Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia-inducible factor 1 (HIF-1). HIF-1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF-1 and mediates the hypoxic repression of a set of nuclear-encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear-encoded mitochondrial genes where it directly antagonizes c-Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short-hairpin RNA (shRNA)-expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia

Prdm5 regulates <i>Decorin</i> through a distal enhancer.

<p>A) ChIP-qPCR validation of Prdm5 peaks on selected ECM genes or negative regions from an independent sample 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) qRT-PCR analysis of <i>Decorin</i> transcript (<i>Dcn</i>) levels upon Prdm5 knockdown as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002711#pgen-1002711-g004" target="_blank">Figure 4E</a>. Results are presented as average of four independent experiments +/− SD; * = p<0.05. (C) qPCR analysis of WT and <i>Prdm5^LacZ/LacZ</i> calvarial osteoblasts for <i>Decorin</i>. Expression values were normalized to the control WT samples. *  = p<0.05; T-test, (+/+ n = 14, LacZ/LacZ n = 19 clones). D) Upper panels. Western blot analysis of Decorin levels upon Prdm5 knockdown in cell layers; Tubulin is used as loading control. Lower panels. Western blot analyses of purified proteoglycans from cell culture media from knockdown cells. Tubulin is used as purity control and Fibronectin for equal protein loading. E) ChIP-qPCR with indicated antibodies for Prdm5 binding site upstream of Dcn gene. Meg3 TSS region is used as negative control.</p

Prdm5 targets all collagen genes and regulates type I collagen expression.

<p>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 <i>Prdm5</i>, <i>Col1a1</i> and <i>Col1a2</i> in MC3T3 cells treated for 72 hours with siRNA oligos against <i>Prdm5</i> (siPrdm5-1 and -2) or controls. Results are presented as average of four independent experiments ± 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 <i>Col1a1</i> gene. IgG control is represented by black and green lines respectively. X-axis = distance (in bp) from <i>Col1a1</i> TSS, * = p<0.05 (unpaired t-test). Lower panel. Genome browser snapshot of the corresponding <i>Col1a1</i> 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.</p