CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

Molecular dissection of CHARGE syndrome highlights the vulnerability of neural crest cells to problems with alternative splicing and other transcription-related processes

“…Of note, the recent discovery of new CHARGE syndrome candidate genes [36,37] also strongly supports the idea that dysregulation of chromatin structure-dependent cotranscriptional alternative splicing plays a fundamental role in CHARGE syndrome pathogenesis. Indeed, direct and/or indirect evidence for a regulatory role in alternative splicing exists for all new five candidate genes (PUF60, EP300, RERE, KMT2D and KDM6A): PUF60 encodes a bona fide splicing factor [38,39]; EP300 codes for the p300 histone acetyltransferase, which has been shown to be directly involved in splicing regulation [40]; RERE codes for a transcriptional co-repressor that notably associates with HDAC1/2 [41][42][43], which are included in the Fam172a/spliceosome interactomes [22,35]; KMT2D encodes a H3K4methyltransferase that is included in the spliceosome interactome [35], and trimethylation of H3K4 is known to trigger the recruitment of splicing factors [33]; and finally, KDM6A codes for a demethylase of H3K27me3, a histone mark associated with AGO-mediated splicing regulation [28]. Considering all of the above, we thus believe that a link with cotranscriptional alternative splicing would help prioritizing potentially damaging variants in CHD7 mutation-negative individuals suspected to have CHARGE syndrome or a related pathology.…”

Transcriptional pulsing of a nucleolar transgene

“…ChIP was performed as described previously (Diermeier et al, 2013;Duskova et al, 2014). In brief, cells were fixed with 1% formaldehyde in 1X PBS, lysed in SDS lysis buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 20 mM EDTA, protease inhibitors) and chromatin was sonicated using a Bioruptor sonicator (Diagenode, Denville, USA).…”

TALE-directed local modulation of H3K9 methylation shapes exon recognition

“…To test whether locally restricted changes in the chromatin context can directly affect splicing of an alternative exon, we altered H3K36 and H3K9 methylation at the EDB exon in human fibronectin ( FN1 ). Methylation of H3K9 and H3K36 were previously proposed to regulate alternative splicing 11 12 15 20 21 , and splicing of the EDB exon was described to be sensitive to the chromatin environment 13 23 24 . To specifically and locally target chromatin around the EDB exon we designed a TALE domain recognizing 23 nt of the plus strand close to the 3′ splice site (SS) of the EDB exon using the TAL Effector Targeter 25 ( https://tale-nt.cac.cornell.edu/node/add/single-tale ).…”

“…In recent years there has been mounting evidence that pre-mRNA splicing is closely coupled with RNA synthesis and the chromatin environment of template DNA 4 36 . The original findings suggested that chromatin modifications modulate alternative splicing 11 13 14 21 23 37 38 and further studies have shown that pre-mRNA splicing reciprocally feeds back on histone modifications 7 8 9 . Most studies have relied on global depletion/inhibition or overexpression of chromatin modifying enzymes.…”

“…This strategy has been successfully utilized to modify chromatin modifications at enhancers 19 . As proof of principle, we chose to investigate the effect of H3K9 and H3K36 methylation 11 12 15 20 21 , histone marks previously shown to modulate alternative splicing, on alternative splicing of the endogenous human fibronectin EDB exon, a widely studied example of a cassette exon 22 23 24 . We further studied the global distribution of H3K9me3 within genes and showed this histone mark plays an unanticipated role in splicing of constitutive exons.…”