Unknown,Transcriptomics,Genomics,Proteomics

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Alternate exon switching establishes a tissue-specific transcription factor to mediate temporal activation of gene expression during differentiation

ABSTRACT: Although splicing occurs in most multi-exon genes, the generation of distinct isoforms through the alternate use of mutually exclusive exons is less prevalent. As exon-switching events have the potential to give rise to isoforms with different cellular functions, we have explored the role of the muscle-specific (Mef2Da2) and ubiquitously expressed (Mef2Da1) isoforms of the transcription factor Mef2D in myogenesis. Here we show that both isoforms of Mef2D bind a largely overlapping subset of genomic loci, yet only the muscle-specific Mef2Da2 isoform can activate the late myogenic gene expression program. This differential ability to activate transcription is modulated by PKA signaling where Mef2Da1 is efficiently phosphorylated by the kinase to enhance its association with repressive HDAC-deacetylase complexes. In contrast, alternate exon usage in Mef2Da2 renders the protein resistant to PKA phosphorylation, allowing it to interact with transcriptionally permissive Ash2L-trithorax complex. Our findings support a model wherein alternative exon usage allows Mef2D to transition from a repressor to activator in a myogenic environment rich in PKA activity. Thus we have identified a novel paradigm in which a ubiquitously expressed transcription factor has evolved to undergo tissue-specific alternative exon usage to permit the proper temporal activation of a gene expression program during differentiation. ChIP-Seq profiling of Mef2Da1 and Mef2Da2 isoforms generated by alternate splicing

ORGANISM(S): Mus musculus

SUBMITTER: Zizhen yao

PROVIDER: E-GEOD-43223 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Human umbilical vein endothelial cells (HUVECs) are a widely-used model system to study many pathological and physiological processes associated with the cardiovascular system. An understanding of genes and proteins that are expressed in any cell type is a fundamental need which facilitates study of molecular changes in disease states and response to various stimuli. In this study, we employed next generation sequencing and mass spectrometry to profile the transcriptome and proteome of primary HUVECs. Analysis of 145 million paired-end reads from next generation sequencing confirmed expression of 12,186 protein-coding genes (FPKM>0.1), 439 novel long non-coding RNAs and revealed 6,089 novel isoforms that were not annotated in GENCODE. A comparison of the transcripts against human gene expression data for 53 tissues catalogued by the Genotype-Tissue Expression (GTEx) project revealed a number of HUVEC-specific genes. Proteomics analysis identified 6,477 proteins including confirmation of N-termini for 1,091 proteins and isoforms for 149 proteins for which transcriptomic evidence was also observed. Alternate translational start sites for seven proteins and alternate splicing in seven proteins were also identified. A database search to specifically identify other post-translational modifications provided evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, mono, di- and tri-methylation. Based on the data from this study and a survey of other databases, we provide evidence for 11 “missing proteins,” which are proteins for which protein level evidence is lacking or insufficient. Proteogenomic analysis identified novel N-termini and exon-exon junctions at both RNA and protein levels by searching against a translated database created from alternate transcript assembly. Peptides supporting the novel events of expressed proteins are further validated by synthesis and spectral comparison. Alternative allelic expression of genes arising from transcription of two alleles of a gene in chromosome pairs is observed. By creating a custom database of proteins incorporated with sample specific single amino acid variations (SAAV) we identified alternative alleles for 207 expressed proteins with 245 peptides with SAAV. Overall, we believe that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.

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Alternate exon switching establishes a tissue-specific transcription factor to mediate temporal activation of gene expression during differentiation

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