Prdm5 Promotes the Transcription of ECM Genes Figure 1. Prdm5 is expressed in osteoblast regions of developing bones. A) Scheme for the generation of the Prdm5LacZ/LacZ mouse strain. B–E) X-gal stainings of Prdm5LacZ/LacZ 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 Prdm5 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 Prdm5LacZ/LacZ newborn skull at P0. Pronounced staining in sutures is indicated with an arrow. Bars = 1 mm, except for (G) where bar = 200 mm. doi:10.1371/journal.pgen.1002711.g001 significance of Prdm5 in chondrogenesis was evaluated by knocking down Prdm5 in the ATDC5 chondrogenic cell line (Figure S2A). In this system, Prdm5 loss did not affect chondrogenic differentiation, as evaluated by measuring glycos- aminoglycan deposition (Figure S2B). To investigate transcriptional changes imposed by Prdm5 loss in primary osteoblasts, expression levels of a series of osteogenic markers in Prdm5 wild type and mutant calvarial osteoblasts were measured by qRT-PCR. Significant reduction was observed for transcripts encoding Osteocalcin (Bglap1) and Bone sialoprotein (Ibsp), both late osteoblast markers, while transcript levels for early markers such as Osteopontin (Spp1), Osterix (Sp7) and Runx2 were unchanged (Figure 2E). When assayed for mineralization activity, no difference in matrix calcification was detected by Alizarin red staining in calvarial osteoblasts from cohorts of WT and mutant animals (Figure S2C and S2D). However, this phenotype may depend on cellular adaptation in culture, since treatment of wild type calvarial osteoblasts with a siRNA oligo which efficiently reduces Prdm5 levels, resulted in decreased matrix mineralization after 14 days of osteogenic stimulation (Figure S2E). Collectively, the data indicate that Prdm5 exerts a cell- autonomous function in the osteogenic pathway. Prdm5 targets gene bodies of transcriptionally active genes via a consensus sequence To unveil the molecular functions of Prdm5 in osteoblastic cells and identify direct target genes, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed for Prdm5 in the MC3T3 cell line. Two different Prdm5 polyclonal antibodies were generated and western blot analysis of Prdm5 wild type and mutant mouse embryonic fibroblasts revealed that these antibodies recognize distinct epitopes (Figure S3A), and both were confirmed as suitable for ChIP experiments (Figure S3B). Data from ChIP-seq analyses with the two antibodies were overlaid resulting in 1712 common loci we defined as high confidence Prdm5 target regions (Figure S3C and Table S1). Interestingly, 29% of Prdm5 peaks resided in promoter regions, while 39% of the peaks resided inside the body of genes (Figure 3A). Across all genes, Prdm5 binding was distributed throughout the length of target genes with the highest density around the TSSs (Figure 3B). A de novo motif finding algorithm for Prdm5 peak centers identified a putative consensus sequence for Prdm5 binding (Figure 3C). This sequence bears strong similarity to a Prdm5 consensus previously identified by in vitro random PLoS Genetics | www.plosgenetics.org 3 May 2012 | Volume 8 | Issue 5 | e1002711