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Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells
Published on: July 29, 2010
Inducible expression of immediate early genes is regulated through dynamic chromatin association by NF45/ILF2 and
Ting-Hsuan Wu1,2, Lingfang Shi1, Anson W Lowe3
1Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
Abstract:
Immediate early gene (IEG) transcription is rapidly activated by diverse stimuli. This transcriptional regulation is assumed to involve constitutively expressed nuclear factors that are targets of signaling cascades initiated at the cell membrane. NF45 (encoded by ILF2) and its heterodimeric partner NF90/NF110 (encoded by ILF3) are chromatin-interacting proteins that are constitutively expressed and localized predominantly in the nucleus. Previously, NF90/NF110 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in K562 erythroleukemia cells revealed its enriched association with chromatin at active promoters and strong enhancers. NF90/NF110 specifically occupied the promoters of IEGs. Here, ChIP in serum-starved HEK293 cells demonstrated that NF45 and NF90/NF110 pre-exist and specifically occupy the promoters of IEG transcription factors EGR1, FOS and JUN. Cellular stimulation with phorbol myristyl acetate increased NF90/NF110 chromatin association, while decreasing NF45 chromatin association at promoters of EGR1, FOS and JUN. In HEK293 cells stably transfected with doxycycline-inducible shRNA vectors targeting NF90/NF110 or NF45, doxycycline-mediated knockdown of NF90/NF110 or NF45 attenuated the inducible expression of EGR1, FOS, and JUN at the levels of transcription, RNA and protein. Dynamic chromatin association of NF45 and NF90/NF110 at IEG promoters are observed upon stimulation, and NF45 and NF90/NF110 contribute to inducible transcription of IEGs. NF45 and NF90/NF110 operate as chromatin regulators of the immediate early response.
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