Global histone modification fingerprinting in human cells using epigenetic reverse phase protein array.
The balance between acetylation and deacetylation of histone proteins plays a critical role in the regulation of genomic functions. Aberrations in global levels of histone modifications are linked to carcinogenesis and are currently the focus of intense scrutiny and translational research investments to develop new therapies, which can modify complex disease pathophysiology through epigenetic control. However, despite significant progress in our understanding of the molecular mechanisms of epigenetic machinery in various genomic contexts and cell types, the links between epigenetic modifications and cellular phenotypes are far from being clear. For example, enzymes controlling histone modifications utilize key cellular metabolites associated with intra- and extracellular feedback loops, adding a further layer of complexity to this process. Meanwhile, it has become increasingly evident that new assay technologies which provide robust and precise measurement of global histone modifications are required, for at least two pressing reasons: firstly, many approved drugs are known to influence histone modifications and new cancer therapies are increasingly being developed towards targeting histone deacetylases (HDACs) and other epigenetic readers and writers. Therefore, robust assays for fingerprinting the global effects of such drugs on preclinical cell, organoid andin vivomodels is required; and secondly, robust histone-fingerprinting assays applicable to patient samples may afford the development of next-generation diagnostic and prognostic tools. In our study, we have used a panel of monoclonal antibodies to determine the relative changes in the global abundance of post-translational modifications on histones purified from cancer cell lines treated with HDAC inhibitors using a novel technique, called epigenetic reverse phase protein array. We observed a robust increase in acetylation levels within 2-24 h after inhibition of HDACs in different cancer cell lines. Moreover, when these cells were treated withN-acetylated amino acids in addition to HDACs, we detected a further increase in histone acetylation, demonstrating that these molecules could be utilized as donors of the acetyl moiety for protein acetylation. Consequently, this study not only offers a novel assay for diagnostics and drug screening but also warrants further research of the novel class of inexpensive, non-toxic natural compounds that could potentiate the effects of HDAC inhibitors and is therefore of interest for cancer therapeutics.
Synthetic Epigenetics Laboratory, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Marina
M
Partolina
Synthetic Epigenetics Laboratory, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Hazel C
HC
Thoms
Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Kenneth G
KG
MacLeod
Synthetic Epigenetics Laboratory, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Giovanny
G
Rodriguez-Blanco
Synthetic Epigenetics Laboratory, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Matthew N
MN
Clarke
Synthetic Epigenetics Laboratory, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; Department of Biosciences, University of Helsinki, PO Box 65 (Viikinkaari 1), 00014, Helsinki, Finland.
Anuroop V
0000-0003-2119-8741
AV
Venkatasubramani
Department of Health Sciences and ANDI Centre of Excellence for Biomedical and Biomaterials Research, Faculty of Science, University of Mauritius , Réduit, Republic of Mauritius.
Rima
R
Beesoo
Developmental Therapeutics Branch, National Cancer Institute , Bethesda, MD 20892, USA.
Vladimir
V
Larionov
Department of Health Sciences and ANDI Centre of Excellence for Biomedical and Biomaterials Research, Faculty of Science, University of Mauritius , Réduit, Republic of Mauritius.
Vidushi S
VS
Neergheen-Bhujun
Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Bryan
B
Serrels
Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology . 4259, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Hiroshi
H
Kimura
Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Neil O
NO
Carragher
Synthetic Epigenetics Laboratory, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh , Edinburgh, UK.
Alexander
0000-0002-6219-6892
A
Kagansky