Impact of TAM67 on AP1 factors in vivo.

<p>TAM67-rTA mice were treated with (+) or without (−) 2 mg/ml doxycycline in drinking water for 3 days. <b>A</b> Murine epidermis was collected free of the dermis by high temperature separation as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036941#pone.0036941-Rorke1" target="_blank">[35]</a>. Total extract was prepared for immunoblot to detect the indicated proteins. TAM67-FLAG was detected with anti-FLAG. <b>B</b> Interaction of TAM67 with AP1 site consensus element. Nuclear extracts were prepared from epidermis and incubated with AP1c-P³² and other probes as indicated. FP indicates free probe, NE indicates nuclear extract. Similar results were observed in each of three experiments. <b>C</b> Impact of TAM67 on interaction of endogenous AP1 factors with AP1 site element. Nuclear extracts were prepared from TAM67-negative and TAM67-expressing epidermis and incubated with the AP1c-P³² and antibodies as indicated. The complexes were then separated on a non-denaturing 6% polyacrylamide gel. FP indicates free probe and NE is nuclear extract. Note the reduction in jun factor binding in the presence of TAM67-FLAG (left panel). We did not observe a significant reduction in fos factor interaction in the presence of TAM67 (right panel).</p

TAM67-FLAG inhibits hINV gene expression.

<p><b>A</b> TAM67 reduces hINV protein and mRNA level. Keratinocytes were infected with indicated MOI of tAd5-EV or tAd5-TAM67-FLAG and after 48 h extracts were prepared to detect hINV protein by immunoblot and mRNA by quantitative PCR. The values are mean ± SD and the asterisks indicate a significant reduction using student’s t-test, n  = 3 (p<0.001). <b>B</b> TAM67 suppresses AP1 factor-dependent promoter activity. Keratinocytes were transfected with the indicated hINV reporter constructs in the presence of empty pcDNA3 vector or pcDNA3-TAM67-FLAG and treated 24 h with or without 50 ng/ml TPA prior to preparation of extracts and assay of luciferase activity. The values are mean ± SEM and the asterisks indicate a significant reduction using student’s t-test, n = 3 (p<0.001).</p

TAM67 binds to the AP1-5 site of hINV gene promoter.

<p>Keratinocytes were infected with 10 MOI tAd5-EV or tAd5-TAM67-FLAG and after 24 h nuclear extracts were prepared for gel shift. <b>A</b> TAM67 interaction with hINV promoter AP1-5 site. Nuclear extracts were incubated with AP1-5-P³² with or without a 50-fold molar excess of AP1-5 or AP1-5 m oligonucleotide, or anti-FLAG antibody, and electrophoresed on a 6% acrylamide non-denaturing gel. FP indicates free probe and NE is nuclear extract. The arrow indicates the major shifted bands and asterisks indicate supershifted bands. AP1-5 is an oligonucleotide encoding the AP1-5 site of hINV promoter. AP1-5 m is an AP1-5 mutant that does not bind AP1 transcription factors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036941#pone.0036941-Welter1" target="_blank">[47]</a>. <b>B</b> TAM67 inhibits AP1 factor interaction with AP1-5. Nuclear extracts were incubated with AP1-5-P³² in the absence or presence of c-jun, junB, junD, Fra-1, Fra-2, c-fos, or fosB specific antibodies, and electrophoresed on a 6% acrylamide non-denaturing gel. Arrows indicate major shifted band and asterisks indicate supershifted bands. FP is free probe. <b>C</b> ChIP analysis reveals TAM67 presence at the hINV upstream regulatory region AP1-5 site <i>in vivo</i>. Nuclear extracts were prepared for ChIP analysis and incubated with anti-IgG or anti-FLAG and the precipitated DNA was analyzed for AP1-5 site encoding sequences. The values are mean ± SD (n = 3, p<0.001) and the asterisk indicates a significant increase compared to all other groups. Nucleotides −2218/−2055 encodes the AP1-5 site and nucleotides −1040/−919 is a region of the hINV upstream regulatory region that lacks an AP1 site.</p

TAM67 suppresses c-jun promoter activity.

<p><b>A</b> TAM67 reduces c-jun mRNA. Keratinocytes were infected with empty (EV) or TAM67-FLAG encoding adenovirus (10 MOI) and after 24 h mRNA was prepared and c-jun mRNA level was measured by quantitative PCR. <b>B</b> TAM67 suppresses c-jun promoter activity. Keratinocytes were transfected with 1 µg of the indicated c-jun promoter luciferase reporter construct and 1 µg of pcDNA3 (EV) or pcDNA3-TAM67-FLAG (TAM67-FLAG). After 24 h the cells were harvested and assayed for luciferase activity. The values in both plots are mean <u>+</u> SD and the asterisks indicate a significant reduction (p<0.005, n = 3). <b>C</b> Map of c-jun promoter region. The promoter constructs encode nucleotides −1780 to +731 with the transcription start site at +1. c-jun(−1780/+731) is the wild-type intact promoter and c-jun(−1780/+731)-AP1m is a construct in which the critical AP1 sites are eliminated by mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036941#pone.0036941-Wei1" target="_blank">[48]</a>. LUC indicates the luciferase gene. The numbers are given in nucleotides.</p

