Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone Giorgio Giacomo Galli1, Kristian Honnens de Lichtenberg1, Matteo Carrara2, Wolfgang Hans3, Manuela Wuelling 4 , Bettina Mentz1, Hinke Arnolda Multhaupt5, Cathrine Kolster Fog 1 , Klaus Thorleif Jensen1, Juri Rappsilber6, Andrea Vortkamp4, Les Coulton 7 , Helmut Fuchs4, Vale´ rie Gailus- Durner3, Martin Hrabeˇ de Angelis3,8, Raffaele Adolfo Calogero2, John Robert Couchman5, Anders 1 Henrik Lund * 1 Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark, 2 Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Torino, Torino, Italy, 3 German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum Mu¨ nchen, German Research Center for Environmental Health GmbH, Neuherberg, Germany, 4 Department of Developmental Biology, Center for Medical Biotechnology, University Duisburg- Essen, Essen, Germany, 5 Department of Biomedical Sciences and BRIC, University of Copenhagen, Copenhagen, Denmark, 6 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom, 7 Academic Unit of Bone Biology, University of Sheffield Medical School, Sheffield, United Kingdom, 8 Chair of Experimental Genetics TUM, Freising-Weihenstephan, Germany Abstract 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. Citation: Galli GG, Honnens de Lichtenberg K, Carrara M, Hans W, Wuelling M, et al. (2012) Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone. PLoS Genet 8(5): e1002711. doi:10.1371/journal.pgen.1002711 Editor: Marshall S. Horwitz, University of Washington, United States of America Received February 22, 2012; Accepted March 29, 2012; Published May 10, 2012 Copyright: 2012 Galli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work in the authors’ laboratory was supported by the Danish National Research Foundation, the Danish National Advanced Technology Foundation, the Novo Nordisk Foundation, the EC FP7 programs (ONCOMIRS, grant agreement number 201102, and APO-SYS: This publication reflects only the authors’ views. The commission is not liable for any use that may be made of the information herein.), the Lundbeck Foundation, and the Danish Cancer Society. This work has also been funded by the German Federal Ministry of Education and Research to the German Mouse Clinic (NGFNplus grant No. 01GS0850), Infrafrontier grant No. 01KX1012), and by an EU grant (EUMODIC, LSHG-2006-037188, German Mouse Clinic) and the Wellcome Trust (senior research fellowship to JR 084229, centre core grants 077707 and 092076, and instrument grant 091020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: anders.lund@bric.ku.dk Introduction PRDM proteins constitute a family of transcriptional regulators characterized by the presence of a N-terminal PR- domain that shares 20–30% similarity to the SET domain of histone methyltransferases and a variable number of zinc-finger domains typically involved in protein-DNA or protein-protein interaction [1]. Members of this family influence tissue specific differentiation as demonstrated for Prdm1 in lymphoid cell maturation [2], and Prdm16 in brown fat development [3]. Moreover, several members of the family are deregulated in pathological settings, most notably cancer, by acting either as oncogenes or tumor suppressors [1]. PRDM5 localizes to human chromosome 4q26 and encodes, aside from the PR domain, 16 C2H2 zinc fingers. PRDM5 has previously been reported to lack intrinsic histone methyltransferase activity but to predominantly repress transcription by recruiting G9a and HDACs enzymes to target genes [4]. Furthermore, PRDM5 has been indicated as a potential tumor suppressor in various cancers [5–7], but its role in mammalian development and normal physiology has not been addressed. In zebrafish, Prdm5 loss induces morphogenic defects due to impairment of convergent extension movements at the gastrulation stage, likely resulting from deregulation of the WNT inhibitor Dkk1 [8]. Recently, mutations in PRDM5 were detected in Brittle Cornea Syndrome (BCS) [9], a connective tissues disease characterized by thinning of the cornea and a wide spectrum of additional symptoms including dermal and skeletal defects [10]. Bone is composed of a highly specialized, mineralized collag- enous matrix that provides tensile strength to the skeletal system. Collagen I is the major component of osteoblasts matrix, composed of a heterotypic triple helix derived from Col1a1 and Col1a2 chains typically in a 2:1 stoichiometric ratio [11,12]. Approximately 40 collagen genes are annotated in mammalian genomes encoding around 28 proteins and, of these, type I collagen is part of the subfamily of fibrillar collagens [13]. Collagen chains are synthesized and assembled as triple helical procollagen molecules. Extracellular proteinase cleavage of N- and PLoS Genetics | www.plosgenetics.org 1 May 2012 | Volume 8 | Issue 5 | e1002711