Structural characterization of the HMG and the dimerization domain of SOX9: Understanding the molecular basis of cooperativity

The SRY-related HMG box 9 (SOX9) transcription factor is involved in the early stages of mammalian sex determination and cartilage formation. Mutations that affect SOX9 function are linked to campomelic displasia, a severe skeletal disease often accompanied by XY female sex reversal. SOX9 DNA binding sites occur both singly and tandemly repeated in many developmental genes which suggest that protein oligomerization may serve an important regulatory role. In a subset of SOX transcription factors (SOX8/9/10), a dimerization region located immediately amino terminal to the HMG DNA binding domain plays an important role in gene regulation. The objective of this research was to map the most essential amino acids that contribute to dimerization and DNA binding in SOX9. The current research demonstrates that (i) dimerization increases the stability of protein-DNA complexes, (ii) sequence and spacing constraints of SOX9 binding sites affect cooperativity, (iii) the dimerization domain lack regular secondary structure, and (iv) a region of hydrophobic amino acids in the dimerization region appear to be important for function and may possibly present on the same face of an amphipathic helix that forms upon DNA binding. This research will also be constructive in the future design and preparation of a SOX9 protein-DNA complex for high-resolution structural studies.