The −1.9 kb element is a NF-κB-dependent LPS inducible promoter and enhancer in transient transfection.

<p>HD11 macrophages were transfected for 18 hrs with Jetpei 2 µl and 1 µg DNA prior to 7 hrs 1 µg/ml LPS stimulation, black bars, or remained untreated for 7 hrs, grey bars. The constructs are illustrated adjacent to the y-axis. (<b>a</b>) <i>Clys</i> promoter is a black arrow, the −1.9 kb CRE is a dark grey arrow, the firefly luciferase coding region is grey. (<b>b</b>) The −1.9 kb element is cloned in antisense orientation, Position of the NF-κB, C/EBP, X unknown protein and AP1 are indicated next to the Y axis. (<b>a</b> and <b>b</b>) The data are plotted as the mean value of two independent experiments, individual experiments had triplicate samples for each condition. Inter sample variation has been corrected by Renilla normalisation. Positive error bars indicate standard deviations.</p

LPS induces recruitment of NF-κB, IKKα, RNAPII and HP1γ with different kinetics.

<p>(<b>a–d</b>) ChIP performed with primary macrophages treated with LPS for the indicated time points in minutes and the following antibodies (<b>a</b>) anti-p65 (NF-κB), (<b>b</b>) anti-RNAPII CTD, (<b>c</b>) anti-IKKα and (<b>d</b>) anti-HP1γ. Horizontal axis indicates primers used for the Real time PCR (distance in kb from the transcription start site of <i>cLys</i>). Data are normalized versus input. Error bars represent SD from three independent qPCR replicates. These data are representative of at least three independent experiments.</p

Rapid nucleosome loss at the−1.9 kb CRE in macrophages after LPS treatment.

<p>(<b>a</b>) Illustration of the chicken lysozyme locus including cis-regulatory elements and the approximate location of LINoCR transcript, grey arrow and <i>cLys</i> mRNA, black arrow <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059389#pone.0059389-Lefevre1" target="_blank">[7]</a>. (<b>b</b>) ChIP assay performed with anti-histone H3 antibody at the −1.9 kb element compared to the −6.1 kb enhancer after LPS treatment in macrophages. Horizontal axis represents time after LPS induction in minutes. Data are normalised versus input and then versus a positive control region. Data are representative of at least three independent experiments.</p

A NF-κB-Dependent Dual Promoter-Enhancer Initiates the Lipopolysaccharide-Mediated Transcriptional Activation of the Chicken Lysozyme in Macrophages

<div><p>The transcriptional activation of the chicken lysozyme gene (c<i>Lys</i>) 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 <i>cLys</i> and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating <i>cLys</i> transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both <i>cLys</i> transcription and NF-κB binding to the CRE. Finally, comparing <i>cLys</i> 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.</p> </div

<i>In vivo</i> MNase and DNase I mapping of the −1.9 kb element reveal chromatin remodelling in response to LPS.

<p>Southern blot of (<b>a</b>) MNase or (<b>b</b>) DNAse I digested genomic DNA from permeabilised nuclei of unstimulated HD11 (lane 1 and 5) or HD11 LPS (5 µg/ml) stimulated (<b>a</b>) for 30 min, 60 min and 240 min (lanes 2–4) or (<b>b</b>) for 45 min, 120 min and 360 min (lanes 6–8). Inferred nucleosome positions are illustrated in grey. Width of arrows indicates the degree of cleavage and the approximate position relative to the <i>clys</i> transcription start site. These experiments are representative of two independent experiments. Quantification of (<b>c</b>) the MNase, LPS stimulated samples are normalised to unstimulated HD11 and (<b>d</b>) the DNAse I southern blots. Arrows are indicating approximate position of cleavages. DNAse I protection domain within the −1.9 Kb element is illustrated by a black box.</p

RNAPII S2p and IKKα occupancies at <i>cLys</i> locus overlap.

<p>(<b>a–c</b>) ChIP performed with primary macrophages treated with LPS for 30 min and the following antibodies (<b>a</b>) anti-p65 (NF-κB) and anti-IKKα, (<b>b</b>) anti-RNAPII S2p and anti-IKKα (<b>a–b</b>) Position of the −1.9 kb element is indicated by an arrow. Horizontal axis indicates distance from <i>cLys</i> transcription start site. Data are normalized versus input and then versus the average of all IP/Input values. These data are representative of at least three independent experiments.</p

<i>In vivo</i> DMS footprinting of the −2000 to −2150 bp region, part of the −1.9 kb CRE, reveals transcription factor occupancy in response to LPS stimulation.

<p>HD11 cells were, in order from left to right, either unstimulated or LPS (1 µg/ml) stimulated for 30 min, 60 min or 240 min. Cells were then treated with DMS before the isolation of genomic DNA for hot piperidine cleavage and LM-PCR analysis. The HD37 erythroid cell line which do not express <i>clys</i> and the naked HD11 genomic DNA, G reaction, reference sequence are also shown. (a) non-coding strand and (b) coding strand. The filled circles represent DMS hyper-methylation and the open circles base protection from DMS. The positions of the selected G bases are indicated relative to the <i>cLys</i> transcription start site. The potential transcription factors are indicated adjacent to a single line encompassing their proposed binding site. LINoCR transcription start site is indicated with an arrow. These images are representative of the result of two independent experiments.</p