A new long form of c-Maf cooperates with Sox9 to activate the type II collagen gene

A new long form of the c-Maf transcription factor (Lc-Maf) was identified and shown to interact specifically with SOX9 in a yeast two-hybrid cDNA library screening. Lc-Maf encodes an extra 10 Amino acids at the carboxyl terminus of c-Maf and contains a different 3'-untranslated region compared with c-Maf. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and Glutathione S-transferase pull-down assays, which mapped the interacting domain of SOX9 to the high mobility group box DNA binding domain and that of Lc-Maf to the basic leucine zipper motif. In situ hybridizations showed that Lc-Maf RNA was coexpressed with Sox9 and Col2a1 RNA in areas of precartilaginous mesenchymal condensations during mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5'-end of a 48-bp Col2a1 enhancer element near the high mobility group binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2a1 enhancer and increased the transcription of the endogenous Col2a1 gene. In summary, Lc-Maf is the first transcription factor shown to interact with Sox9, to be coexpressed with Sox9 during an early step of chondrogenesis and to cooperate with Sox9 in activating a downstream target gene of Sox9.