TAM67-FLAG expression in keratinocytes. A

<p>Comparison of c-jun and TAM67 structure. The numbers are indicated in amino acids. The transactivation, DNA binding and leucine zipper domains are indicated. The TAM67 truncated protein is FLAG epitope tagged as indicated. <b>B/C</b> TAM67-FLAG is expressed in keratinocytes. Normal human keratinocytes were infected with 10 MOI of tAd5-EV or tAd5-TAM67-FLAG with 5 MOI of Ad5-TA. After 24 h the cells were fixed for immunostaining and extracts were prepared for immunoblot with anti-FLAG. Similar results were observed in each of three repeated experiments.</p

Mechanism of TAM67 action in keratinocytes.

<p><b>A</b> Wild-type regulation involves the binding of fos:jun heterodimers (and jun:jun hetero and homodimers, not shown) to AP1 response element to drive differentiation-associated gene expression. Blocking occurs when the concentration of TAM67 present in the cells is high enough that TAM67 homodimers comprise the major complex bound to DNA and this complex blocks interaction of endogenous AP1 factors with the element. Quenching occurs when TAM67 complexes with endogenous jun and fos factors and this complex, which is transcriptionally inactive, binds to DNA. We propose that blocking is a major mechanism of TAM67 action in our system, but that quenching is also important. <b>B</b> TAM67 interaction at the promoter elements leads to blocking and quenching to reduce AP1 factor interaction and activity at AP1 binding sites. This leads to reduced expression of jun factors and ultimately reduced target gene (involucrin, loricrin) expression.</p

TAM67-FLAG inhibits AP1 factor binding to AP1 consensus DNA binding element.

<p>Keratinocytes were infected with 10 MOI tAd5-EV or tAd5-TAM67-FLAG and after 24 h nuclear extracts were prepared. <b>A</b> AP1 factors interact with AP1 consensus DNA element. Nuclear extracts were incubated with AP1c-P³² without or with a 50-fold molar excess of Sp1c or AP1c oligonucleotides, or anti-FLAG antibody and electrophoresed on a 6% acrylamide non-denaturing gel. FP indicates free probe and NE is nuclear extract. The arrow indicates the major shifted band and asterisks indicate migration of supershifted complexes. AP1c and Sp1c encode consensus AP1 and Sp1 binding elements. <b>B</b> TAM67-FLAG reduces AP1 factor binding to DNA. Nuclear extracts were incubated with AP1c-P³² in the absence or presence of c-jun, junB, junD, Fra-1, Fra-2, c-fos, or fosB antibodies, and electrophoresed on a 6% acrylamide non-denaturing gel. Arrows indicate shifted band and asterisks supershifted bands. FP indicates free probe. <b>C</b> TAM67-FLAG forms homodimers and heterodimers. Nuclear extracts were treated with or without DSS crosslinker prior to electrophoresis on a denaturing 8% polyacrylamide gel and TAM67-FLAG was detected by anti-FLAG immunoblot. Identical results were observed in three repeated independent experiments.</p

Characterization of lncRNAs involved in cold acclimation of zebrafish ZF4 cells

<div><p>Long non-coding RNAs (lncRNAs) are increasingly regarded as a key role in regulating diverse biological processes in various tissues and species. Although the cold responsive lncRNAs have been reported in plants, no data is available on screening and functional prediction of lncRNAs in cold acclimation in fish so far. Here we compared the expression profile of lncRNAs in cold acclimated zebrafish embryonic fibroblast cells (ZF4) cultured at 18°C for 30 days with that of cells cultured at 28°C as control by high-throughput sequencing. Totally 8,363 novel lncRNAs were identified. Including known and novel lncRNAs, there are 347 lncRNAs up-regulated and 342 lncRNAs down-regulated in cold acclimated cells. Among the differentially expressed lncRNAs, 74 and 61 were detected only in control cells or cold-acclimated cells, respectively. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses of adjacent genes to the differentially expressed lncRNAs showed that the enriched genes are involved in electron transport, cell adhesion, oxidation-reduction process, and so on. We also predicted the target genes of the differentially expressed lncRNAs by looking for interactions between lncRNAs and mRNAs, and constructed an interaction network. In summary, our genome-wide systematic identification and functional prediction of cold responsive lncRNAs in zebrafish cells suggests a crucial role of lincRNAs in cold acclimation in fish.</p></div

Variation of DNA Methylome of Zebrafish Cells under Cold Pressure

<div><p>DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure.</p></div

FDRs of the methylation level comparisons from three different samples (28°C, 18°C /5d and 18°C /30d).

<p>FDRs of the methylation level comparisons from three different samples (28°C, 18°C /5d and 18°C /30d).</p