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

Analysis of bivalently modified genes.

<p>Bivalent (H3K4me3 and H3K27me3) modifications were observed for a subset of genes at 23 d. These genes were classified into those that lost both modifications at 52 d (K4+K27- None, dark blue), those that retained the H3K27me3 mark at 52 d (K4+K27 – K27, light blue), those that retained the H3K4me3 mark at 23 d (K4+K27- K4, yellow) and those that retained the bivalent marks (K4+K27- K4+K27, brick red). Genes thus classified were then placed into the five groups shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033151#pone-0033151-g004" target="_blank">Figure 4</a>. Genes that retained the H3K27me3 mark were restricted to groups A and B while genes that retained the H3K4me3 mark were restricted to groups C–E.</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

Expected patterns of H3K4me3 and H3K27me3 histone modifications on two representative genes.

<p>Genes with exons represented as green thick lines and introns shown as green thin lines are shown at the top of this Genome Browser image, and the green arrow shows the direction of transcription. The number of sequence reads for 23 d are shown in blue and for 52 d in brown. H3K4me3 (K4) is shown on the top two tracks, H3K27me3 (K27) on the next two tracks. Gray rectangles below tracks indicate regions with sequence reads significantly above relative input. A) <i>ACT2</i> (<i>At3g18780</i>) shows significant enrichment above respective input samples for the H3K4me3 mark. This constitutive gene is associated with H3K4me3 marks, but lacks H3K27me3 marks. B) <i>FLC</i> (<i>At5g10140</i>) shows significant levels of silencing H3K27me3 marks, which is expected in the <i>fri</i> mutant Columbia ecotype that does not require vernalization for flowering. <i>FLC</i> has some H3K4me3 marks near the first and last exons.</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

Genic distributions of differential histone marks.

<p>Regions with significantly different levels of histone modification were identified by ChIPnorm and placed into 200 bp bins relative to the transcription start site (TSS). A) Differential regions for the H3K4me3 mark are most abundant in the five bins downstream of the TSS. B) Differential regions for the H3K27me3 mark are most abundant at the TSS and the first three bins downstream of the TSS.</p

Mature (23 d) and senescent (52 d) leaf tissue used for ChIP-seq and RNA isolation.

<p>Fully-expanded, green rosette leaves were harvested from mature and senescent plants similar to those shown above.</p