Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5589337 | Gene | 2017 | 10 Pages |
Abstract
Epigenetic mechanisms control phenotypic commitment of mesenchymal stromal/stem cells (MSCs) into osteogenic, chondrogenic or adipogenic lineages. To investigate enzymes and chromatin binding proteins controlling the epigenome, we developed a hybrid expression screening strategy that combines semi-automated real-time qPCR (RT-qPCR), next generation RNA sequencing (RNA-seq), and a novel data management application (FileMerge). This strategy was used to interrogate expression of a large cohort (n > 300) of human epigenetic regulators (EpiRegs) that generate, interpret and/or edit the histone code. We find that EpiRegs with similar enzymatic functions are variably expressed and specific isoforms dominate over others in human MSCs. This principle is exemplified by analysis of key histone acetyl transferases (HATs) and deacetylases (HDACs), H3 lysine methyltransferases (e.g., EHMTs) and demethylases (KDMs), as well as bromodomain (BRDs) and chromobox (CBX) proteins. Our results show gender-specific expression of H3 lysine 9 [H3K9] demethylases (e.g., KDM5D and UTY) as expected and upregulation of distinct EpiRegs (n > 30) during osteogenic differentiation of MSCs (e.g., HDAC5 and HDAC7). The functional significance of HDACs in osteogenic lineage commitment of MSCs was functionally validated using panobinostat (LBH-589). This pan-deacetylase inhibitor suppresses osteoblastic differentiation as evidenced by reductions in bone-specific mRNA markers (e.g., ALPL), alkaline phosphatase activity and calcium deposition (i.e., Alizarin Red staining). Thus, our RT-qPCR platform identifies candidate EpiRegs by expression screening, predicts biological outcomes of their corresponding inhibitors, and enables manipulation of the human epigenome using molecular or pharmacological approaches to control stem cell differentiation.
Keywords
HDAC7HDAC4HDAC9WDR5ALPLHDAC2Hdac5UTYHDAC1histone deacetylase 9CBXEpigenetic regulatorHDAC11Epigenetic regulatorsBRDKMTHDACMSCRT-qPCRMesenchymal stromal/stem cellRNA-seqKDMAlkaline phosphataseEpigeneticsBromodomainRNA sequencingdeacetylaseMesenchymal stem celllysine demethylaselysine methyltransferaseMethyltransferaseHistonehistone acetyl transferasehistone deacetylasehistone deacetylase 1Histone deacetylase 2Histone deacetylase 5Histone deacetylase 4PanobinostatHAT
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Authors
Amel Dudakovic, Martina Gluscevic, Christopher R. Paradise, Halil Dudakovic, Farzaneh Khani, Roman Thaler, Farah S. Ahmed, Xiaodong Li, Allan B. Dietz, Gary S. Stein, Martin A. Montecino, David R. Deyle, Jennifer J. Westendorf, Andre J. van Wijnen,