School of BioSciences, The University of Melbourne, Melbourne, Victoria, Australia.
BMC Mol Cell Biol. 2020 Apr 15;21(1):27. doi: 10.1186/s12860-020-00270-5.
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.
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.
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.
基因调控的变化被广泛认为是适应表型进化的重要驱动因素。然而，这种变化背后的具体分子机制仍知之甚少。染色质状态在基因调控中起着至关重要的作用，它影响编码基因座对转录机制的可及性。因此，染色质重塑因子功能的改变是驱动与表型适应相关的基因表达变化的有力候选者。在这里，我们在已灭绝的有袋动物袋狼和真兽类狼之间发现了染色质重塑因子 CHD9 中的氨基酸同型性，它们的颅骨表现出显著的趋同进化。CHD9 参与成骨作用，但它在该过程中的作用仍知之甚少。我们研究了 CHD9 是否能够调节成骨靶基因的表达，并研究了 CHD9 DNA 结合域中一个关键取代的功能。
我们研究了 CHD9 是否能够上调其成骨靶基因，即 HEK293T 细胞中的 RUNX2、骨钙素（OC）和碱性磷酸酶（ALP）。我们发现，CHD9 的过表达上调了 RUNX2，即成骨细胞命运的主调控因子，但不是下游基因 OC 或 ALP，这支持了 CHD9 调节成骨前体细胞而不是终末成骨细胞的观点。我们还发现，CHD9 DNA 结合域中的进化取代不会改变蛋白质二级结构，但能够驱动 RUNX2 激活的微小但无统计学意义的增加。最后，CHD9 无法激活 RUNX2 启动子报告载体构建体，表明 CHD9 需要完整的染色质成分才能发挥其功能。
我们通过新发现的 CHD9 上调 RUNX2 表达的能力，为 CHD9 在成骨分化中的作用提供了新的证据。虽然我们未能在 HEK293T 细胞中确定进化取代的显著功能后果，但我们的研究为蛋白同型性及其在发育过程中的作用的功能研究提供了重要的步骤。编码基因突变可能是驱动基因表达适应性变化的一种机制，其验证对于确定进化同型性的功能后果至关重要。
