Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix

Assessment of mixture toxicity of (tri)azoles and their hepatotoxic effects in vitro by means of omics technologies.

Seeger B, Mentz A, Knebel C, et al. Assessment of mixture toxicity of (tri)azoles and their hepatotoxic effects in vitro by means of omics technologies. Archives of toxicology. 2019;93(8):2321-2333.Consumers are constantly exposed to chemical mixtures such as multiple residues of different pesticides via the diet. This raises questions concerning potential combination effects, especially because these substances are tested for regulatory purposes on an individual basis. With approximately 500 active substances approved as pesticides, there are too many possible combinations to be tested in standard animal experiments generally requested for regulatory purposes. Therefore, the development of in vitro tools and alternative testing strategies for the assessment of mixture effects is extremely important. As a first step in the development of such in vitro tools, we used (tri)azoles as model substances in a set of different cell lines derived from the primary target organ of these substances, the liver (human: HepaRG, rat: H4IIE). Concentrations were reconciled with measured tissue concentrations obtained from in vivo experiments to ensure comparable effect levels. The effects of the substances were subsequently analyzed by transcriptomics and metabolomics techniques and compared to data from corresponding in vivo studies. The results show that similar toxicity pathways are affected by substances and combinations, thus indicating a similar mode of action and additive effects. Two biomarkers obtained by the approach, CAR and Cyp1A1, were used for mixture toxicity modeling and confirmed the concentration-additive effects, thus supporting the selected testing strategy and raising hope for the development of in vitro methods suitable to detect combination effects and prioritize mixtures of concern for further testing

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 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

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