Characterization of NF-κB as a regulatory element in distal part of promoter.

<p><b>A)</b> Luciferase reporter gene assay for the effect of NF-κB and Twist-1 on the activity of <i>KIAA1199</i> promoter: Lysates of COS-1 cells transfected with cDNAs as indicated were examined by Dual Luciferase activity assay. Wild type and substitute mutation (pro-1.4 kb swap NF-κB^4th) at distal region of NF-κB binding site were used. <b>B)</b> ChIP assay for identification of the interaction between NF-κB (p65) and <i>KIAA1199</i> promoter. Top panel: A schematic diagram of four putative NF-κB binding sites relative to +1 site. Primer sets A, B, and C were designed for NF-κB binding site IV and I + II, and an area of the first <i>KIAA1199</i> intron, respectively. Middle and low panels: ChIP PCR in HeLa cells transfected with GFP control and P65 cDNAs. Anti-p-65 antibody and normal IgG (control) were used for immunoprecipitation. Results were calculated according to the bound/input ratio.</p

Elevated expression of <i>KIAA1199</i> in human cancers.

<p>A) By mining Oncomine and GEO databases, <i>KIAA1199</i> expression pattern in more than 40 microarray data sets shows significant alteration (P<0.01). Representative data are presented. High <i>KIAA1199</i> expression in various human cancers.1-breast cancer n:27, normal n:7, p = 6.97E-4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Radvanyi1" target="_blank">[41]</a>; 2-colon cancer n:36, normal n:24, p = 1.7E-4<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Skrzypczak1" target="_blank">[42]</a>; 3-gastric cancer n:26, normal n:31, p = 3.69E-13<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-DErrico1" target="_blank">[43]</a>; 4-lung cancer n:45, normal n:65, p = 8.59E-9<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Hou1" target="_blank">[44]</a>; 5-head&neck cancer n:41, normal n:13, p = 2.35E-7<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Ginos1" target="_blank">[45]</a>; 6-ovarian cancer n:6, normal n:4, p = 5.92E-4<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Adib1" target="_blank">[46]</a>; 7-pancreatic cancer n:11, normal n:11, p = 0.001 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone.0044661-Grutzmann1" target="_blank">[47]</a>. <b>B)</b> Expression of <i>KIAA1199</i> in human cancer cell lines: Human prostate (LNCaP and Du145), and breast cancer (MCF-7 and MDA-MB-231) cell lines were examined by real time RT-PCR using <i>KIAA1199</i> specific primers. The expression of <i>KIAA1199</i> was normalized by house-keeping genes (HPRT-1 and GAPDH). The relative levels of genes were determined using the ΔΔCt method. Each bar represents the mean ± S.E (*<0.05).</p

Transcriptional and Epigenetic Regulation of <em>KIAA1199</em> Gene Expression in Human Breast Cancer

<div><p>Emerging evidence has demonstrated that upregulated expression of <em>KIAA1199</em> in human cancer bodes for poor survival. The regulatory mechanism controlling <em>KIAA1199</em> expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human <em>KIAA1199</em> promoter in terms of regulation of <em>KIAA1199</em> gene expression. A 3.3 kb fragment of human genomic DNA containing the 5′-flanking sequence of the <em>KIAA1199</em> gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic <em>KIAA1199</em> promoter containing a TATA-box close to the transcription start site. A combination of 5′-primer extension study with 5′RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human <em>KIAA1199</em> gene. Bioinformatics analysis suggested that the 1.4 kb <em>KIAA1199</em> promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for <em>KIAA1199</em> gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these <em>cis</em>- and <em>trans</em>-acting elements in controlling <em>KIAA1199</em> gene expression. Finally, we found that upregulated <em>KIAA1199</em> expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of <em>KIAA1199</em> expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling <em>KIAA1199</em> gene expression in human cancer.</p> </div

Determination of the transcription start site of the human <i>KIAA1199</i>. A)

<p>5′ primer extension analysis: Mapping of the <i>KIAA1199</i> mRNA start site by non-radioactive 5′ primer extension analysis was performed using the reverse primer located between +254 and +234 in the first exon (shown by underlie arrow in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044661#pone-0044661-g002" target="_blank">Figure 2D</a>). <b>B)</b> Identification of the transcription start site(s) of the <i>KIAA1199</i> mRNA: 5′ RLM-RACE was performed followed by DNA sequencing analysis. <i>DNA sequencing chromatogram</i> represents a longest transcript and percentages of the alternative transcription start sites are given. C) CAGE data analysis: One major transcription start site was identified in the CAGE analysis viewer (CTSS:Cage Transcription Start Site). <b>D)</b> Nucleotide sequence of the 5′-flanking region of the <i>KIAA1199</i>: +1 was given to the transcription start site of <i>KIAA1199</i>. According to the +1, TATA-box was identified between −31 and −27; GC-box was identified between −248 and −243. Start site of CpG island was shown with gray box in the upstream region of promoter and nucleotides in the CpG island are shown by italics.</p

Sequence alignment of the <i>KIAA1199</i> promoter between human and mouse genomes and putative transcription factor-binding sites.

