Foxp2/FOXP2 expression in murine bone and association with reduced growth of osteosarcoma cell lines.

<p>(A,B) Real-time PCR analysis of gene expression in primary murine tissues from 12-week old male C57Bl/6 mice using SyBr-green, specifically whole bone marrow (BM), spleen (Spl), thymus (Thy), whole long bones after flushing and 2 rounds of collagenase digestion (Bone) and bone-associated cells from the collagenase fraction (CF). Data represent mean +/- SD of three mice. While <i>Foxp1</i> and <i>Foxp4</i> are most expressed in <i>CD45</i>+ haematopoietic tissues, <i>Foxp2</i> expression is highest in bone as are the established osteoblast genes <i>Ibsp</i> and <i>Sp7</i>. Expression normalised to <i>Hprt</i>, expressed relative to highest sample (100%); (C) Immunoblot analysis of COS-1 fibroblast-like cells transiently transfected with CMV-driven mammalian expression plasmids containing murine full-length Foxp cDNAs or pCDNA4 empty vector (control) as indicated top. All antibodies exhibited specificity for appropriate ectopically-expressed proteins, low level endogenous FOXP4 expression was detectable in all lysates; (D) Immunohistochemical detection of Foxp2 protein in murine E17.5 long bone, detail (middle panels) showing variation in Foxp2 positivity along the periosteum, staining with the anti-rabbit murine monoclonal antibody MR12 was performed on serial sections as negative control (same regions, right panels)</p

Confluent growth arrest is associated with increased FOXP2 expression.

<p>(A) Cell cycle analysis of 143B subjected to increasing confluence (approximate percentage confluence indicated top), numbers within plots from left to right indicate percentage of cells in G1/G0, S, and G2/M phase respectively, representative of three experiments; (B) Quantitation of apoptotic cell death in 143B cell populations by flow cytometric analysis of Annexin V positivity, in cultures either exponentially growing (Exp. Growth), subjected to overnight culture with 20μg/ml cyclohexmide as a positive control that induces apoptosis (cycloheximide), or subjected to 4 days growth arrest at confluence (late arrest, as per <i>A</i>). Numbers represent mean % annexin positive ± SD from three experiments; (C) Real-time PCR analyses of <i>FOXP</i> expression in MG-63, 143B and U2-OS cultured to increasing confluence, expressed as 2^-δβCT, relative to growing culture, <i>N</i> = 3 ± SD; (D) Immunoblot analyses of nuclear extracts from cells cultured as in <i>C</i>, including nucleophosmin (NPM) as a loading and transfer control, representative of two experiments, (E) Real-time PCR analyses of <i>p21</i>, <i>p27</i> and <i>IL-6</i> expression in 143B cultured to increasing confluence, expressed as 2^-δδCT, relative to growing culture, <i>N</i> = 3 ± SD.</p

Growth arrest-induced FOXP2 transcription is upstream of the cell cycle machinery.

<p>(A, C) Cell cycle analyses of 143B grown for 6 days in reduced serum or 1 day in the CDK4/6 inhibitor Palbociclib 100 nM or 1 μM as indicated; (B, D) Real-time PCR analyses of gene expression in 143B cultured as in <i>A</i> and <i>C</i>, expressed as 2^-δδCT, relative to growing or vehicle-treated culture, <i>N</i> = 3 ± SD; (E) Real-time PCR analyses of gene expression in 143B reduced serum experiments similar to <i>A</i> over a shorter timecourse, <i>N</i> = 3 ± SD; (F) Real-time PCR analysis of <i>FOXP2</i> expression in 143B cultured at subconfluence or confluence for 24hrs in the presence of inhibitors/vehicle as indicated, (PD-98059 ERK1/2 inhibitor and LY-294002 PI3K inhibitor at 50μM, IKK inhibitor 7, Bay117082 NFκB inhibitor, and DBZ Notch pathway inhibitor at 1μM), expressed as fold change induced by confluence, inhibitors had minimal effect on subconfluent <i>FOXP2</i> expression, <i>N</i> = 5 ± SD; (G) Immunoblot analysis of nuclear extracts from 143B treated at confluence as in <i>F</i>, including nucleophosmin (NPM) as a loading and transfer control.</p

FOXP2 induction is required for efficient 143B growth arrest and p21 ^CIP1/WAF1 control.

