Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing

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

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)28-37
Number of pages10
JournalGene
Volume609
DOIs
StatePublished - Apr 20 2017

Fingerprint

Mesenchymal Stromal Cells
Epigenomics
Lysine
Histone Code
RNA Sequence Analysis
Methyltransferases
Transferases
Histones
Chromatin
Alkaline Phosphatase
Cell Differentiation
Carrier Proteins
Protein Isoforms
Up-Regulation
Stem Cells
Pharmacology
Staining and Labeling
Calcium
Bone and Bones
Messenger RNA

Keywords

  • Adipose-tissue derived stromal cells
  • Deacetylase
  • Epigenetic regulators
  • Epigenetics
  • Histone
  • Mesenchymal stem cell
  • Methyltransferase

ASJC Scopus subject areas

  • Medicine(all)
  • Genetics

Cite this

Dudakovic, A., Gluscevic, M., Paradise, C. R., Dudakovic, H., Khani, F., Thaler, R., ... van Wijnen, A. J. (2017). Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing. Gene, 609, 28-37. https://doi.org/10.1016/j.gene.2017.01.019

Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing. / Dudakovic, Amel; Gluscevic, Martina; Paradise, Christopher R.; Dudakovic, Halil; Khani, Farzaneh; Thaler, Roman; Ahmed, Farah S.; Li, Xiaodong; Dietz, Allan B; Stein, Gary S.; Montecino, Martin A.; Deyle, David R; Westendorf, Jennifer J; van Wijnen, Andre J.

In: Gene, Vol. 609, 20.04.2017, p. 28-37.

Research output: Contribution to journalArticle

Dudakovic, A, Gluscevic, M, Paradise, CR, Dudakovic, H, Khani, F, Thaler, R, Ahmed, FS, Li, X, Dietz, AB, Stein, GS, Montecino, MA, Deyle, DR, Westendorf, JJ & van Wijnen, AJ 2017, 'Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing', Gene, vol. 609, pp. 28-37. https://doi.org/10.1016/j.gene.2017.01.019
Dudakovic, Amel ; Gluscevic, Martina ; Paradise, Christopher R. ; Dudakovic, Halil ; Khani, Farzaneh ; Thaler, Roman ; Ahmed, Farah S. ; Li, Xiaodong ; Dietz, Allan B ; Stein, Gary S. ; Montecino, Martin A. ; Deyle, David R ; Westendorf, Jennifer J ; van Wijnen, Andre J. / Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing. In: Gene. 2017 ; Vol. 609. pp. 28-37.
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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.",
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AU - Khani, Farzaneh

AU - Thaler, Roman

AU - Ahmed, Farah S.

AU - Li, Xiaodong

AU - Dietz, Allan B

AU - Stein, Gary S.

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