Author Summary Bone provides the essential tensile strength of the skeletal system, constitutes an important storage for minerals, and hosts the initial differentiation stages of the hematopoietic system. In addition, bones are important endocrine organs affecting organismal metabolism. Consequently, many human diseases arise from defects in bone formation or homeostasis. Hence, deciphering the molecular mecha- nisms underlying bone formation is essential for under- standing the basis of bone and extracellular matrix- associated diseases. Here, we provide a detailed charac- terization of the cellular and molecular functions of the transcription factor Prdm5 during murine bone formation in vivo and find that Prdm5 is expressed in skeletal structures during development and that its loss impacts the ossification process, leading to a decrease in bone mineral density. A genome-wide mapping of Prdm5 binding sites in pre-osteoblastic cells reveals an unprece- dented role for a transcription factor in targeting virtually all members of the Collagen and SLRP gene families. Interestingly, Prdm5 predominantly binds exonic regions of collagen genes and associates with RNA Polymerase II to sustain Collagen I transcription. C-terminal telopeptides leads to mature tropocollagen that is further assembled into fibrils and fibers [14]. The latter process is regulated by other extracellular macromolecules including pro- teoglycans from the Small Leucine Repeat family (SLRP), such as Decorin and Fibromodulin [15,16]. A number of transcription factors have been discovered as regulators of collagen I genes (reviewed in [17,18]), such as Sp1 [19], Cebpb [20] or members of the AP1 family [21]. Furthermore, a number of transcription factors are known to be key regulators of bone development, such as Runx2 [22], which controls the expression of a multitude of extracellular matrix (ECM) genes essential for both the chondrogenic and osteogenic programs [23]. We present here a novel molecular function for Prdm5 in sustaining transcription of key ECM genes. Prdm5 is highly expressed in the osteoblast region of developing bones in vivo and genome wide mapping of Prdm5 occupancy in osteoblastic cells identifies all collagens and a number of SLRP genes as direct targets for Prdm5. Interestingly, Prdm5 binds predominantly within the exonic regions of collagen genes and its presence dictates the amount of intragenic RNA polymerase II. Indeed, Prdm5 sustains transcription of Collagen I genes by maintaining RNA polymerase II occupancy throughout the Col1a1 gene, while the binding to a distal enhancer element upstream of Decorin gene suggests a further role in chromatin organization. Osteoblasts lacking Prdm5 display decreased Collagen I and Decorin expression leading to reduced Collagen I fiber assembly in vivo. Downregulation of these key extracellular matrix genes likely participates in the delayed ossification and decreased bone mineral density observed in Prdm5 mutant mice. Our data defines novel roles for Prdm5 as a transcriptional modulator of collagen genes by influencing RNA polymerase II occupancy, as well by binding to enhancer-like elements in osteoblastic cells. Results Prdm5 is expressed in developing bones of mouse embryos To address a possible role in mammalian development for Prdm5, a gene-trap mouse model featuring the integration of a b- Prdm5 Promotes the Transcription of ECM Genes galactosidase-neomycin (b-geo) cassette in intron 2 of the Prdm5 gene was generated (Prdm5LacZ). This cassette is preceded by a splice acceptor site to direct exon 2-b-geo splicing and ends with a poly-A site to terminate transcription (Figure 1A). In mutant cells, expression of the Prdm5 locus results in the production of a fusion transcript between the first two exons of Prdm5 (<60 amino acids) and the b-geo cassette with a resulting fusion protein of approximately 135 kDa in size (Figure S1A). To validate the effectiveness of the gene-trap system, we quantified the levels of the wt Prdm5 allele in Prdm5LacZ/LacZ embryonic fibroblasts and found it to reach a maximum of 10% relative to the expression in wt littermates (Figure S1A). In adult organs, except for brain, testis and lung, we observed Prdm5 levels reduction to be at least 85% in the tissues tested (Figure S1B). In contrast to the essential role in zebrafish [8], Prdm5LacZ/LacZ mice are viable and fertile and the mutant allele segregates according to Mendelian ratios (Figure S1C). Gross pathology analysis did not reveal obvious abnormal- ities and no notable differences in weight were detected between mutant mice and wild type littermates up to the age of 56 weeks (Figure S1D). To characterize the expression pattern of Prdm5, we tracked the b-galactosidase expression driven by the endogenous Prdm5 promoter by whole mount X-gal staining at various developmental stages. At E10.5–12.5 the LacZ reporter was expressed in a diffuse staining pattern along meso-endodermal derived regions with higher intensity in the heart (Figure 1B and 1C). At E14.5 LacZ staining accumulated in limbs and snout regions and in particular in cartilaginous templates (Figure 1D). From E16.5 a specific staining pattern was observed in skeletal elements, particularly in long bones and ribs (Figure 1E). In these tissues LacZ was highly expressed in the osteoblastic regions including the trabecular compartment and periosteum/perichondrium (arrows in Figure 1F). LacZ staining on sections from E16.5 embryo tibia revealed that the Prdm5 promoter is active in a subpopulation of cells in the osteoblast region (periosteum and trabecular area) (Figure 1G), whereas no signal was detected in the hypertrophic or proliferative chondrocytes zones (Figure 1G). Moreover, LacZ staining of calvariae of Prdm5LacZ/LacZ mice at P0 indicated that Prdm5 expression in osteoblasts is not restricted to long bones but can be detected also in skull sutures and weakly in calvariae (Figure 1H). Robust PRDM5 expression in osteoblasts was further confirmed in primary human osteoblast cells isolated from healthy donors, compared to a panel of human immortalized cell lines of different origins (Figure S1E). Moreover we found comparable Prdm5 expression levels in mouse primary calvarial osteoblasts, the osteoblastic MC3T3 cell line and primary embryonic fibroblasts (Figure S1F). In summary, in vivo expression analyses confirmed Prdm5 as consistently expressed in osteoblastic com- partments in the mouse. Acute Prdm5 deregulation affects osteogenic differentiation in vitro Since Prdm5 is highly expressed in osteoblastic cells, we chose the MC3T3 cell line model to investigate the roles of Prdm5 during osteogenic differentiation. Cells were transduced with lentiviral shRNA constructs against Prdm5, which resulted in efficient reduction of both Prdm5 transcript and protein levels (Figure 2A). Prdm5 depletion did not significantly affect proliferation of MC3T3 cells as assessed by BrdU labeling (data not shown) but led to a significant reduction in matrix mineralization as measured by Alizarin red staining of calcium nodules upon induction of osteogenic differentiation (Figure 2B). In line with this, overex- pression of PRDM5 induced the opposite phenotype resulting in increased nodule formation (Figure 2C and 2D). In parallel, the PLoS Genetics | www.plosgenetics.org 2 May 2012 | Volume 8 | Issue 5 | e1002711