DNA methylation of the Trip10 promoter accelerates mesenchymal stem cell lineage determination.

Epigenetic regulation of gene expression by DNA methylation and histone modification controls cell fate during development and homeostasis in adulthood. Aberrant epigenetic modifications may lead to abnormal development, even diseases. We have found that Trip10 (thyroid hormone receptor interactor 10), an adaptor protein involved in diverse functions, is epigenetically regulated during lineage-specific induction of human bone marrow-derived mesenchymal stem cells (MSCs). To determine whether DNA methylation-induced gene silencing is sufficient to restrict cell fate changes, we applied an invitro method to specifically methylate the promoter of Trip10. Our hypothesis was that the methylation status of the Trip10 promoter in MSCs alters the differentiation preference of MSCs. Transfection of in vitro-methylated Trip10 promoter DNA into MSCs resulted in progressive accumulation of cytosine methylation at the endogenous Trip10 promoter, reduced Trip10 expression, and accelerated MSC-to-neuron and MSC-to-osteocyte differentiation. A two-component EGFP reporter gene system was established to confirm the level of transcriptional silencing and visualize the targeted DNA methylation. EGFP expression induced in the reporter system by targeted Trip10 methylation was reversed by adding 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, confirming that the suppressed Trip10 expression and disrupted MSC differentiation resulted from the in vitro-introduced methylations in the Trip10 promoter. With this targeted DNA methylation and reporter system, we are able to monitor the progression of locus-specific DNA methylation in vivo and correlate such changes with potential functional changes. Using this approach, we have established a new role for Trip10, showing that the level of Trip10 expression is associated with the maintenance and differentiation of MSCs.

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