AP-2α Inhibits c-MYC Induced Oxidative Stress and Apoptosis in HaCaT Human Keratinocytes

AP-2α and c-MYC are important transcription factors involved in multiple cellular processes. They each display the paradoxical capacities to stimulate both cell proliferation and apoptosis under different conditions. In the present study we found that over expression of c-MYC was associated with accumulation of reactive oxygen species (ROS) and apoptosis in human keratinocytes, both of which were significantly inhibited by co-expression of AP-2. The effects of AP-2 on c-MYC were active at several levels. First, AP-2 and c-MYC were confirmed to interact at the protein level as previously described. In addition, forced expression of AP-2 significantly decreased steady state levels of c-MYC mRNA and protein. These findings suggested that AP-2 may have a direct effect on the c-myc gene. Chromatin immunoprecipitation assays demonstrated that AP-2 proteins bound to a cluster of AP-2 binding sites located within a 2 kb upstream regulatory region of c-myc These results suggest that the negative regulation of AP-2 on c-MYC activity was achieved through binding of AP-2 protein to the c-myc gene. The effects of AP-2 on c-MYC induced ROS accumulation and apoptosis in epidermal keratinocytes are likely to play an important role in cell growth, differentiation and carcinogenesis of the skin

Genome-Wide Evaluation of Histone Methylation Changes Associated with Leaf Senescence in Arabidopsis

Leaf senescence is the orderly dismantling of older tissue that allows recycling of nutrients to developing portions of the plant and is accompanied by major changes in gene expression. Histone modifications correlate to levels of gene expression, and this study utilizes ChIP-seq to classify activating H3K4me3 and silencing H3K27me3 marks on a genome-wide scale for soil-grown mature and naturally senescent Arabidopsis leaves. ChIPnorm was used to normalize data sets and identify genomic regions with significant differences in the two histone methylation patterns, and the differences were correlated to changes in gene expression. Genes that showed an increase in the H3K4me3 mark in older leaves were senescence up-regulated, while genes that showed a decrease in the H3K4me3 mark in the older leaves were senescence down-regulated. For the H3K27me3 modification, genes that lost the H3K27me3 mark in older tissue were senescence up-regulated. Only a small number of genes gained the H3K27me3 mark, and these were senescence down-regulated. Approximately 50% of senescence up-regulated genes lacked the H3K4me3 mark in both mature and senescent leaf tissue. Two of these genes, SAG12 and At1g73220, display strong senescence up-regulation without the activating H3K4me3 histone modification. This study provides an initial epigenetic framework for the developmental transition into senescence

Expression of Arabidopsis <i>KDM5B</i>–like genes in senescent leaf tissue.

<p><i>KDM5B</i> genes encode H3K4me3 demethylases, and eight KDM5B-like genes were identified in Arabidopsis. The same tissue used in the ChIP-seq analysis was analyzed for expression of the <i>KDM5B-like</i> genes using real-time qPCR. <i>ACT2</i> was the reference gene and relative expression at 52 d compared to 23 d is shown. Two <i>KDM5B-like</i> genes affect flowering time <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone.0033151-Jeong1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone.0033151-Noh1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone.0033151-Yang2" target="_blank">[43]</a>, and their published names as well as known target gene are shown. <i>At2g34880</i> mRNA was undetectable in both RNA samples, and is not shown.</p

Real-time qPCR measurement of senescence-up-regulated genes (SURGs) and senescence-down-regulated genes (SDRGs) mRNA levels in tissue used for ChIP-seq.

<p>Total RNA was harvested from the same leaves used in the ChIP-seq analysis, and real-time qPCR was performed using <i>ACT2</i> as a reference. SURGs and SDRGs were identified from published microarray data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone.0033151-vanderGraaff1" target="_blank">[4]</a>. Fold-induction at 52 d for SURGs and at 23 d for SDRGs is reported. Eight of 8 tested SURGs were up-regulated by at least 15-fold, while 6 of 7 tested SDRGs were down-regulated by at least 24-fold.</p

Correlation between regions with differential histone modification and senescence regulated gene expression.

<p>4090 genes were equally partitioned into five groups depending on differential expression in mature (23 d) vs. senescent (52 d) tissue. Group A genes have the highest differential expression at 23 d, group E genes have the highest differential expression at 52 d while group C genes have approximately equal expression at both time points. Genes that were identified as having different levels of histone modification (H3K4me3 in panel A, H3K27me3 in panel B) were then placed into the groups. Genes showing a decrease in the specific histone methylation at 52 d (K4-None or K27-None) are shown in blue while those that showed an increase in the specific histone methylation at 52 d (None-K4 or None-K27) are shown in brick red.</p

Genome Browser view of two genes that are down-regulated in senescent leaves and show differential histone modification.

<p>These GBrowse views are similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone-0033151-g005" target="_blank">Figure 5</a>, but a new track, K7_diff (pink font), is now shown. The red bars in the K4_diff track indicate significantly higher histone methylations at 23 d while the green bars in K7_diff indicate significantly higher histone methylations at 52 d. A) <i>At3g27690</i> or Lhcb2.3, shows a 25-fold decrease at 52 d which is accompanied by a decrease in H3K4me3 marks. B) <i>At2g10940</i> shows a 14,000-fold decrease at 52 d which is accompanied by a decrease in H3K4me3 marks and an increase in H3K27me3 marks.</p

Two genes that are expressed at high levels in senescent tissue lack activating H3K4me3 marks.

<p>A) <i>SAG12</i> (<i>At5g45890</i>) is up-regulated 90,000-fold at 52 d, but is devoid of H3K4me 3 marks. B) <i>At1g73220</i> is up-regulated 113-fold at 52 d and likewise shows no H3K4me3 marks. For both genes, H3K27me3 marks are present, but do not show a significant difference between 23 d and 52 d.</p