Epigenetics and Gene Expression in Cancer, Inflammatory and Immune Diseases

202 Pages·2017·4.164 MB·English

by  Barbara Stefanska, David J. MacEwan (eds.)

Methods in Pharmacology
and Toxicology
Barbara Stefanska
David J. MacEwan Editors
Epigenetics and
Gene Expression
in Cancer,
Infl ammatory and
Immune Diseases
M P
ethods in harMacology
t
and oxicology
Series Editor
Y. James Kang
University of Louisville
School of Medicine
Prospect, Kentucky, USA
For further volumes:
http://www.springer.com/series/7653
Epigenetics and Gene Expression
in Cancer, Inflammatory
and Immune Diseases
Edited by
Barbara Stefanska
Department of Nutrition Science, Purdue University, West Lafayette, IN, USA
David J. MacEwan
Department of Molecular and Clinical Pharmacology,
Institute of Translational Medicine, University of Liverpool, Liverpool, UK
Editors
Barbara Stefanska David J. MacEwan
Department of Nutrition Science Department of Molecular
Purdue University and Clinical Pharmacology
West Lafayette, IN, USA Institute of Translational Medicine
University of Liverpool
Liverpool, UK
ISSN 1557-2153 ISSN 1940-6053 (electronic)
Methods in Pharmacology and Toxicology
ISBN 978-1-4939-6741-4 ISBN 978-1-4939-6743-8 (eBook)
DOI 10.1007/978-1-4939-6743-8
Library of Congress Control Number: 2016959003
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Preface
Epigenetics refers to alterations in gene expression without changes in the underlying DNA
sequence and consists of three main components: DNA methylation, histone covalent modi-
fications, and noncoding RNA mechanisms. Aberrant epigenetic patterns have been linked
to chronic inflammation in numerous studies, which consequently leads to the development
of many diseases including cancer, diabetes, multiple sclerosis and other autoimmune dis-
eases, psychiatric, and neurodegenerative disorders. Due to the inherent reversibility of epi-
genetic states, epigenetic modifications constitute an excellent target for prevention and
treatment of these various illnesses. The last two decades of scientific efforts brought about
a remarkable move forward in understanding epigenetics in human disease and health, which
was made possible with novel advanced methodologies. One of the milestones was introduc-
ing genome-wide approaches to studying epigenetics which opened a new emerging field of
epigenomics that is useful to a wide range of researchers in different areas.
The vision for Epigenetics and Gene Expression in Cancer, Inflammatory and Immune
Diseases is to provide pharmacologists, molecular biologists, bioinformaticians, and toxi-
cologists with a background on epigenetics and state-of-the-art techniques in epigenomics.
Although the focus of the book is cancer, inflammatory and autoimmune disorders, the
presented methodologies can find applications in areas outside of these fields. Chapters
discuss three main components of the epigenome and their role in the regulation of gene
expression and present a detailed method section specific to studying each component,
including data analyses, troubleshooting, and feasibility in different experimental settings.
The main topics are high-throughput and targeted methods for DNA methylation analysis,
nucleosome position mapping, studying epigenetic effects of gut microbiota, optical imag-
ing for detection of epigenetic aberrations in living cells, methods for microRNA, and his-
tone code profiling.
The book begins with three chapters detailing the methods for DNA methylation pro-
filing. Genome-wide approaches include methylated DNA immunoprecipitation (MeDIP)
paired with microarray technology or next generation sequencing, Infinium
HumanMethylation450 BeadChip (Illumina 450K), and Reduced Representation Bisulfite
Sequencing (RRBS). The protocols are compared and advantages and disadvantages of
each are discussed in Chap. 1. RRBS and Illumina 450K are both bisulfite-based methods
that measure site-specific methylation, whereas MeDIP-seq and MeDIP-ChIP are
enrichment-b ased methods that provide information on the relative abundance of DNA
methylation. Thus, they differ with regard to coverage, sample size, resolution, and dis-
crimination toward CpG rich and CpG poor regions. One must consider which method
best suits a particular study in order to generate robust and accurate data. Given the het-
erogeneity in cell populations, which is of special interest in neuroscience, development of
single-cell techniques exploring the epigenome is of high interest. As RRBS requires low
starting input DNA, this approach can be applied to single-cell analysis of DNA methyla-
tion patterns. Chapter 2 discusses the current issue of cell population heterogeneity in
epigenetic profiling and describes how dividing cells into their distinct subpopulations
v
vi Preface
using fluorescence-activated cell sorting (FACS) can help to address this problem. Genome-
wide experimental approaches in DNA methylation profiling lead to the discovery of spe-
