Hmgb1 can facilitate activation of the matrilin-1 gene promoter by Sox9 and L-Sox5/Sox6 in early steps of chondrogenesis Tibor Szénási a,1 , Erzsébet Kénesi a,1 , Andrea Nagy a,b , Annamária Molnár a , Bálint László Bálint c , Ágnes Zvara b , Zsolt Csabai a , Ferenc Deák a , Beáta Boros Oláh c , Lajos Mátés b , László Nagy c,d , László G. Puskás b,e , Ibolya Kiss a,e, ⁎ a Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, Hungary b Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, Hungary c Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, H-4032 Debrecen, Hungary d MTA-DE “Lendulet” Immunogenomics Research Group, University of Debrecen, H-4032 Debrecen, Hungary e Avidin Ltd., H-6726 Szeged, Hungary abstract article info Article history: Received 23 November 2012 Received in revised form 8 July 2013 Accepted 9 July 2013 Available online 13 July 2013 Keywords: Growth plate Cartilage-specific regulation Matrilin Transgenic mice Chromatin immunoprecipitation Silencing The architectural high mobility group box 1 (Hmgb1) protein acts as both a nuclear and an extracellular regulator of various biological processes, including skeletogenesis. Here we report its contribution to the evolutionarily conserved, distinctive regulation of the matrilin-1 gene (Matn1) expression in amniotes. We previously demonstrated that uniquely assembled proximal promoter elements restrict Matn1 expression to specific growth plate cartilage zones by allowing varying doses of L-Sox5/Sox6 and Nfi proteins to fine-tune their Sox9-mediated transactivation. Here, we dissected the regulatory mechanisms underlying the activity of a conserved distal promoter element 1. We show that this element carries three Sox-binding sites, works as an enhancer in vivo, and allows promoter activation by the Sox5/6/9 chondrogenic trio. In early steps of chondrogenesis, declining Hmgb1 expression overlaps with the onset of Sox9 expression. Unlike repression in late steps, Hmgb1 overexpression in early chondrogenesis increases Matn1 promoter activation by the Sox trio, and forced Hmgb1 expression in COS-7 cells facilitates induction of Matn1 expression by the Sox trio. The conserved Matn1 control elements bind Hmgb1 and SOX9 with opposite efficiency in vitro. They show higher HMGB1 than SOX trio occupancy in established chondrogenic cell lines, and HMGB1 silencing greatly increases MATN1 and COL2A1 expression. Together, these data thus suggest a model whereby Hmgb1 helps re- cruit the Sox trio to the Matn1 promoter and thereby facilitates activation of the gene in early chondrogenesis. We anticipate that Hmgb1 may similarly affect transcription of other cartilage-specific genes. © 2013 Elsevier B.V. All rights reserved. 1. Introduction SRY-related high-mobility-group (HMG) box (Sox) proteins and canonical high-mobility-group box (HMGB) proteins have distantly re- lated DNA-binding domains that regulate gene expression by diverse mechanisms during development and disease. Several Sox proteins control cell-fate decisions in early steps of endochondral bone formation [1], whereas the Hmgb1 protein was shown to regulate later steps [2]. We determined here the role of Hmgb1 in early steps by focusing on the control region of the matrilin-1 gene (Matn1) [3]. The Sox and Hmgb protein families have similar as well as distinct features [1,4,5]. Their HMG boxes show only 20% identity, but both interact with the minor groove of the DNA helix and induce a sharp bend of this helix upon binding. While Hmgb proteins are abundant and widely expressed non-histone chromatin components, Sox pro- teins are expressed at a low level and only in certain cell types. Hmgb proteins bind distorted DNA transiently and without sequence specific- ity [4,6]. Lacking a transactivation domain, they act only as architectural Biochimica et Biophysica Acta 1829 (2013) 1075–1091 Abbreviations: CEC, chicken embryo chondrocyte; CEF, chicken embryo fibroblast; ChIP, chromatin immunoprecipitation; Dpe1 and Dpe2, distal promoter upstream ele- ments 1 and 2; ECM, extracellular matrix; EMSA, electrophoretic mobility shift assay; GP, growth plate; GST, glutathione S-transferase; HDM, high density mesenchyme; HMG, high-mobility-group; Hmgb, HMG box; Ine, initiator element; LDM, low density mesenchyme; LM-PCR, ligation-mediated PCR; Nfi, nuclear factor I; Pe1, promoter element 1; RCS, rat chondrosarcoma; SI and SII, silencer elements I and II ⁎ Corresponding author at: Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári krt. 62., H-6726 Szeged, Hungary. Tel.: +36 62 599 782; fax: +36 62 432756. E-mail addresses: sztibor@brc.hu (T. Szénási), kenesie@gmail.com (E. Kénesi), nagya@brc.hu (A. Nagy), molnar.annamaria@gmail.com (A. Molnár), lbalint@med.unideb.hu (B.L. Bálint), zvara@brc.hu (Á. Zvara), csabai0911@gmail.com (Z. Csabai), deak.ferenc@brc.mta.hu (F. Deák), bejjus0102@gmail.com (B. Boros Oláh), mates.lajos@brc.mta.hu (L. Mátés), nagyl@med.unideb.hu (L. Nagy), pusi@brc.hu (L.G. Puskás), kiss@brc.hu (I. Kiss). 1 These authors contributed equally to the work and should be considered as equal first authors. 1874-9399/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagrm.2013.07.004 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm