LHP1 occupancy across the <i>Arabidopsis</i> genome.

<p><b>(A)</b> Correlation between the genome-wide distribution of H3K27me3, RNA Pol II and LHP1. Tag density distribution of LHP1 in WT along with H3K27me3 and RNA Pol II across the genes with 2kb flank, as indicated in the scheme below de graph, gene in grey, flanking regions as black lines. Regions are clustered using K-means linear clustering according to tag density profiles. Density profiles were generated using density array method in seqMINER. Darker red indicates higher density of reads. On the Y-axis there is the list of all genes in TAIR10 annotation. Ten clusters are shown here, revealing the co-occupancy of LHP1 and H3K27me3 and a negative relationship between LHP1 and RNA Pol II. <b>(B)</b> Density plot showing overlap of LHP1 and RNA Pol II using Hexagonal binning routine. As large number of data points may overlap, Hexagonal binning gives additional dimension of differentiation of overlapping points based on count. Each point represents the distance of midpoint of peak to nearest gene. On the Y-axis is location of midpoint of LHP1 peak in comparison to gene position; X-axis is location of midpoint of RNA Pol II in comparison to mid-point of gene. This reveals peaks of RNA Pol II and LHP1 do not co-occur physically in the genome. <b>(C)</b> Hexagonal binning plot showing the association of LHP1 peak region to that of H3K27me3. Each point represents the distance of midpoint of peak to nearest gene. Most of the peaks overlap on the coding region. Large number of points occurs along the positive correlation line, showing the co-occurrence pattern of LHP1 and H3K27me3. (<b>D)</b> Average ChIP-seq enrichment profiles plot of H3K27me3 and LHP1 in WT, stratified by gene length. Normalization of coverage using spline algorithm was performed over the genes and flanking 2 kb region. (<b>E)</b> Average enrichment profile of LHP1 is correlated with gene expression variations. Gene expression is categorized from low (first quantile) to high expression (fourth quantile). Mean-normalized ChIP-Seq densities of equal bins along the gene and 2-kb region flanking the TSS or the TES were plotted. Highly expressed genes show lower enrichment for binding of LHP1. (<b>F)</b> Boxplot showing the comparison of expression levels in RPKM of LHP1-targeted genes and non-targeted genes in WT. LHP1 targeted genes show lower expression levels. (*) represents Mann–Whitney–Wilcoxon test between LHP1 target and non-target with a p-value < 2.2e-16.</p

Genome topology is globally altered in the <i>lhp1</i> backgorund.

<p><b>(A)</b> 2D interaction map showing significant interactions in WT and <i>lhp1</i>. Highly significant interactions are marked as red dots in their corresponding boxes. The color scale represents log2 (interaction) values. Lower panel in red (marked “LHP1”) are peaks from the LHP1 WT ChIP-seq. The two LHP1 panels are identical as they correspond to LHP1 binding in WT. The second LHP1 panel is shown here to correlate this dataset with the Hi-C in the mutant. (<b>B)</b> A screenshot of zoomed 2D interaction map showing intra-chromosomal interaction and LHP1 binding region for Chromosome 1. Centromeric interactions in those regions are masked. WT specific interactions are marked as black dots and <i>lhp1</i> specific interactions are marked as purple dots. The color scale represents the log2 (interaction), which is calculated against the background (taken as <i>lhp1</i> mutant). Lower panel in black (marked “LHP1”) shows the peaks of LHP1 deposition in WT ChIP-seq. (<b>C)</b> 2D interaction map showing the loss of interaction in <i>lhp1</i> when compared to the same region in WT (top panels). Lower panels show the loss of H3K27me3 in the same region. In red it is highlighted the region in WT where LHP1 and H3K27me3 co-occur, exhibiting interaction changes in <i>lhp1</i> mutants, along with the loss of H3K27me3.</p

Global changes in chromatin interactions are observed in <i>lhp1</i>, impacting gene transcription.