<p>(A) Immunoblot analysis of FOXP expression in nuclear extracts from confluent 143B cells 48hr after siRNA transfection, including TATA-binding protein (TBP) as a loading and transfer control, representative of three experiments; (B) Quantitation of changes in cell cycle fractions induced by confluence as per <i>A</i>, relative to dividing cells, <i>N</i> = 3 ± SD; (C) Phase contrast images of cells as in <i>A</i>, to show frequently increased saturation density following FOXP2 depletion;; (D) Real-time PCR analysis of <i>p21/CDKN1A</i> expression in 143B cultured as in <i>A</i>, expressed as 2^-δδCT, relative to subconfluent culture (T = 0), control is mean of four different control siRNAs. <i>N</i> = at least 4 ± SD; (E) Immunoblot analyses of whole cell lysates from cells cultured as in <i>A</i>; (F) Real-time PCR analysis of <i>p21CDKN1A</i> expression in cells transfected at subconfluence (T = 0) with siRNAs as shown and harvested still at subconfluence after 48 hr culture in 1% serum, <i>N</i> = 3 ± SD; (G) Real-time PCR analysis of <i>p21CDKN1A</i> expression in and phase contrast images of cells cultured as in <i>A</i>, in the presence of either 10ng/ml hIL-6 (+ IL-6) or PBS/BSA carrier alone (—IL-6); (H) Real-time PCR analyses of cells transfected at subconfluence with p53 cDNA plasmid or empty control plasmid (vector), then treated with siLGC or each FOXP2 siRNA and harvested still at subconfluence after 48hr culture in 10% serum. Expression is shown as percentage relative to highest (100%).</p

Analyses of Prdm5 chromatin interactions.

<p>A) Diagram illustrating the overall distribution of Prdm5 binding sites categorized according to the distance from the nearest TSS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen.1002711.s010" target="_blank">Text S1</a>). B) The mean distribution of tags across gene bodies for Prdm5 ChIP-seq (Prdm5-ab1 in blue, Prdm5-ab2 in red and IgG in black). Vertical dashed line at x = 0 represents the TSS. Positions after the TSS are represented as % of the length of the gene. C) Upper panel. Slogos plot produced using the motifs detected by the Weeder program from Prdm5 “shrunk” peaks (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen.1002711.s010" target="_blank">Text S1</a>). Lower panel. DNA pulldown assay from nuclear extract of 293 cells overexpressing HA-PRDM5 using biotinylated oligos representing the <i>Col1a1</i> exon 33 (WT) and a mutated control sequence (G-A/T Mut). D) Histogram showing the percentage of H3K9me3 (left panel), H3K4me3 (middle panel) and RNA Polymerase II (right panel) positivity for “Random sampling” (mean value of 100 iterations for 1446 random genes sets) or for Prdm5 target genes. (E) Q-Q plot comparing the quantile distribution of Prdm5 target genes' expression (on Y axis) and all genes (on X axis). Red line is reference line representing equal quantile distribution.</p

Prdm5 deregulation impairs osteogenic differentiation <i>in vitro</i>.

<p>A) Upper panel. qRT-PCR of <i>Prdm5</i> levels in MC3T3 cells transduced with lentiviral shRNA constructs against <i>Prdm5</i> (shPrdm5-a and shPrdm5-b) and control construct. Lower panel. Prdm5 western blot from the same experiment. Tubulin is included as loading control. B) Upper panel. Quantification of Alizarin red staining after 21 days of osteogenic differentiation in MC3T3 cells. Data are presented as mean of 3 independent experiments ± SEM. ** = p<0.01 and *** = p<0.005 (t-test). Lower panel. Representative image from osteogenic differentiation experiments. C) Western blot showing overexpression of human PRDM5 (marked with star) in MC3T3 cells (filled circle = endogenous Prdm5). GFP overexpression is used as a negative control and Vinculin western blot for equal protein loading. D) Quantification of Alizarin red staining from osteogenic differentiation experiments of MC3T3 cells overexpressing GFP or PRDM5. A representative experiment is shown and data are presented as average ± standard deviation. E) qRT-PCR analysis of WT and <i>Prdm5^LacZ/LacZ</i> calvaria osteoblasts for osteogenic markers as indicated. Expression values were normalized to a panel of housekeeping genes (<i>Rps18, Ubc, Actb, Rpl0</i>) and indexed to the average expression value of wild type clones. * = p<0.05 and *** = p<0.001, by unpaired T-test, +/+ (n = 14), LacZ/LacZ (n = 19).</p

Prdm5 loss results in decreased Collagen I and Decorin levels and leads to reduced bone formation <i>in vivo</i>.

<p>A) Immunofluorescence staining of E16.5 tibiae for Collagen I (upper panel, bar = 200 µm), Decorin (middle panel, bar = 100 µm, asterisk indicates periosteum area) and Picrosirius red staining of WT and <i>Prdm5^LacZ/LacZ</i> E16.5 tibiae under polarized light (lower panel, bar = 100 µm). B) H&E staining of <i>Prdm5</i> WT and <i>Prdm5^LacZ/LacZ</i> E16.5 bones. The bars indicate the length of the osteoblast region (bar = 100 µm). C) Von Kossa staining of WT and <i>Prdm5^LacZ/LacZ</i> E16.5 tibiae. D) μCT measurements of E18.5 embryos. BV/TV = Bone volume/Total embryo volume. Values for <i>Prdm5^LacZ/LacZ</i> embryos are normalized to littermate controls. * = p<0.05; T-test with Welch correction. E) Representative pictures from pQCT scans of femoral metaphysis of a 5 weeks old <i>Prdm5^LacZ/LacZ</i> mouse and a littermate control. Color bar represents the density scale. F) Quantification of total bone mineral density in femoral metaphysis from 5 weeks old WT and <i>Prdm5^LacZ/LacZ</i> animals. Each dot represents the average of two measurements on each animal tested (n = 9/10 per group).</p

Prdm5 targets all collagen genes and regulates type I collagen expression.

