Changes in DNA methylation In cultered WI-38 cells over passages
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Amir Eden
PROVIDER: E-GEOD-30640 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In humans, there are four Ago proteins (Ago1–4) and AGO1- and 2 were previously implicated in TGS induced by exogenous siRNAs and microRNAs (miRs) directed against gene promoter transcripts via promotion of changes in histone covalent modifications and DNA methylation. Not-with-standing, many mechanistic details of this process remain poorly defined in human cells, and very little is known about the identity of possible endogenous signals, which may drive this process in human cells. Given the evolutionary conserved role of siRNAs and AGO proteins in TGS and heterochromatin formation, we set out to analyse their possible involvement in senesence-associated repression of E2F target genes. To determine, in an unbiased manner, which genes might be under the control of AGO proteins we perfomed genome-wide promoter profiling in senescent and presenescent control WI38 primary fibroblasts applying “ChIP-on-chip” technology using 4 an anti-pan-AGO antibody. DNA from matched genomic inputs and chromatin immunoprecipitation (ChIP) samples were purified, amplified by PCR, and labeled either with Cy3 or Cy5 fluorophores. The labeled DNA from input and ChIP fractions were then combined and hybridised to human promoter arrays containing ~59,000 promoters. determination of AGO binding sites in WI-38 pre-senescent human fibroblast and in senescent human fibroblast
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Project description:In humans, there are four Ago proteins (Ago1–4) and AGO1- and 2 were previously implicated in TGS induced by exogenous siRNAs and microRNAs (miRs) directed against gene promoter transcripts via promotion of changes in histone covalent modifications and DNA methylation. Not-with-standing, many mechanistic details of this process remain poorly defined in human cells, and very little is known about the identity of possible endogenous signals, which may drive this process in human cells. Given the evolutionary conserved role of siRNAs and AGO proteins in TGS and heterochromatin formation, we set out to analyse their possible involvement in senesence-associated repression of E2F target genes. To obtain a detailed picture of AGO-immunoprecipitating miRs (RIP) in senescent cells, we used next-generation sequencing (NGS)(RIP-Seq). We also included histone H3 dimethylated on lysine 9 (H3K9me2) in this analysis to assign potential AGO2-interacting miRs to a repressive chromatin state and unfractionated, cellular RNA from senescent cells for normalisation.
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