Silencing p300 in MCF7 cells to study expression and alternative splicing
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ABSTRACT:
PROVIDER: PRJNA432418 | ENA |
REPOSITORIES: ENA
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Project description:Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing (AS) regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of AS. P300 promotes genome-wide exon inclusion in both a transcription-dependent and ‑independent manner. Using CD44 as a paradigm, we found that p300 regulates AS by modulating the binding of splicing factors to pre-mRNA. Employing a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates AS by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. P300-mediated CD44 AS reduces cell motility and promotes epithelial features. Our findings reveal a mechanism through which chromatin-related proteins regulate AS and show the impact of this mechanism on cell function.
2019-10-31 | GSE109957 | GEO
Project description:Silencing KDM3A in MCF7 cells to study expression and alternative splicing
| PRJNA595373 | ENA
Project description:This SuperSeries is composed of the following subset Series: GSE39746: Argonaute proteins couple chromatin silencing to alternative splicing (exon array) GSE39748: Argonaute proteins couple chromatin silencing to alternative splicing (RNA IP-Seq) Refer to individual Series
2012-08-22 | E-GEOD-39749 | biostudies-arrayexpress
Project description:Alternative splicing analysis was performed in MCF7
2016-10-01 | E-MTAB-5104 | biostudies-arrayexpress
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. Deep sequencing of small RNAs (approx. 15-80 nucleotides) bound to either cytoplasmic or chromatin-associated AGO2 complex.
2012-09-10 | E-GEOD-39748 | biostudies-arrayexpress
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.
2012-09-10 | E-GEOD-39746 | biostudies-arrayexpress
Project description:MCF7 cells were transfected with non-targeting siRNA (siNT) and siKDM3A for 72 h. RNA was extracted, libraries prepared and RNA-seq was conducted.
2020-02-26 | GSE141948 | GEO
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
2012-09-10 | GSE39746 | GEO
Project description:We used splicing-sensitive microarrays to detect differences in alternative splicing between two breast cancer cell lines MCF7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative), as well as cultured human mammary epithelial cells (HMEC). Several splicing alterations in genes, including CD44, FAS, RBM9, HnRNPA/B, APLP2, and MYL6, were detected by the microarray, and verified by RT-PCR. We also compared splicing in these breast cancer cells cultured in either two-dimensional flat dishes (2-D) or in three-dimensional Matrigel (3-D) conditions. Only a subset of the splicing differences that distinguish MCF7 cells from MDA-MB-231 cells under 2-D culture condition is retained under 3-D conditions, suggesting that alternative splicing events are influenced by the geometry of the culture conditions of these cells. Keywords: Splicing-sensitive microarray
2005-12-30 | GSE3042 | GEO
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.
2012-09-10 | GSE39748 | GEO