Identification and Validation of a Putative Polycomb Responsive Element in the Human Genome

<div><p>Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The <i>cis</i>-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human <i>PIK3C2B</i> gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG). We demonstrate that PRE-PIK3C2B interacts directly with YY1 <i>in vitro</i> and recruits PRC2 complex <i>in vivo</i>. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either <i>YY1</i> or <i>SUZ12</i>. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic <i>Drosophila</i>, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS), which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.</p></div

Interaction of PcG proteins with PRE-PIK3C2B in HEK cells.

<p>The experiments were carried out in HEK293 ells. A-Diagrammatic representation of the endogenous PRE-PIK3C2B region with primers (arrows) used for PCR and the 25 mer repeating unit (filled box). B and C- ChIP with antibodies indicated. Input is 20% of sonicated chromatin. D-Quantitative PCR for PRE-PIK3C2B following ChIP in control cells and cells transfected with siRNAYY1 and siRNA SUZ12. E-Interaction of EZH2 with PRE-PIK3C2B estimated by qPCR in absence and presence of YY1siRNA: F-Localization of EED and the H3K4me3 at PRE-PIK3C2B in absence and presence of siRNAYY1. Error bars: S.E.M of assay in triplicate, * p<0.05, **p-value<0.005,n = 3. The qPCR profile for positive and negative controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067217#pone.0067217.s003" target="_blank">Figs. S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067217#pone.0067217.s004" target="_blank">S4</a>.</p

Effect of Pho mutation on PRE-PIK3C2B mediated repression.

<p>A- Expression of miniwhite mRNA by qPCR. The genetic background is indicated below the histograms.* p<0.05, **p<0.005. B- comparison of eye pigmentation between transgenic flies with and without PHO mutation. ΔPI indicates the lines where PRE-PIK3C2B is flipped out.</p

<i>Hottip</i> localizes to 5′ Hoxa genes and deletion of <i>Hottip</i> reduces <i>5</i>′ Hoxa expression.

<p>(A) Schematics showing the mouse <i>Hoxa13</i> and <i>Hottip</i> loci. The CpG Island (CGI) at the <i>Hoxa13</i> promoter is shown in a grey bar. Genome co-ordinates are from the mm9 assembly of the mouse genome. Guide RNA binding sites for deletion of <i>Hottip</i> are shown as arrow heads, primers used for genotyping are shown in arrows (p1 to p4). The deletion product of <i>Hottip</i> (<i>HottipΔ</i>) is shown below. Agarose gel image showing genotyping PCR, first two lanes are amplicons of primers (p1 and p2) within the deleted <i>Hottip</i> region, second two lanes are for amplicons from primers (p3 and p4) 3’ of deleted region. (B) Mean (± s.e.m) expression, assayed by RT-qPCR and normalized to <i>Gapdh</i>, of Hoxa genes and <i>Hottip</i>, in wild-type (black bars, WT), and <i>Hottip</i> knock out (gray bars, <i>HottipΔ</i>) limb mesenchymal cells, (n = 3 biological replicates). * p < 0.05, ** p <0.01. (C) RT-qPCR showing mean (± s.e.m) ± percentage (%) enrichment over input for <i>Hottip</i>, <i>7SK</i> and <i>Gapdh</i> RNAs from <i>Hottip</i> ChIRP pulldown from two experiments. (D) qPCR showing mean (± s.e.m) percentage (%) enrichment over input of ChIRPed DNA at promoters of <i>Actb</i>, <i>Hoxa1</i>, <i>Hoxa7</i>, <i>Hoxa9</i>, <i>Hoxa10</i>, <i>Hoxa11</i>, and <i>Hoxa13</i> from Hottip ChIRP experiments in wild type (black bars, WT) and <i>Hottip</i> knock out limb mesenchymal cells (grey bars, <i>HottipΔ</i>).</p

Artificial induction of <i>Hottip</i> is sufficient to activate 5’ Hoxa genes.

<p>(A) Schematics showing UCSC genomic coordinates of <i>Hottip</i>, <i>Hoxa13</i>, CpG Islands (CGI) in the mouse (top, mm9) and human (bottom, hg19) genomes. Schematics of guide RNA mediated recruitment of dCas9-VP160 to the <i>Hottip or Hoxa13</i> promoters is also shown. Direction of transcription is indicated as arrow marks. (B) Heat map showing the log2 mean fold change in expression of Hoxa, Hoxd and pluripotency associated genes (control genes) from expression microarray experiment, upon co-transfection of guide RNAs recognizing the <i>Hottip</i> promoter (<i>Hottip</i> gRNAs + dcas9-VP160). dCas9-VP160 was also co transfected with guide-RNAs recognizing <i>Hoxa13</i> promoter (<i>Hoxa13</i> gRNAs + dcas9-VP160) (n = 3 or 4 biological replicates). (C) Similar to (B) RT-qPCR data showing mean (± s.e.m) log2 fold change in expression of <i>Hottip</i>, <i>Hoxa13</i>, <i>a11</i>, <i>a10</i>, <i>a9</i>, <i>a7</i>, <i>a1</i>, <i>Pou5f1</i> and <i>Hoxb9</i> upon guide RNA mediated recruitment of dCas9-VP160 to the <i>Hottip</i> promoter (A) in mouse ES cells. Data were normalized to those from a dcas9 control (n = 3 biological replicates). (D) Similar to (C) mean log2 fold change in <i>Hottip and Hoxa13</i> expression in wild type ES cells co-transfected with guide-RNAs recognizing the <i>Hottip</i> promoter and dCas9-VP160 (Black bars, WT). <i>Hoxa13</i> expression in <i>Hottip</i> knock out limb mesenchymal cells is also shown (grey bar, <i>HottipΔ</i>). * p < 0.05, ** p <0.01 throughout.</p