<p><b>(A)</b> Gene pairs showing altered chromatin interactions and reduced levels of H3K27me3 in <i>lhp1</i> compared to WT. <b>(B)</b> Hi-C interactions between the pair of gene loci <i>PID</i> and <i>APOLO</i> (chr2:14588900–14599067). Several interactions can be detected between these two loci in WT. These interactions are diminished and even lost (the one in dark blue) in the <i>lhp1</i> mutant. (<b>C</b>) Genome browser screenshot showing gene pairs revealing loss of chromatin interactions and reduced levels of H3K27me3 in <i>lhp1</i> compared to WT. For <b>A</b> to <b>C</b>, colors indicate different interactions (red to blue) in <i>cis</i> and in <i>trans</i> (not adjacent <i>Hind</i> III sites). (<b>D)</b> Expression level changes in<i>l hp1</i> compared to WT of significantly interacting pairs of genes which are LHP1-targeted. Colors were attributed according to the fold-change (FC) observed in the expression level: genes with a log 2 fold change of 2 or above are coloured in red while genes with a log 2 fold change of -2 or lower are coloured in green and genes with a log 2 fold change between -2 and 2 are in black. Interacting pairs show similar transcriptional behavior in the <i>lhp1</i> background.</p

H3K27me3 spreading is affected in the <i>lhp1</i> and <i>clf</i> mutants.

<p><b>(A)</b> Venn diagram showing differential marking of H3K27me3 deposition and LHP1 binding. Hyper (Higher enrichment) and Hypo-H3K27me3 (lower enrichment) refers to differential marking of H3K27me3 in the <i>lhp1</i> mutant compared to WT. (<b>B)</b> Venn diagram showing that Hyper-methylated (H3K27me3) genes are predominantly down-regulated in <i>lhp1</i> and Hypo-H3K27me3 genes are up-regulated in <i>lhp1</i>, as highlighted by the red boxes. (<b>C)</b> H3K27me3 distribution pattern (tag density) over the CDS and flanking regions for WT and <i>lhp1</i>. Mean-normalized ChIP-Seq densities of equal bins along the gene and 5 kb region flanking the TSS or the TES were plotted. (<b>D)</b> Boxplot showing differential peak lengths of H3K27me3 in WT and <i>lhp1</i> over Hyper-H3K27me3 region (Higher enrichment of H3K27me3 in <i>lhp1</i>, compared to WT). (<b>E)</b> Boxplot showing differential peak lengths of H3K27me3 in WT and <i>lhp1</i> over Hypo-H3K27me3 region (Lower enrichment of H3K27me3 in <i>lhp1</i>, compared to WT). (<b>F)</b> H3K27me3 distribution pattern (tag density) over the CDS and flanking regions for WT and <i>clf</i>. Normalizaton of coverage densities of equal bins using spline algorithm was performed over the genes and flanking 5 kb region. (<b>G)</b> Boxplot showing differential peak lengths of H3K27me3 in <i>clf</i> and WT over Hyper-H3K27me3 region (Higher enrichment of H3K27me3 in <i>clf</i>, compared to WT). (<b>H)</b> Boxplot showing differential peak lengths of H3K27me3 in <i>clf</i> and WT over Hypo-H3K27me3 region (Lower enrichment of H3K27me3 in <i>clf</i>, compared to WT). (<b>I)</b> LHP1 binding andH3K27me3 deposition in WT, <i>lhp1</i> and <i>clf</i> across two genes. Decreased level of H3K27me3 towards the 3’-end of both genes is observed in <i>lhp1</i> and <i>clf</i> compared to WT. (<b>J)</b> Venn diagram showing differential H3K27me3 deposition in <i>lhp1</i> and <i>clf</i>. A high overlapping of Hypo and Hyper-methylated genes can be observed between <i>lhp1</i> and <i>clf</i>, as indicated in the red boxes.</p

BAF60 controls histone modifications and RNA Pol II occupancy at the <i>IPT3</i> and <i>IPT7</i> loci.

<p>(A) Schematic representation of the regions of the <i>IPT3</i> and <i>IPT7</i> loci analysed. Black boxes correspond to exons, the arrow indicates the site of translation initiation, numbers indicate the positions of primer pairs used. (B) Quantification data of the chromatin immunoprecipitation results. Nuclei were extracted from 14-day-old roots and immunoprecipited with antibodies specific for H3K27me3, or H3K4me3 or RNA polymerase II. Average relative quantities ± sd are shown for each sample. Data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138276#pone.0138276.ref012" target="_blank">12</a>]significant difference from the wild type (WT) (P < 0.05, Student’s t test).</p

BAF60 binds <i>IPT3</i> and <i>IPT7</i> loci to regulate gene loops formation.

