title: Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing.
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.
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Amel
authors.initials: A
authors.lastname: Dudakovic
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Martina
authors.initials: M
authors.lastname: Gluscevic
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Paradise
authors.affiliation: Information Technology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Halil
authors.initials: H
authors.lastname: Dudakovic
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Khani
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Roman
authors.initials: R
authors.lastname: Thaler
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Farah S
authors.initials: FS
authors.lastname: Ahmed
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Li
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Allan B
authors.initials: AB
authors.lastname: Dietz
authors.affiliation: Department of Biochemistry, University of Vermont Medical School, Burlington, VT, USA.
authors.forename: Gary S
authors.initials: GS
authors.lastname: Stein
authors.affiliation: Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago, Chile.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Montecino
authors.affiliation: Medical Genetics, Mayo Clinic, Rochester, MN, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Deyle
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA; Biochemistry & Molecular Biology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Westendorf
authors.affiliation: Departments of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA; Biochemistry & Molecular Biology, Mayo Clinic, Rochester, MN, USA; Physiology & Biomedical Engineering, Mayo Clinic, Rochester, MN, USA. Electronic address: vanwijnen.andre@mayo.edu.
authors.forename: Andre J
authors.initials: AJ
authors.lastname: van Wijnen
mesh_terms: D002454:Cell Differentiation; D044127:Epigenesis, Genetic; D057890:Epigenomics; D059014:High-Throughput Nucleotide Sequencing; D042421:Histone Code; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA
keywords: Adipose-tissue derived stromal cells; Deacetylase; Epigenetic regulators; Epigenetics; Histone; Mesenchymal stem cell; Methyltransferase