exclusively in the gene body with approx. 60% of peaks centered in exonic regions (Figure 4C). Importantly, Prdm5 occupancy in the body of collagen genes correlated with the amount of bound RNA polymerase II in the same regions (Figure 4D), suggesting a role for Prdm5 in sustaining transcriptional activity of the collagen gene family. Very high enrichment for Prdm5 binding was observed in the two genomic loci corresponding to the collagen I genes Col1a1 and Col1a2 (Figure 4D and Figure S4C). Prdm5 knockdown by means of two siRNA oligos demonstrated that Prdm5 occupancy in Col1a1 and Col1a2 genes was functionally relevant, as reduction in Prdm5 levels led to a decrease in transcript and protein levels of both type I collagen genes (Figure 4E and Figure S4D). This effect was observed also in primary osteoblasts upon genetic ablation of Prdm5 (Figure S4E). Given the close correlation between Prdm5 occupancy and RNA polymerase II presence, we measured the occupancy of the latter along the whole length of the Col1a1 gene in Prdm5 wild type and mutant osteoblasts. While RNA polymerase II levels were unchanged in LacZ/LacZ cells at the Col1a1 TSS, we observed a significant drop in RNA polymerase II levels in mutant Prdm5 osteoblasts between +6.2 kb and the end of the Col1a1 gene (Figure 4F). Towards understanding the mech- anism, we hypothesized that Prdm5 could affect RNA polymerase II by direct interaction. Indeed, overexpressed HA-PRDM5 co- immunoprecipitated with endogenous RNA polymerase II and with higher affinity to the elongating form of RNA polymerase II as evident from analysis using a phospho-serine 2 specific RNA polymerase II antibody (Figure 4G). In summary, Prdm5 targets virtually all the collagen genes in the mouse genome and high Prdm5 occupancy inside the Col1a1 gene body promotes RNA polymerase II occupancy and transcription. Prdm5 regulates Decorin via a distal element with an enhancer-like chromatin signature Analyses of the annotation of Prdm5 bound loci revealed that the Prdm5 target repertoire extends to genomic regions encoding other ECM genes involved in collagen fibrillogenesis, such as Periostin (Postn) and genes from the SLRP family, such as Decorin (Dcn), Fibromodulin (Fmod), Biglycan (Bgn) and Epiphycan (Epc). Also in this case, Prdm5 occupancy on selected targets was validated in independent samples from MC3T3 cells (Figure 5A), as well as primary calvarial osteoblasts (Figure S5A and S5B). Since Decorin is well known to regulate collagen fibrillogenesis, we analyzed the influence of Prdm5 on Decorin expression. Indeed, we observed that Prdm5 knockdown resulted in decreased Decorin transcription (Figure 5B). This effect could be reproduced in Prdm5LacZ/LacZ calvarial osteoblasts (Figure 5C). While cell- associated Decorin protein levels were only mildly decreased upon Prdm5 knockdown (Figure 5D), the amount of secreted Decorin detected in cell culture media from Prdm5 siRNA treated cells was strongly reduced (Figure 5D). A closer inspection revealed that the Prdm5 binding site assigned to Decorin gene was 100 kilobases distant from its TSS, suggesting the binding to a distal enhancer (Figure S5C). Chromatin immunoprecipitation for H3K4me1 and H3K27ac confirmed that Prdm5 bound a site upstream of Dcn with a chromatin signature corresponding to an enhancer element (Figure 5E). Our data show that Prdm5 targets SLRP family members and likely regulates Decorin via a distal enhancer. Delayed ossification and impairment of collagen fibrillogenesis in Prdm5LacZ/LacZ embryos Collagen I is the main component of osteoblastic ECM [11] and Decorin is known to regulate collagen I fibrillogenesis [16]. The Prdm5 Promotes the Transcription of ECM Genes observed Prdm5 regulation of Collagen I and Decorin genes prompted us to characterize their deregulation in vivo and the resulting phenotype in Prdm5LacZ/LacZ mice. qRT-PCR analyses revealed that the Col1a1 and Col1a2 transcripts were significantly reduced upon Prdm5 loss in E16.5 limbs (Figure S6A). Moreover, decreased Collagen I was observed by in situ hybridization in the periosteum at E16.5 (data not shown), as well as by immunoflu- orescence microscopy (Figure 6A). While Decorin transcript levels were unchanged in whole E16.5 mutant limbs (Figure S6B), immunofluorescence staining displayed reduction of Decorin protein, particularly in the periosteum and invading osteoblasts region (Figure 6A), indicating an osteoblast-restricted Prdm5 regulation of Decorin. Given that both molecules are involved in collagen fiber formation, fibrillar collagen levels were evaluated in Prdm5LacZ/LacZ embryonic limbs by picrosirius red staining. Using bright field microscopy, a decreased staining of collagen could be appreciated in the mutants (Figure S6C). Moreover, using polarized light to visualize specifically assembled collagen fiber birefringency, a marked decrease in the presence and organization of collagen fibers was observed in Prdm5LacZ/LacZ embryonic limbs (Figure 6A). Histological examination revealed a shorter osteoblast com- partment in mutant animals (Figure 6B), suggesting a delay in the ossification process. Likewise, Von Kossa stainings revealed decreased calcification in mutant limbs further pointing to an impaired ossification process in Prdm5 mutants (Figure 6C). To quantitate the delayed ossification, wild type and Prdm5LacZ/LacZ E18.5 embryos were analyzed by micro-CT (mCT) scanning. Quantification of total bone volume demonstrated a significant reduction in the ossification process in mutant embryos (Figure 6D). However, at E16.5, no overt differences in the expression of various bone formation markers were observed (Figure S6D). To measure the impact of the embryonic ossification defect in young mice hind limbs from cohorts of WT and Prdm5LacZ/LacZ mice of both genders were analyzed by peripheral Quantitative Computed Tomography (pQCT). Images of distal metaphyseal sections from CT-scans of femurs at 5 weeks of age demonstrated a decrease in bone mineral density (Figure 6E). Quantification of total bone mineral density, total mineral content and total bone area in large cohorts of mice demonstrated a statistically significant reduction in all three parameters in Prdm5LacZ/LacZ mice of both genders (Figure 6F and Table S2). Separate measurements of the cortical and trabecular compart- ments revealed a more robust reduction in all measured parameters in cortical regions than trabecular areas (Table S2), coinciding with the areas where downregulation of Collagen I and Decorin was predominantly observed in mutant embryo limbs. In summary, Prdm5LacZ/LacZ animals display a significant reduction in collagen levels and fibrillogenesis, likely resulting in the observed osteopenic phenotype. Discussion In this study, we uncover a novel function for Prdm5 in promoting the transcription of key extracellular matrix genes in osteoblastic cells. We find that Prdm5 is specifically expressed in the osteoblastic compartment of developing bones and exerts its function along the osteogenic lineage by promoting osteogenic differentiation in culture. Mechanistically, we demonstrate that Prdm5 targets ECM gene families such as collagens and small leucine-rich proteoglycans. By association with RNA polymerase II, Prdm5 sustains the transcription of collagen I genes, while the regulation of Decorin expression is mediated via binding to a distal enhancer-like element. Prdm5 regulation of these genes occurs in PLoS Genetics | www.plosgenetics.org 7 May 2012 | Volume 8 | Issue 5 | e1002711