<p>A) ChIP-qPCR validation of Prdm5 peaks in collagen genes or negative control regions from an independent chromatin preparation immunoprecipitated with IgG, Prdm5-Ab1 (in black and blue respectively, plotted on left Y axis) or Prdm5-Ab2 (in red, plotted on right Y axis). Orange horizontal line represents the highest “noise” value obtained by ChIP-qPCR on a set of negative regions. B) Biological processes enrichment from gene ontology annotation of Prdm5 target regions. C) Distribution of the “peak centre” position for collagen genes targeted by Prdm5 or random sampling of Prdm5 target genes according to genetic feature. D) Correlation between Prdm5-Ab1 coverage inside the gene body of all mouse collagen genes (X-axis) and Pol II coverage in the same regions (Y-axis) normalized by base pairs. E) qRT-PCR for <i>Prdm5</i>, <i>Col1a1</i> and <i>Col1a2</i> in MC3T3 cells treated for 72 hours with siRNA oligos against <i>Prdm5</i> (siPrdm5-1 and -2) or controls. Results are presented as average of four independent experiments ± SD; * = p<0.05, ** = p<0.01. F) Upper panel. ChIP-qPCR for RNA Pol II in WT and mutant (blue and red respectively) calvarial osteoblasts along the <i>Col1a1</i> gene. IgG control is represented by black and green lines respectively. X-axis = distance (in bp) from <i>Col1a1</i> TSS, * = p<0.05 (unpaired t-test). Lower panel. Genome browser snapshot of the corresponding <i>Col1a1</i> genomic region displaying MC3T3 tracks for: qPCR amplicons, IgG, RNA Pol II and Prdm5-Ab1 coverage. G) Western blot from co-immunoprecipitation experiment of HA-PRDM5 in HEK293 cells; endogenous interacting proteins or IgG are indicated.</p

Prdm5 is expressed in osteoblast regions of developing bones.

<p>A) Scheme for the generation of the <i>Prdm5^LacZ/LacZ</i> mouse strain. B–E) X-gal stainings of <i>Prdm5^LacZ/LacZ</i> embryos at E10.5 (B), E12.5 (C) E14.5 (D) and E16.5 (E). F) E16.5 embryo image detail. LacZ reporter expression in the perichondrium and growth plate of femur and ribs is marked by arrows. G) X-gal staining of tibiae section from E16.5 <i>Prdm5</i> mutant embryo. Juxtaposition of three pictures (separated by white lines) to represent the whole length of a tibia. Indicated are different compartments: PC = proliferative chondrocytes, HC = hypertrophic chondrocytes, OB = osteoblasts. Periosteum is marked by asterisks. H) Whole mount X-gal staining of <i>Prdm5^LacZ/LacZ</i> newborn skull at P0. Pronounced staining in sutures is indicated with an arrow. Bars = 1 mm, except for (G) where bar = 200 µm.</p

Prdm5 regulates <i>Decorin</i> through a distal enhancer.

<p>A) ChIP-qPCR validation of Prdm5 peaks on selected ECM genes or negative regions from an independent sample immunoprecipitated with IgG, Prdm5-Ab1 (in black and blue respectively, plotted on left Y axis) or Prdm5-Ab2 (in red, plotted on right Y axis). Orange horizontal line represents the highest “noise” value obtained by ChIP-qPCR on a set of negative regions. B) qRT-PCR analysis of <i>Decorin</i> transcript (<i>Dcn</i>) levels upon Prdm5 knockdown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen-1002711-g004" target="_blank">Figure 4E</a>. Results are presented as average of four independent experiments +/− SD; * = p<0.05. (C) qPCR analysis of WT and <i>Prdm5^LacZ/LacZ</i> calvarial osteoblasts for <i>Decorin</i>. Expression values were normalized to the control WT samples. *  = p<0.05; T-test, (+/+ n = 14, LacZ/LacZ n = 19 clones). D) Upper panels. Western blot analysis of Decorin levels upon Prdm5 knockdown in cell layers; Tubulin is used as loading control. Lower panels. Western blot analyses of purified proteoglycans from cell culture media from knockdown cells. Tubulin is used as purity control and Fibronectin for equal protein loading. E) ChIP-qPCR with indicated antibodies for Prdm5 binding site upstream of Dcn gene. Meg3 TSS region is used as negative control.</p