<p>(A) Top: Schematic representation of the <i>IPT3</i> and <i>IPT7</i> loci, the position of each primer pair used for ChIP-qPCR is indicated. The arrow indicates the position of the transcription start site. Exons are represented as black boxes and introns as black lines. Bottom: Quantification data of chromatin immunoprecipitation results. Nuclei were extracted after cross-linking from 14-day-old roots expressing the BAF60-CFP transgene. Chromatin-protein complexes were isolated and immuno-precipitated with antibodies specific for GFP or IgG. Average relative quantities ± sd are shown for each sample. Data presented are representative of three biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.05, Student’s t test). (B) Quantitative 3C of the <i>IPT3</i> and <i>IPT7</i> loci using region I as the anchor region in 14-day-old WT and <i>BAF60</i> RNAi roots. Relative interaction frequencies were calculated as described in Materials and Methods. Data are the average of three biological replicates (each performed on three technical replicates). In the graph, NheI-HF and BglII restriction sites are indicated with vertical dotted lines for <i>IPT3</i> and <i>IPT7</i> respectively. A schematic representation of these loci is shown above with the position of primers used for the 3C analysis represented by grey arrowheads. Grey Asterisks indicate significant difference between the crosslinked and the not crosslinked (P < 0.05, Student’s t test). Black Asterisks indicate significant difference from the wild type (WT) (P < 0.05, Student’s t test).</p

BAF60 regulates root development.

<p>(A) Fourteen-day-old wild type (WT) and <i>BAF60</i> RNAi lines grown vertically under LD conditions. Bar = 10 mm. (B) Time course analysis of root length (left) and lateral root density (right) in wild type (black) and <i>BAF60</i> RNAi plantlets (red) grown vertically on MS medium. Values are average +/- standard deviation (n = 100). Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (C) Quantification of root meristem length of fourteen-day-old wild type and <i>BAF60</i> RNAi lines grown vertically on half MS medium. Values are average +/- standard deviation (n > 15). (D) Confocal images of mPS-PI–stained root tips of fourteen-day-old wild type and <i>BAF60</i> RNAi plantlets growth vertically on MS medium. Bars = 50 μm.</p

BAF60 regulates cytokinin production.

<p>(A) Zeatin content in the roots of 14-day-old wild-type and <i>BAF60</i> RNAi plantlets grown vertically on MS medium. Values are average +/- standard deviation of four biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (B) qRT-PCR data show the relative expression of the indicated genes in wild-type and <i>BAF60</i> RNAi lines. Total RNA samples were collected from 14-day-old roots. mRNA abundance was quantified by qRT-PCR and expressed relative to the abundance of <i>UBQ10</i> transcripts. Values are average +/- standard deviation of triplicates. Data presented are representative of three biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test).</p

BAF60 control cell cycle progression.

<p>(A) Mitotic index in root tips of 7-day-old wild-type and <i>BAF60</i> RNAi plantlets grown vertically on MS medium. Values are average +/- standard deviation of biological triplicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (B) Histochemical staining of the GUS activity in wild-type and <i>BAF60_2</i> RNAi roots harbouring the CYCB1;1::DB-GUS construct. The number of stained cells reflects the number of cells in G2/M. Plantlets were grown vertically for 7 days on MS medium. Bars = 100 μm. (C) Proportion of EdU positive cells in the root tips of seven-day-old wild-type and <i>BAF60</i> RNAi lines grown vertically on MS medium. Values are average +/- standard deviation of biological triplicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.01, Student’s t test). (D) Cell cycle progression was examined by flow cytometry. Histograms show the percentage of cells in G1, S and G2/M phases in wild-type and <i>BAF60</i> RNAi root-derived protoplasts. Plantlets were grown vertically for 7 days on MS medium before protoplast preparation. Data presented are representative of three biological replicates. Asterisks indicate significant difference from the wild type (WT) (P < 0.05, Student’s t test).</p

Model for the role of BAF60 in root development.

<p>In the wild-type root tip, BAF60 binds <i>IPT3</i>, <i>IPT7</i> and <i>KRP7</i> and inhibits their expression during root development. In contrast, in <i>BAF60</i> RNAi lines, these genes are overexpressed due to formation of gene loops and augmentation of H3K4me3 level. This blocks the cell cycle in G1 phase and inhibits root development.</p