Prdm5 Promotes the Transcription of ECM Genes Figure 4. Prdm5 targets all collagen genes and regulates type I collagen expression. A) ChIP-qPCR validation of Prdm5 peaks in collagen genes or negative control regions from an independent chromatin preparation immunoprecipitated with IgG, Prdm5-Ab1 (in black and blue respectively, plotted on left Y axis) or Prdm5-Ab2 (in red, plotted on right Y axis). Orange horizontal line represents the highest ‘‘noise’’ value obtained by ChIP-qPCR on a set of negative regions. B) Biological processes enrichment from gene ontology annotation of Prdm5 target regions. C) Distribution of the ‘‘peak centre’’ position for collagen genes targeted by Prdm5 or random sampling of Prdm5 target genes according to genetic feature. D) Correlation between Prdm5-Ab1 coverage inside the gene body of all mouse collagen genes (X-axis) and Pol II coverage in the same regions (Y-axis) normalized by base pairs. E) qRT-PCR for Prdm5, Col1a1 and Col1a2 in MC3T3 cells treated for 72 hours with siRNA oligos against Prdm5 (siPrdm5-1 and -2) or controls. Results are presented as average of four independent experiments 6 SD; * = p,0.05, ** = p,0.01. F) Upper panel. ChIP-qPCR for RNA Pol II in WT and mutant (blue and red respectively) calvarial osteoblasts along the Col1a1 gene. IgG control is represented by black and green lines respectively. X-axis = distance (in bp) from Col1a1 TSS, * = p,0.05 (unpaired t-test). Lower panel. Genome browser snapshot of the corresponding Col1a1 genomic region displaying MC3T3 tracks for: qPCR amplicons, IgG, RNA Pol II and Prdm5-Ab1 coverage. G) Western blot from co-immunoprecipitation experiment of HA-PRDM5 in HEK293 cells; endogenous interacting proteins or IgG are indicated. doi:10.1371/journal.pgen.1002711.g004 PLoS Genetics | www.plosgenetics.org 6 May 2012 | Volume 8 | Issue 5 | e1002711