In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

• MeSH
• Alternative Splicing genetics   MeSH
• Chromatin metabolism   MeSH
• Exons genetics   MeSH
• Fibronectins genetics   MeSH
• Transcription, Genetic MeSH
• HeLa Cells MeSH
• Histones metabolism   MeSH
• Humans MeSH
• Lysine metabolism   MeSH
• Methylation MeSH
• Transcription Activator-Like Effectors metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Fibronectins MeSH
• Histones MeSH
• Lysine MeSH
• Transcription Activator-Like Effectors MeSH

Histone acetylation modulates alternative splicing of several hundred genes. Here, we tested the role of the histone acetyltransferase p300 in alternative splicing and showed that knockdown of p300 promotes inclusion of the fibronectin (FN1) alternative EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as p300 knockdown. Next we showed that p300 controls histone H4 acetylation along the FN1 gene. Consistently, p300 depletion and CRE deletion/mutation both reduced histone H4 acetylation on mini-gene reporters. Finally, we provide evidence that the effect of CRE inactivation on H4 acetylation and alternative splicing is counteracted by the inhibition of histone deacetylases. Together, these data suggest that histone acetylation could be one of the mechanisms how promoter and promoter binding proteins influence alternative splicing.

• Keywords
• alternative splicing, fibronectin, histone acetylation, p300, promoter,
• MeSH
• Acetylation MeSH
• Alternative Splicing * MeSH
• Fibronectins genetics   metabolism   MeSH
• Gene Knockdown Techniques MeSH
• HeLa Cells MeSH
• Histones metabolism   MeSH
• Integrases genetics   MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Promoter Regions, Genetic MeSH
• E1A-Associated p300 Protein genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cre recombinase MeSH Browser
• EP300 protein, human MeSH Browser
• Fibronectins MeSH
• FN1 protein, human MeSH Browser
• Histones MeSH
• Integrases MeSH
• RNA, Messenger MeSH
• E1A-Associated p300 Protein MeSH

Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.

• MeSH
• Alternative Splicing MeSH
• Chromatin metabolism   MeSH
• Transcription, Genetic MeSH
• Genome, Human * MeSH
• HeLa Cells MeSH
• Histones genetics   metabolism   MeSH
• Humans MeSH
• Promoter Regions, Genetic MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Signal Transduction MeSH
• Protein Structure, Tertiary MeSH
• Transcription Factors MeSH
• Protein Binding MeSH
• Microscopy, Video MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BRD2 protein, human MeSH Browser
• Chromatin MeSH
• Histones MeSH
• Protein Serine-Threonine Kinases MeSH
• Recombinant Fusion Proteins MeSH
• Transcription Factors MeSH