Evolutionarily Conserved, Growth Plate Zone-Specific Regulation of the Matrilin-1 Promoter: L-Sox5/Sox6 and Nfi Factors Bound near TATA Finely Tune Activation by Sox9

Abstract

To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer. The proximal elements include a Pe1 element binding the chondrogenic L-Sox5, Sox6, and Sox9 proteins, a SI element binding Nfi proteins, and an initiator Ine element binding the Sox trio and other factors. Sox9 binding to Pe1 is indispensable for functional interaction with the distal promoter. Binding of L-Sox5/Sox6 to Ine and Nfib to SI modulates Sox9 transactivation in a protein dose-dependent manner, possibly to enhance Sox9 activity in early stages of chondrogenesis and repress it at later stages. Hence, our data suggest a novel model whereby Sox and Nfi proteins bind to conserved Matn1 proximal elements and functionally interact with each other to finely tune gene expression in specific zones of the cartilage growth plate.

ACKNOWLEDGMENTS

We are grateful to B. de Crombrugghe for providing plasmid p3000i3020Col2a1 and SOX9, L-Sox5, and Sox6 antisera, to P. Berta for the GST-SOX9 plasmid, to R. Gronostajski for Nfia, Nfib, Nfic, and Nfix expression plasmids, and to N. Mermod for the CTF1 expression plasmid. We thank P. Szabó for introducing O.R. to genomic footprinting, A. Simon, E. Horváth, I. Kravjár, and K. Kávai for excellent technical assistance, and M. Tóth for the artwork.

This work was supported by grant OTKA PD50006 to E.K., by grants OTKA T049608 from the Hungarian National Scientific Research Foundation, ETT 008/2006 from the Medical Research Council of Hungary, and GVOP-3.1.1.-2004-05-0290/3.0 from the Economic Competitiveness Operative Program of the National Development Plan to I.K., and by NIH/NIAMS grant AR60016 to V.L. Á. Zvara and E. Barta were supported by János Bolyai fellowships from the Hungarian Academy of Sciences (BO/00381/07 and BO/00383/08). This work was also partly supported by grant AVINOMID from the Ányos Jedlik Programme of the National Office for Research and Technology (NKTH) to L.G.P.