Prdm5 Promotes the Transcription of ECM Genes Figure 6. Prdm5 loss results in decreased Collagen I and Decorin levels and leads to reduced bone formation in vivo. A) Immunofluorescence staining of E16.5 tibiae for Collagen I (upper panel, bar = 200 mm), Decorin (middle panel, bar = 100 mm, asterisk indicates periosteum area) and Picrosirius red staining of WT and Prdm5LacZ/LacZ E16.5 tibiae under polarized light (lower panel, bar = 100 mm). B) H&E staining of Prdm5 WT and Prdm5LacZ/LacZ E16.5 bones. The bars indicate the length of the osteoblast region (bar = 100 mm). C) Von Kossa staining of WT and Prdm5LacZ/LacZ E16.5 tibiae. D) mCT measurements of E18.5 embryos. BV/TV = Bone volume/Total embryo volume. Values for Prdm5LacZ/LacZ 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 Prdm5LacZ/LacZ 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 Prdm5LacZ/LacZ animals. Each dot represents the average of two measurements on each animal tested (n = 9/10 per group). doi:10.1371/journal.pgen.1002711.g006 vivo in developing limbs resulting in the decreased bone mineral density observed in Prdm5LacZ/LacZ animals. Members of the PRDM family display tissue specific patterns of expression [24], in agreement with a role in tissue specific differentiation. In line with this, Prdm1 is known to be a master regulator of lymphoid differentiation [2], Prdm16 to control brown fat development [3], Prdm14 to regulate embryonic stem cell pluripotency and germ cell differentiation [25,26] and Prdm9 to determine meiotic recombination hotspots [27]. So far Prdm5 has not been characterized in the context of mammalian development. Our study thus provides the first evidence of a role for Prdm5 in tissue specific differentiation extending the concept of PRDM proteins as regulators of specific tissues and in agreement with the idea of functional specialization of PRDM members during expansion of the family in vertebrates [28]. The identification of high Prdm5 expression in osteoblasts and its function in osteogenic differentiation in vitro permitted us to evaluate its molecular functions in a relevant cellular context. Indeed, it is noteworthy that Prdm5 silencing does not restrict chondrogenic differentiation in vitro suggesting cell type specificity in Prdm5 action. By genome wide mapping of Prdm5 binding sites in a pre- osteoblastic cell line, we demonstrate a unique capacity of Prdm5 to bind the whole family of collagen genes and especially to bind these genes within the gene body. This is an unprecedented feature for a transcriptional regulator, i.e. binding the gene bodies of all the members of such a large gene family. Moreover, we observed Prdm5 occupancy in genomic regions encoding for non-collagen proteins with collagenous domains (e.g. C1q family), suggesting that either Prdm5 consensus sequence is overrepresented in genomic regions encoding for collagenous domains, or that simply Prdm5 binds common regulatory elements shared by specific gene families. Indeed we detected Prdm5 binding to several members of the SLRP family as well as a number of genes encoding for essential extracellular matrix components (complete list in Table S1), demonstrating that Prdm5 potentially regulates a wide but specific transcriptional program necessary for proper extracellular matrix formation. In this study, we focus on fibrillar collagens, particularly Collagen I genes; these were highly enriched for Prdm5 binding and are essential constituent of osteoblastic matrix. A number of transcription factors have been shown to regulate Collagen I genes and the location of their binding sites on Col1a1 and Col1a2 promoters have been shown to determine the expression in different osteoblast subtypes [29,30]. Very little is known concerning the regulation of Col1a1 and Col1a2 gene expression by factors binding downstream of their promoter, except for a repressor region located within intron 1 that can recruit GATA and IRF proteins to block enhancer stimulation of promoter activity [31,32]. To our knowledge, Prdm5 is the first example of a transcriptional modulator able to bind a consensus sequence found inside collagen genes, particularly Col1a1 and Col1a2. Interestingly, upon Prdm5 loss in osteoblasts, we observe decreased RNA polymerase II occupancy throughout the gene body of the Col1a1 gene, while at the TSS, RNA polymerase II occupancy remains unchanged. This suggests that Prdm5 is sustaining transcription of the Col1a1 gene by affecting polymerase processivity. This hypothesis is further supported by the observed interaction between Prdm5 and RNA polymerase II. The higher enrichment for the processive, ‘‘elongating’’, form of RNA polymerase II phosphorylated on Serine 2 of its C-terminal domain, argues in favor of Prdm5 directly sustaining RNA polymerase II processivity although further analyses are required to clearly define the underlying mechanism. Since Prdm5 binds predominantly exonic regions of collagen genes, and splicing is known to occur co-transcriptionally [33], there may be a role for Prdm5 in the coupling of splicing to transcription. However, we failed to detect an altered Col1a1 splice pattern in Prdm5 mutant osteoblasts (data not shown), making this hypothesis less likely. The correlation between Prdm5 binding levels within collagen genes and RNA polymerase II occupancy suggests that Prdm5 in certain contexts may act as a transcriptional activator in contrast to the previously described role of PRDM5 acting predominantly as a transcriptional repressor [4]. Interestingly, we observe major enrichment for Prdm5 target genes to be transcriptionally active, presenting both H3K4me3 and RNA Polymerase II (Figure 3D), suggesting a predominant role for Prdm5 in transcriptional activation in osteoblastic cells. The Prdm5 target repertoire extends also to SLRP genes, including Decorin (Dcn), Fibromodulin (Fmod), Biglycan (Bgn) and Epiphycan (Epc). Members of this family are involved in correct type I collagen fibers assembly [15]. Specifically, we found a Prdm5 binding site residing 100 kb upstream from the Dcn TSS. Decorin is the closest gene within a range of 760 kb and the Prdm5 binding site displays the chromatin signature of an enhancer element, suggesting a previously undiscovered role for Prdm5 in transcriptional enhancement or chromatin organization. Decorin has been shown to bind directly collagen I [34] and, in bone tissues, mice lacking Decorin display decreased collagen fiber diameter, although they do not show pronounced skeletal defects [35]. Our data show a marked decrease in fibrillar collagen staining, as evaluated by collagen fibers birefringency, indicating that downregulation of both Collagen I transcripts as well as Decorin might contribute to the skeletal phenotype observed. Of direct relevance, mutations in PRDM5 were recently detected in Brittle Cornea Syndrome [9]. Brittle Cornea Syndrome is a generalized connective tissue disease characterized by impairment of ECM formation and patients develop a number of other symptoms, aside from ocular defects, such as skin hyperelasticity, joint hypermobility and skeletal defects [10,36]. While no studies so far have causally linked PRDM5 to this disease, our data demonstrating that Prdm5 regulates the expression of fibrillar collagens are in line with the defects in ECM production and assembly characteristically observed in BCS patients. The distinct expression pattern observed for Prdm5 in developing skeleton prompted us to evaluate a bone phenotype but future studies will be needed to understand if Prdm5LacZ/LacZ mice develop also corneal, skin and joint defects resembling BCS or PLoS Genetics | www.plosgenetics.org 10 May 2012 | Volume 8 | Issue 5 | e1002711