1. An Intronic Enhancer Driven by NF-κB Contributes to Transcriptional Regulation of Peptidylarginine Deiminase Type I Gene in Human Keratinocytes 
Shibo Ying, Toshio Kojima, Akira Kawada, Rachida Nachat, Guy Serre, Michel Simon, Hidenari Takahara 
Journal of Investigative Dermatology 
Volume 130, Issue 11, Pages (November 2010)
DOI: /jid
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2. Figure 1
CNSi enhances the activity of the PADI1 minimal promoter in cultured keratinocytes (KCs). Normal human epidermal keratinocytes (NHEKs) cultured in 1.5mM (black bars) or 0.2mM (hatched bars) calcium-containing medium were cotransfected with firefly luciferase (luc) reporter plasmids containing the entire CNSi or fragments of CNSi upstream of either the PADI1 minimal promoter or the SV40 promoter as indicated. Luciferase activities were measured 48hours after transfections. Luciferase activities are expressed as fold-increase over promoterless vector, pGBasic (set as 1). (a) CNSi and some fragments of CNSi enhance the activity of PADI1 core promoter in cultured KCs. (b) Orientation-independent effects of the CNSi fragment 2 (nucleotides 310–576). Values were normalized for transfection efficiency by cotransfection with the Renilla expression plasmid, and are expressed as mean ± SD from four separate experiments. **P<0.01 versus the difference between luciferase activity of reporter plasmids containing PADI1 core promoter (or SV40 promoter) alone and others in each condition. (Student's t-test).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
An intronic NF-κB cis-element enhances the activity of the PADI1 minimal promoter. (a) Sequence and putative transcription factor-binding sites of the CNSi fragment 2 (nucleotides 310–576) in human PADI1 and mouse PADI1 genes. Putative transcription factor-binding sites predicted using the BIOBASE P-Match-public v1.0 ( are underlined. (b) Characterization of the transcription factor-binding sites in the PADI1 CNSi fragment 2 by site-directed mutagenesis. Shown is the schematic diagram of serial mutation constructs and their luciferase activities in normal human epidermal keratinocyte (NHEKs) cultured in high-calcium-containing medium. The targeted putative transcription factor-binding sites are shown on the constructs with a solid cross. Values were corrected for transfection efficiency by cotransfection with the Renilla expression plasmid, and are expressed as mean ± SD from four separate experiments. *P<0.05, significantly different from wild type (WT) (Student's t-test). (c) Site-directed mutagenesis of putative NF-κB-binding sites and its inhibitive effect on the binding shown by the NoShift transcription factor assay. Nuclear extracts were harvested from NHEKs cultured in high-calcium-containing medium. Binding activity assessed by biotinylated NF-κB wild-type or mutant NF-κB consensus-binding motif double-stranded DNA (Mut) probes plus their nonbiontinylated competitor DNA in a 50-fold molar excess. A positive control with no competitor DNA was also included (lanes 1 and 4). Negative control (Neg) consisted of reactions carried out in the absence of a binding sequence. All assays were carried out in triplicate.
Journal of Investigative Dermatology  , DOI: ( /jid )
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4. Figure 3
p50 and p65 bind to the intronic NF-κB cis-element of PADI1 in vivo. Chromatin immunoprecipitation (ChIP) assays using anti-p50, anti-p65, or IgG control were carried out on chromatin from normal human epidermal keratinocytes (NHEKs) cultured in low- (L) or high- (H) calcium-containing medium. (a) The positions of the ChIP primer sets are indicated above the schematic organization of the PADI1 gene, including the core promoter with transcription factor-binding sites (Dong et al., 2008) and the putative NF-κB binding site identified in the CNSi. (b) Binding of p50 and p65 was detected by gel staining after PCR amplifications using primers corresponding to the PADI1 core-promoter region (upper panel), CNSi (medium panel), and PADI1 exon 2 (lower panel, negative control). (c) For quantitative analyses of p50 and p65 binding to the CNSi, as well as core promoter, the samples from NHEKs cultured in low- (hatched bars) and high- (black bars) calcium-containing medium were analyzed using real-time PCR. The relative DNA levels were calculated as described in the Materials and Methods section.