<p>Putative transcription factor-binding motifs are underlined and TATA/GC boxes and transcription start site are shown in the box. The asterisks mark the fully conserved sequences across the species.</p

Suppression of <i>KIAA1199</i> expression in less invasive cancer cells by DNA methylation.

<p><b>A)</b> Bioinformatics analysis of the CpG island of <i>KIAA1199</i>: Two major subregions within the CpG island were examined by MSP method for MCF-7 and MDA-MB-231 cells. F: forward primers and R: reverse primers. <b>B)</b> Pyrosequencing analysis for quantitation of methylation of cytosine residues in the second subregion of the CpG island between MCF-7 and MDA-MB-231 cells: The percentage of methylation for 36 CG pairs between +525 and +1059 was shown individually (Left panel). Average methylation rate was calculated for MCF-7 and MDA-MB-231 cells (p<0.001) (Right panel). <b>C)</b> Effect of 4 days treatment with 5′-azaon <i>KIAA1199</i> expression: Real time RT PCR was performed in MCF-7 and MDA-MB-231 cells treated with 5′-aza using <i>KIAA1199</i> specific primers. Housekeeping genes were used to normalize the gene expression. <b>D)</b> Methylation status of <i>KIAA1199</i> in human breast cancer specimens: Human invasive breast cancer cells as well as normal breast epithelial cells were harvested by LCM and pyrosequencing was performed using bisulfite-treated DNA. The average methylation level in normal cells was found higher than in cancer cells. A pair of normal breast epithelial cells and breast cancer cells from patients A and B was labeled. n: case number. <b>E)</b><i>KIAA1199</i> expression in human breast cancer specimens: Total RNA from micro-dissected human breast cancer cells as well as normal breast epithelial cells was examined by real time RT-PCR using <i>KIAA1199</i> specific primers. Housekeeping genes were used to normalize the gene expression. A pair of normal breast epithelial cells and breast cancer cells from patients A and B was labeled. n: case number. <b>F)</b> Hypomethylation of <i>KIAA1199</i> in invasive human breast cancer specimens: Bisulfite-treated DNA from paired normal and cancer cells of breast cancer specimens harvested by laser microdissection technique was examined by a pyrosequencing approach. Representative methylation profile between the +932 and +1025 was shown for the benign and invasive cells of single patients.</p

Requirement of AP-1 binding element in the <i>KIAA1199</i> promoter.

<p><b>A)</b> A schematic diagram of mutations at the AP-1 binding site: A site-directed mutagenesis was carried out to generate either a deletion mutant by removing the AP-1 consensus sequence (GAGT) or a substitute mutation within the pro-1.4 promoter construct. The relative promoter activities of the mutations (ratio of firefly luciferase over Renilla luciferase) were then compared to the activity of the wild type pro-1.4 promoter construct (defined as arbitrary value of 100) in COS-1, MDA-MB-231, and MCF-7 cells. <i>Error bars indicate</i> mean +/− S.E. <b>B)</b> Binding of nuclear proteins to the AP-1 site in the <i>KIAA1199</i> promoter: EMSA was carried out using a biotinylated double-stranded oligonucleotide (50 bp) containing the AP-1 binding site and nuclear extracts from MDA-MB-231 cells. Where indicated, binding was competed with 50–200 fold excess amounts of unlabeled probe. DNA-protein complexes formed are indicated as Shift-1 and Shift-2. <b>C)</b> Determination of specific binding between AP1 and the <i>KIAA1199</i> promoter: EMSA was performed using a biotinylated double-stranded oligonucleotide (50 bp) containing the AP-1 binding site and nuclear extracts from MDA-MB-231 cells. Where indicated, biotinylated probe along with anti-C-Jun antibody or biotinylated probe containing mutated site of AP-1 consensus sequence were incubated with nuclear extracts from MDA-MB-231 cells. <b>D)</b> ChIP assay for analysis of association between endogenous AP-1 and the KIAA199 promoter sequence: A strong relation between AP-1 and DNA sequence was shown by 20% bound/input ratio as compared to the unrelated intron region. Normal rabbit IgG was used as a negative control. Results were calculated according to the bound/input ratio.</p

In Vivo Tumorigenicity and Spontaneous Metastasis Assays.

<p>“n.d.” = not done.</p