Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing
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Fecha
2017-04
Profesor/a Guía
Facultad/escuela
Idioma
en
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Editor
Elsevier B.V.
Nombre de Curso
Licencia CC
ATRIBUCIÓN-NOCOMERCIAL-SINDERIVADAS 4.0 INTERNACIONAL
CC BY-NC-ND 4.0
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Licencia CC
https://v2.sherpa.ac.uk/id/publication/16823
Resumen
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. © 2017 Elsevier B.V.
Notas
Indexación: Scopus
Palabras clave
Adipose-tissue derived stromal cells, Deacetylase, Epigenetic regulators, Epigenetics, Histone, Mesenchymal stem cell, Methyltransferase
Citación
Gene Volume 609, Pages 28 - 3720 April 2017
DOI
10.1016/j.gene.2017.01.019