Reduced <i>Hottip</i> expression and Mll occupancy in <i>Psip1</i>^{<i>–/–</i>}.

<p>(A) Mean Log2 ChIP/input for Psip1/p75, Mll1, Menin and H3K4me3 in WT and <i>Psip1</i>^{<i>–/–</i>}MEFs over <i>Hoxa</i> clusters from custom tiling arrays[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006677#pgen.1006677.ref035" target="_blank">35</a>]. Annotated noncoding transcripts (grey, top) and Hox gene transcripts (black) are shown below. (n = 2 biological replicates). Genome co-ordinates are from the mm9 assembly of the mouse genome. Direction of transcription for <i>Hoxa13</i> and <i>Hottip</i> genes are indicated with arrow below. (B) Mean (± s.e.m) expression, assayed by RT-qPCR and normalized to <i>Gapdh</i>, of <i>Hoxa13</i> and <i>Hottip</i> in <i>WT</i> and <i>Psip1</i>^{<i>–/–</i>}MEFs, (n = 3 biological replicates). (C) Nimblegen tiling microarray data showing log2 ratio of <i>Psip1</i>^{<i>–/–</i>}/ <i>WT</i> run-on transcribed RNA (nascent RNA) over posterior Hoxa genes n = 2 technical replicates.</p

Directinteraction of YY1 with PRE-PIK3C2B.

<p>Labeled 25 merOligo corresponding to repeating unit of PRE-PIK3C2B (5′AGTGAA<u>GCCATCAT</u>GTGAGAATACC3′) was used as the probe in gel mobility shift assay with purified His-YY1. Cold probe 1 is competing unlabeled cognate (Oligo25 mer), 2- (OligoΔYY1) repeating unit without YY1 recognition sequence (5′AGTGAAGTGAGAATACC3′). Each competing oligo was used at a concentration 100× higher than the labeled probe. The super shift with anti-YY1 is shown in the last lane (arrow).</p

Genetic interactions of PRE-PIK3C2B with PRC genes in transgenic flies.

<p>Effect of PRE-PIK3C2B in the background of different PcG mutations on rescue of eye pigmentation is shown: cases of rescue of eye colour are marked with an arrow; trans-heterozygotes show better recovery of eye pigmentation. We have used same conditions of image processing in all the cases.</p

Effect of PRE-PIK3C2B on transcription.

<p>A. Part of the vectors used in transfection assays: CMV represents the promoter, GFP is the reporter present in all the constructs. pcDNA3.1-GFP is control plasmid without PRE-PIK3C2B, pc(1 kb)UP is pPRE-PIK3C2B/UP-GFP and pc(1 kb)DN is pPRE-PIK3C2B/DN-GFP with PRE-PIK3C2B (inverted triangle) cloned upstream and downstream of the reporter respectively. B. GFP expression indicated as ratio relative to that from pcDNA3.1GFP. pc(25 mer)11UP and pc(25 mer)11DN are 25 mer oligo corresponding to repeating unit of PRE-PIK3C2B cloned upstream and downstream of the GFP reporter respectively. pcΔYY1UP and pcΔYY1DN are repeating unit without YY1 motif cloned upstream and downstream of the GFP reporter respectively. Error bars, S.E.M of assay triplicate is shown. (**) p-value<0.005, n = 3</p

YY1 and SUZ12 dependent transcriptional repression by PRE-PIK3C2B.

<p>A-Expression of endogenous <i>PIK3C2B</i> in HEK cells analysed by qPCR. The expression is increased in presence of siRNAYY1 and siRNASUZ12. Expression in untransfected HEK cells is taken as control. B- Effect on reporter gene expression. The nomenclature for vectors is same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067217#pone-0067217-g001" target="_blank">Figure 1</a>. Ratio of GFP expression in pcDNA-GFP, pc(1 kb)UP or pc(1 kb)DN with and without co-transfection with siRNA YY1 and siRNA SUZ12 is shown on the Y-axis. Error bars, S.E.M of assay in triplicate is shown * p-value<0.05, ** p-value<0.005, n = 3.</p