Prdm5 Promotes the Transcription of ECM Genes osteogenic markers as indicated. Expression values were normalized to a panel of housekeeping genes (Rps18, Ubc, Actb, Rpl0) 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). doi:10.1371/journal.pgen.1002711.g002 oligonucleotide selection experiments (Figure S3D) [4]. To confirm whether DNA fragments containing the identified sequence motif were directly recognized by Prdm5, in vitro pulldown assays using biotinylated DNA oligonucleotide probes were performed. Overexpressed HA-PRDM5 readily bound to DNA probes containing the consensus motif (a region of Col1a1 exon 33, containing 3 motifs with p-score 0.96), whereas the binding was impaired by mutation of the first and last two guanines of the consensus motifs (Figure 3C). Gene activity in MC3T3 cells was also estimated by performing ChIP-seq on the same chromatin preparation for total RNA polymerase II, histone H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 9 trimethylation (H3K9me3), allowing for a correlation of Prdm5 binding to gene activity. Prdm5 bound genes were enriched either for the presence of H3K9me3 or H3K4me3, with a strong preference for Prdm5 target genes to present H3K4me3 peaks around their TSS, when compared to the average of 100 permutations of a size-matched set of random genes (Figure 3D). These results indicate that Prdm5 may act both as a transcriptional repressor and activator in a promoter-dependent fashion and that in MC3T3 cells the majority of Prdm5 target genes are actively transcribed. Indeed, Prdm5 target genes were also associated with RNA polymerase II occupancy (to a similar extent as H3K4me3) (Figure 3D) and expression analysis from microarray data of MC3T3 cells showed that genes bound by Prdm5 are character- ized by a general increase in expression signal with respect to the total of the genes represented on the microarray (Figure 3E). Prdm5 targets genomic loci encoding collagen genes and sustains collagen I transcription in osteoblasts Annotation of the Prdm5 bound loci and detailed analysis followed by ChIP-qPCR validation on independent samples with both Prdm5 antibodies strikingly revealed that 42 of 43 collagen genes in the mouse genome contained at least one Prdm5 peak (Figure 4A). Moreover, the results were validated in primary wt and LacZ/LacZ calvarial osteoblasts, where Prdm5 enrichment was strongly reduced in mutant cells (Figure S4A and S4B). When genomic regions bound by Prdm5 were subsequently annotated according to biological processes, a strong enrichment was observed for genes associated with collagen fibril and extracellular matrix organization, as well as bone development (Figure 4B). Prdm5 binding to collagen genes occurred almost Figure 3. Analyses of Prdm5 chromatin interactions. A) Diagram illustrating the overall distribution of Prdm5 binding sites categorized according to the distance from the nearest TSS (see Text S1). 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 Text S1). Lower panel. DNA pulldown assay from nuclear extract of 293 cells overexpressing HA-PRDM5 using biotinylated oligos representing the Col1a1 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. doi:10.1371/journal.pgen.1002711.g003 PLoS Genetics | www.plosgenetics.org 5 May 2012 | Volume 8 | Issue 5 | e1002711