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.
Highlights
The rapid cellular response that occurs upon recognition of biological or environmental signals is crucial for adaptation and survival of the organism [1,2,3]
Successful chromatin fragmentation was assayed by reverse cross-linking and electrophoresis through 0.8% agarose gel to confirm enrichment of small chromatin fragments at the target range. 15% of chromatin fragments were designated as input, and the rest were pre-cleared with untreated protein A/G agarose beads (Santa Cruz), incubated in protein A/G agarose beads that were pre-conjugated overnight at 4 ̊C with monoclonal antibodies to NF90/NF110 (BD mAB DRBP76), NF45 (Santa Cruz sc-365283), or the large subunit of RNA polymerease II (8WG16; Covance)
Increased NF90/NF110 chromatin occupancy occurred at active promoters and strong enhancers, and NF90/NF110 clustered with transcription factors exhibiting preference for promoters over enhancers (POLR2A, MYC, YY1)
Summary
The rapid cellular response that occurs upon recognition of biological or environmental signals is crucial for adaptation and survival of the organism [1,2,3]. The subset of genes that are rapidly expressed upon induction are termed immediate early genes (IEG) [4]. Immediate Early Gene Regulation by NF45/ILF2 and NF90/NF110/ILF3 expression of IEGs in response to diverse regulatory signals underlies acute inflammation [5,6,7,8], neuronal activity [9], cell proliferation, and differentiation [1, 10, 11]. The earliest protein products of these IEGs critically include ‘forward-driving’ transcription factors such as EGR1, FOS, and JUN, followed by expression of delayed primary response genes (D-PRG), followed by protein synthesis-dependent expression of secondary response genes (SRG) [15]. The AP-1 components FOS and JUN regulate transcription of many cytokine genes and acute inflammation. The intensity and duration of signaling is attenuated through IEG induction of the family of dual-specificity phosphatases/ MAPK phosphatases [16]
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