HP1gamma Knock Down in Human cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE25282 | GEO | 2011/02/27
SECONDARY ACCESSION(S): PRJNA134825
REPOSITORIES: GEO
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Project description:Study of HP1 Knock Down on gene expression and splicing regulation in Human HeLa cells Maturation of pre-mRNAs is initiated co-transcriptionally. It is therefore conceivable that chromatin-borne information participates in alternative splicing. Here, we find that elevated levels of tri-methylation of histone H3 on lysine 9 (H3K9me3) are a characteristic of the alternative exons of several genes including CD44. On this gene, the chromodomain protein HP1gamma, frequently defined as a transcriptional repressor, facilitates inclusion of the alternative exons via a mechanism involving decreased RNA polymerase II elongation rate. In addition, accumulation of HP1gamma on the variant region of the CD44 gene stabilizes association of the pre-mRNA with the chromatin. Altogether, our data provide evidence for localized histone modifications impacting alternative splicing. They further implicate HP1gamma as a possible bridging molecule between the chromatin and the maturating mRNA, with a general impact on splicing decisions. Transcriptome analysis of siRNA anti-HP1gamma transfected HeLa cells on GeneChip® Human Exon 1.0 ST Arrays (Affymetrix). Control HeLa cells have been transfected with the same concentration of siRNA anti-GAPDH. Experiment has been done in experimental triplicates.
2011-02-27 | E-GEOD-25282 | biostudies-arrayexpress
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Project description:While Argonaute (AGO) proteins play a major role in transcriptional gene silencing (TGS) in many organisms, their role in the nucleus of somatic mammalian cells remains elusive. Here, we have purified AGO1 and AGO2 chromatin-embedded complexes, and found these proteins associated with previously described partners, but also with chromatin modifiers and, rather unexpectedly, with different splicing factors. Using the CD44 gene as a model for alternative splicing, we show that both AGO1 and AGO2 are required for Protein Kinase C (PKC)-dependent variant exon inclusion. AGO proteins facilitate the spliceosome recruitment and modulate the elongation rate of RNA polymerase II (RNAPII). The recruitment of AGO proteins to CD44 transcribed region is dependent on both the endonuclease Dicer and the chromodomain-containing protein HP1g, and results in locally increased levels of histone H3 lysine 9 (H3K9) methylation on variant exons. Genome wide analysis of splicing in either AGO2 or Dicer null cells showed that the two proteins have similar effects on many splicing events. Finally, sRNAs associated with nuclear AGO2 are mostly in sense orientation relative to protein-coding genes, supporting a role for intragenic antisense non-coding RNAs in the recruitment AGO and splicing factors. Together, our data demonstrate for the first time that the endogenous RNAi pathway is involved in alternative splicing decisions, unravelling a new model in which AGO proteins couple RNAPII elongation and chromatin modification. Study of AGO2 or Dicer knock-out on gene expression and splicing regulation in MEF cells Transcriptome analysis of AGO2 and Dicer null MEF cells on GeneChipM-BM-. Mouse Exon 1.0 ST Arrays (Affymetrix). Dicer null MEF cells and wild-type MEF cells were from M. Otsuka. AGO2 null MEF cells were from A. Tarakhovsky. Experiment has been done in experimental triplicates. 9 Total samples were analyzed.
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