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Expression of NF-κB is crucial for PADI1 endogenous expression in normal human epidermal keratinocytes (NHEKs). NHEKs were transfected with 100nM of the indicated siRNA and cultured for 36h, or stimulated by tumor necrosis factor (TNF)-α (10ngml−1) for 2hours. Nuclear proteins, total RNA, and cell lysates were extracted for western blotting (a) with the indicated antibodies (anti-NF-κB p50, anti-NF-κB p65, anti-PAD1, or anti-Histone H1), quantitative reverse-transcription PCR analysis (b), and luciferase (luc) reporter analysis (c), respectively. (a) Inhibition of expression of NF-κB p50 and p65 with their respective specific siRNA, including combined si-p50/p65, was confirmed by western blotting, as compared with si-Control. Activation of expression of NF-κB p50 and p65 stimulated by TNF-α was also confirmed by western blotting, as compared with mock. Histone H1 served as a loading control. Similar results were obtained with low- or high-calcium treatments. (b) Effects of NF-κB p50 and p65 on endogenous PADI1 expression. PADI1 expression levels in NHEKs cultured in low (hatched bars) and high (black bars) calcium-containing medium were normalized to the levels of expression of the internal control glyceraldehyde-3-phosphate dehydrogenase, and expressed relative to the nontreated NHEKs cultured in low-calcium-containing medium. Results are mean ±SD from four experiments. *P<0.05, **P<0.01 versus si-Control group (Student's t-test). (c) Relative luciferase activity in NHEKs transfected with the indicated siRNA and cotransfected with the reporter gene vector CNSi/Fragment2. NHEKs cultured in low (hatched bars) and high (black bars) calcium-containing medium. Results are mean ±SD from four experiments. *P<0.05, **P<0.01 versus si-Control group (Student's t-test).
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Interaction between the promoter and CNSi of human PADI1 gene indicating intrachromatin looping. (a) The chimeric DNAs containing CNSi and the promoter region of PADI1 were explored by chromosome conformation capture analysis using specific primers. The positions of the two BglII restriction sites (perpendicular lines) are indicated along the 2kb region between the promoter and CNSi of the human PADI1 gene. Arrows represent the primers used in PCR. Exon 1 and 2 are shown as boxes. F and R mean forward and reverse primer, respectively. (b) High potential interaction between the promoter and CNSi indicating chromatin looping. Chromatin from cross-linked normal human epidermal keratinocytes (NHEKs) was digested with excessive amounts of BglII restriction enzyme and diluted for intramolecular ligation. Purified DNA fragments were assayed by conventional PCR. NHEKs were mock treated (lanes 2 and 5), transfected by combined p50/p65-siRNA (lanes 3 and 6), or stimulated for 2hours by tumor necrosis factor (TNF)-α (10ngml−1; lanes 4 and 7). Random ligation control templates were generated by amplifying the human genomic DNA fragment with primers that span the BglII sites. Equimolar amounts of PCR products were mixed and digested with BglII overnight. Ligated DNA was used to generate control PCR products by using a combination of the primer pairs to monitor the efficiency of ligation (lane 1). BglII-digested chromosomal DNA was used as a template for PCR to examine the efficiency of BglII digestion (lanes 2, 3, and 4). Ligated templates after dilution were used to PCR amplify after BglII digestion and ligation (lanes 5, 6, and 7). Input DNA was used as an internal control for PCR on CNSi. All PCR products were examined on 2% agarose gel stained with ethidium bromide and confirmed by sequencing as indicated in Supplementary Figure S2. All primers used are listed in Supplementary Table S1. See Materials and Methods section for additional information. (c) Relative efficiency of looping from mock-transfected, siRNA-transfected, and TNF-α-treated cells. The PCR products (lanes 5, 6, and 7) were quantified after scanning using the “Quantity One” software package (version 4.2; Bio-Rad). The values are expressed relative to that obtained from mock-transfected keratinocytes. They correspond to the averages from three experiments with standard deviations.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Hypothetical model of the transcriptional regulation of human PADI1 gene by cross talk between its promoter and an NF-κB-mediated signaling pathway in normal human epidermal keratinocytes (NHEKs). This study shows that both p65 and p50 bind to a cis-element in a conserved noncoding sequence of the first intron of the PADI1 gene (CNSi), and interact with the transcription complex on the gene core promoter in the upper domain. It suggests that this interaction contributes to the control of PADI1 transcription in human keratinocytes. The lower domain of the schematic representation denotes PADI3 transcription regulation through a long-range chromatin loop and a long-distance enhancer, as reported previously (Adoue et al., 2008; Chavanas et al., 2008). Please note that the distance between genes and the length of PADI1 are approximations. Please refer to text for details. TBP, TATA-box binding protein; pol II, RNA polymerase II complex.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions