Abstract
BACKGROUND: Changes in gene regulation are widely recognized as an important driver of adaptive phenotypic evolution. However, the specific molecular mechanisms that underpin such changes are still poorly understood. Chromatin state plays an essential role in gene regulation, by influencing the accessibility of coding loci to the transcriptional machinery. Changes in the function of chromatin remodellers are therefore strong candidates to drive changes in gene expression associated with phenotypic adaptation. Here, we identify amino acid homoplasies in the chromatin remodeller CHD9, shared between the extinct marsupial thylacine and eutherian wolf which show remarkable skull convergence. CHD9 is involved in osteogenesis, though its role in the process is still poorly understood. We examine whether CHD9 is able to regulate the expression of osteogenic target genes and examine the function of a key substitution in the CHD9 DNA binding domain. RESULTS: We examined whether CHD9 was able to upregulate its osteogenic target genes, RUNX2, Osteocalcin (OC) and ALP in HEK293T cells. We found that overexpression of CHD9 upregulated RUNX2, the master regulator of osteoblast cell fate, but not the downstream genes OC or ALP, supporting the idea that CHD9 regulates osteogenic progenitors rather than terminal osteoblasts. We also found that the evolutionary substitution in the CHD9 DNA binding domain does not alter protein secondary structure, but was able to drive a small but insignificant increase in RUNX2 activation. Finally, CHD9 was unable to activate an episomal RUNX2 promoter-reporter construct, suggesting that CHD9 requires the full chromatin complement for its function. CONCLUSIONS: We provide new evidence to the role of CHD9 in osteogenic differentiation through its newly observed ability to upregulate the expression of RUNX2. Though we were unable to identify significant functional consequences of the evolutionary substitution in HEK293T cells, our study provides important steps forward in the functional investigation of protein homoplasy and its role in developmental processes. Mutations in coding genes may be a mechanism for driving adaptive changes in gene expression, and their validation is essential towards determining the functional consequences of evolutionary homoplasy.