Higashihori N, Lehnertz B, Sampaio A, Underhill T M, Rossi F, Richman J M
1 Department of Oral Health Sciences, Life Sciences Institute, Faculty of Dentistry, The Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
2 Department of Medical Genetics, Faculty of Medicine, The Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
J Dent Res. 2017 Sep;96(10):1136-1144. doi: 10.1177/0022034517716438. Epub 2017 Jun 23.
Here we investigate the role of epigenetic factors in controlling the timing of cranial neural crest cell differentiation. The gene coding for histone H3 lysine 9 methyltransferase G9A was conditionally deleted in neural crest cells with Wnt1-Cre. The majority of homozygous-null animals survived to birth but thereafter failed to thrive. Phenotypic analysis of postnatal animals revealed that the mutants displayed incomplete ossification and 20% shorter jaws as compared to their wild-type littermates. At E13.5, patterns of expression of the osteogenic transcription factor RUNX2 and the mesenchymal transcription factor TWIST are similar in controls and mutants; both overlap in areas of future intramembranous bone formation. At E14.5, the nonosteogenic mesenchyme expressed TWIST, whereas the ossification center had strong RUNX2 and osteopontin expression. In the mutants, TWIST protein was present in the osteogenic mesenchyme, while osteopontin was not expressed until E15.5. In addition, in mutants, small regions of TWIST-positive osteogenic mesenchyme were visible until E15.5. The delay in ossification and reduction in size of the ossification centers were correlated with an earlier decrease in proliferation. We used micromass cultures of the face to investigate the direct effects of G9A inhibition on skeletal differentiation. Addition of a small molecule inhibitor for G9A, BIX-01294, to wild-type cells upregulated Twist genes similar to what was observed in vivo. The inhibitor also caused decreases in several osteogenic markers. Chromatin immunoprecipitation analysis of primary osteogenic mesenchyme from calvaria revealed that Twist1 and Twist2 regulatory regions contain the repressive H3K9me2 marks catalyzed by G9A, which are removed when BIX-01294 is added. Our results establish a role for G9A and H3K9me2 in the regulation of Twist genes and provide novel insights into the significance of epigenetic mechanisms in controlling temporal and tissue-specific gene expression during development.
在此，我们研究表观遗传因子在控制颅神经嵴细胞分化时间方面的作用。利用Wnt1-Cre在神经嵴细胞中条件性敲除编码组蛋白H3赖氨酸9甲基转移酶G9A的基因。大多数纯合缺失动物存活至出生，但此后生长不良。对出生后动物的表型分析显示，与野生型同窝仔相比，突变体表现出不完全骨化且颌骨短20%。在胚胎第13.5天（E13.5），成骨转录因子RUNX2和间充质转录因子TWIST的表达模式在对照组和突变体中相似；两者在未来膜内骨形成区域重叠。在E14.5时，非成骨间充质表达TWIST，而成骨中心有强烈的RUNX2和骨桥蛋白表达。在突变体中，TWIST蛋白存在于成骨间充质中，而骨桥蛋白直到E15.5才表达。此外，在突变体中，直到E15.5才能看到TWIST阳性成骨间充质的小区域。骨化延迟和骨化中心大小减小与增殖的早期下降相关。我们利用面部的微团培养来研究G9A抑制对骨骼分化的直接影响。向野生型细胞中添加G9A的小分子抑制剂BIX-01294会上调Twist基因，类似于在体内观察到的情况。该抑制剂还导致几种成骨标志物减少。对来自颅骨的原代成骨间充质进行染色质免疫沉淀分析表明，Twist1和Twist2调控区域含有由G9A催化的抑制性H3K9me2标记，添加BIX-01294时这些标记会被去除。我们的结果确立了G9A和H3K9me2在Twist基因调控中的作用，并为表观遗传机制在发育过程中控制时间和组织特异性基因表达的重要性提供了新的见解。
