Sequences of oligonucleotides and primers used.

<p>Sequences of oligonucleotides and primers used.</p

Schematic model depicting the potential mechanism that might contribute to regulation of the <i>CFTR</i> gene.

<p>In this model, coloured bubbles correspond to the transcription factors characterized in this study. Double arrow shows the functional antagonism between C/EBPβ and YY1. The upper representation corresponds to the transcriptional activation of the <i>CFTR</i> gene when C/EBPβ is phosphorylated and the lower representation corresponds to the decrease of the transcription following overexpression of a C/EBPβ form not phosphorylatable on its 235T residue.</p

NAF treatment stimulates the <i>CFTR</i> activity induced by C/EBPβ.

<p>(A) Left panel: When indicated, Beas2B cells were incubated in the presence of NAF (left panel) at the indicated concentrations. Immunoblots showing either CFTR or LaminA/C expression are represented below the graph. Right panel: Endogenous mRNA level of either <i>CFTR</i> or <i>C/EBPβ</i> following NAF incorporation at indicated concentrations. (B) Upper panel: Combinatorial effect of C/EBPβ and NAF treatment. Cells were transfected either with C/EBPβ plasmid after NAF treatment (left panel) or with LAPT235A expression vector (right panel). Middle panel: Representative immunoblots are shown below the graphs. Lower panel: Endogenous <i>CFTR</i> mRNA level. *P<0.05.</p

YY1 antagonizes the positive effect of C/EBP-LAP through mutually exclusive DNA binding.

<p>(A) Beas2B cells were cotransfected with a fixed amount of both <i>CFTR</i> (0.072 µg) reporter and C/EBPβ (0.04 µg) expression vectors and increasing amount of plasmid encoding YY1 (0.004 to 0.08 µg) as indicated (left panel). Endogenous <i>CFTR</i> mRNA level following C/EBPβ (0.04 µg) expression vectors and increasing amounts of plasmid encoding YY1 (0.004 and 0.08 µg) (right panel). *P<0.05. (B) Left panel: ChIP experiment was performed on cells transfected or not with either C/EBPβ plasmid or respective siRNA. Protein extracts were immunoprecipitated IP with the indicated antibody. Input (IN) corresponds to total lysate used as a control for PCR amplification of total DNA. CFTR, represents <i>CFTR</i> promoter amplification and negative control, ChIP analysis of <i>CFTR</i> sequence which lacks the YY1 binding motif. Right panel: DNA from immunoprecipitates and input DNA (which represents 5% of total chromatin) were analyzed by quantitative PCR using primers amplifying the minimal <i>CFTR</i> promoter. Data are defined as fold enrichment of DNA associated with indicated immunoprecipitated antibody relative to input chromatin and specific binding was expressed as a function of non-sepcific (NS) antibody binding set as 1. (C) Functional interplay between C/EBPβ and YY1. Mutually exclusive DNA-binding activity of YY1 and C/EBPβ at the C/EBPb3 binding site. The labelled b3WT probe was incubated with C/EBPβ-transfected Beas2B nuclear extracts in the presence of increasing amounts of purified C/EBPβ. Arrows indicate the position of C/EBPβ and YY1.</p

Phosphorylation of C/EBPβ affects the YY1 DNA occupancy and favours YY1/USF2 interaction.

<p>(A) Left panel: ChIP experiments were performed using NAF-treated Beas2B cells when indicated. Protein extracts were immunoprecipitated IP with the indicated antibodies. Input (IN), corresponds to total lysate used as a control for PCR amplification of total DNA and served to normalize <i>CFTR</i> amplification as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060211#s2" target="_blank">Materials and Methods</a> Section. CFTR, represents <i>CFTR</i> promoter amplification and negative control, ChIP analysis of <i>CFTR</i> sequence which lacks both C/EBPβ and YY1 binding motifs. Right panel: DNA from immunoprecipitates and input DNA were analyzed by quantitative PCR. Data are defined as fold enrichment relative to input chromatin and specific binding was expressed as a function of non-specific (NS) antibody (anti-HA) binding set as 1. (B) Beas2B cells were treated when indicated with NAF and protein extracts were immunoprecipitated with either an anti-YY1 (lanes 2 and 3) or an irrelevant antibody (lane 4). Immunoprecipitated proteins were then analyzed by western blotting using either an anti-USF2a or an anti-YY1 antibody. Lane 1 corresponds to whole cell extracts used for IP.</p

C/EBPβ activates <i>CFTR</i> promoter activity.

<p>(A) Assessment of C/EBPβ binding to the minimal <i>CFTR</i> promoter by ChIP analysis using quantitative PCR. Cells were transfected or not with C/EBPβ plasmid as indicated. Extracts were immunoprecipitated IP with either an anti-C/EBPβ or a non-specific antibody (HA also denoted NS). DNA from immunoprecipitates and input DNA (which represents 5% of total chromatin) were analyzed by quantitative PCR using primers amplifying the minimal <i>CFTR</i> promoter. Input (IN) corresponds to the amplification of total DNA and serves to normalize <i>CFTR</i> amplification as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060211#s2" target="_blank">Materials and Methods</a> section. Data are expressed as fold enrichment of DNA associated with the indicated immunoprecipitated antibody relative to a 1/20 dilution of IN and specific binding was determined by subtracting binding with NS antibody. The asterisk (*) indicates that the value is statistically significant (<i>p</i><0.05) (B) The <i>CFTR</i> promoter construct (−226 to +135) in the Luc reporter vector was co-transfected with 0.04 µg of C/EBPβ LAP or LIP, or A-CEBP expression vectors. The position of the C/EBPβ isoforms LAP (35 kDa and 38 kDa) and LIP (20 kDa) are indicated on immunoblots. Flag antibody was used for revealing A-CEBP form over-expression. (C) The <i>CFTR</i> promoter construct was co-transfected with either a non-specific (NS) siRNA or specific C/EBPβ siRNA. Densitometric Analysis (DA) was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060211#s2" target="_blank">Materials and Methods</a> section. (D) Effect of C/EBPβ on <i>CFTR</i> mRNA level in Beas2B epithelial cell lines. Cells were transfected with the different forms of C/EBPβ (left panel) and the C/EBPβ-specific siRNA (right panel<b>)</b>. The mRNA expression level following transfection of either an empty vector or a control siRNA was then set as 1. The asterisk reflects the statistical significance set at P<0.05.</p

Two-loci haplotypes with a frequency higher than 3% (2% for HLA-A); obs: estimated frequency; exp: expected frequency; stdres: standardized residual–in grey if significant, i.e. corresponding to an absolute value above 2.

<p>Two-loci haplotypes with a frequency higher than 3% (2% for HLA-A); obs: estimated frequency; exp: expected frequency; stdres: standardized residual–in grey if significant, i.e. corresponding to an absolute value above 2.</p

HLA-A~HLA-H~ HLA-G UTRs haplotypes with a frequency greater than 1%.

<p>HLA-A~HLA-H~ HLA-G UTRs haplotypes with a frequency greater than 1%.</p

HLA-E, -H, -G, -F and HFE relative location and distance from HLA-A on chromosome 6.

<p>HLA-E, -H, -G, -F and HFE relative location and distance from HLA-A on chromosome 6.</p

HLA-A, HLA-G UTRs, -G, -E, -H and -F allelic frequencies in 191 voluntary blood donors from southeastern France.

<p>HLA-A, HLA-G UTRs, -G, -E, -H and -F allelic frequencies in 191 voluntary blood donors from southeastern France.</p