Abstract
The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-κB, AP1 and C/EBPβ controls this CRE, which is strictly dependent on NF-κB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKα, known to be recruited by NF-κB to NF-κB-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-κB binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKKα detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3′ end of the gene.
Highlights
Genes transcription is controlled by cisregulatory element (CRE), which are, when activated, nucleosome free regions occupied by transcription factors and identified in vivo as DNAse I hypersensitives sites (DHS) [1]
We have previously established that the eviction of the insulatorassociated protein CCCTC-binding factor (CTCF) from its binding site and subsequent nucleosome movement over this site was dependent on transcription of LPS-Inducible Non-Coding RNA (LINoCR) at the chicken lysozyme gene (cLys) locus in activated macrophages [7]
LINoCR is firing from a CRE located 21.9 kb upstream of cLys transcription start site (TSS), this element being previously described as a hormone response enhancer in the oviduct [9,10]
Summary
Genes transcription is controlled by CREs, which are, when activated, nucleosome free regions occupied by transcription factors and identified in vivo as DNAse I hypersensitives sites (DHS) [1]. We have reported that cLys expression activation was preceded by the transient transcription of LINoCR from a promoter 21.9 kb upstream of cLys TSS, this transcription being necessary for nucleosome reorganisation and eviction of the enhancer blocker protein CTCF from the silencer element [7]. This promoter was originally identified as a hormone response enhancer element functional in the oviduct and bound by estrogen, glucocorticoid and progesterone receptors [9,10]. We did not fully establish that this 21.9 kb element was driving any enhancer activity in macrophages
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