional start site. CHD1 then recruits U2 snRNP (via the SF3a sub-complex) (more ...)