cific CpG sites, regions, and genes that may play a functional role and require further
validation with targeted DNA methylation analysis methods. Chapter 3 describes and com-
pares pyrosequencing, quantitative methylated DNA immunoprecipitation (qMeDIP), and
methylation-sensitive high-resolution melting (MS-HRM) analysis. These methods replace
nowadays bisulfite standard sequencing that is time-consuming, labor intensive, and often
underpowered. While pyrosequencing quantitatively measures the percentage of methyla-
tion at single CpG resolution, qMeDIP and MS-HRM provide semi-quantitative results
often at the region-based resolution.
Components of the epigenome exert effects over each other and participate in the for-
mation of specific patterns of chromatin structure such as condensed or open chromatin
states. Epigenetic modifications determine the chromatin structure partially through alter-
ing the basic subunit of DNA, the nucleosome. Accessibility of a given genomic region for
active transcription can strictly depend on nucleosome positioning. Thus, mapping nucleo-
somes can deliver new mechanisms of regulation of gene transcription, which is discussed
in Chap. 4 along with a detailed description of a methodology to determine nucleosome
position and occupancy using scanning qPCR. Furthermore, nucleosome assembly is tightly
associated with histone covalent modifications that have the potential to alter nucleosome
positioning and occupancy. Methods for delineating histone marks and changes in histone-
modifying enzymes are detailed in Chap. 5. Oncometabolites generated in cancer cells due
to disrupted metabolic pathways affect the activity of histone-modifying enzymes, includ-
ing Jumonji histone demethylases. This chapter elaborates on a workflow of how to assess
oncometabolites’ tremendous consequences for histone methylation in mammalian cells.
It becomes apparent that even active chromatin contains regions that are not tran-
scribed. These silenced regions may be occupied by specific proteins, e.g., Polycomb group,
which mediate specific histone modifications and gene silencing. On the other hand,
Polycomb group-mediated gene repression can be antagonized by chromatin remodelers,
e.g., BRAHMA (BRM). Hundreds of small molecule epigenetic regulators exist including
derivatives of the intermediary metabolism such as adenosine triphosphate (ATP), acetyl
coenzyme A (AcCoA), S-adenosyl methionine (SAM), nicotinamide adenine dinucleotide
(NAD), and inositol polyphosphates (IPs). Chapter 6 presents a method for using qRT-
PCR to assay the regulation of multiple genes in a 384-well format. This technology can be
potentially utilized in screening for transcriptional regulators without well-defined func-
tions that are endogenous or synthetically developed.
With Chap. 7’s description of approaches for profiling expression of microRNAs, we
conclude the methodology for assessing all the components of the epigenome. MicroRNAs
are small noncoding RNAs that participate in post-transcriptional regulation of gene expres-
sion. Depending on their targets, microRNAs play a tumor suppressive or oncogenic role.
In addition to their potential use as anticancer agents, rising evidence indicates the role of
miRNAs as biomarkers for cancer. To explore their therapeutic and diagnostic potential,
expression profiling in different tissues and body fluids is needed. Many miRNA profiling
methods have been developed, including target-based techniques (Northern blotting,
qRT-PCR, in situ hybridization [ISH]) and high-throughput methodology (microarray,
RNA-Seq platforms). Chapter 7 describes target-based techniques and provides details on
ISH. While Northern blotting and qRT-PCR are robust in quantifying miRNA expression
in a mixture of cells from different specimens, ISH is the only imaging-based technique that
Preface vii
takes into account expression levels along with expression heterogeneity and tissue- and
cell-type specificity. Advantages and disadvantages of the methods in various applications
are further discussed.
Epigenetics constitutes the interface between the environment and the genome.
Numerous environmental factors have been shown to trigger changes in the epigenome
including recently reported effects of gut microbiota. Gut microbial metabolites, such as
butyrate or lipopolysaccharide (LPS), are known to influence the epigenome of the host
and thereby regulate expression of genes involved in inflammation and fat metabolism. The
workflow for compositional evaluation using qPCR and diversity analysis of microbial flora
using denaturing gradient gel electrophoresis (DGGE) is detailed in Chap. 8, along with
methods for studying epigenetic alterations associated with specific microbial patterns.
Most of the methods for evaluating epigenetic modifications described in Chaps. 1–8
are optimized for cell pellets, tissues, isolated nucleic acids, chromatin, etc. using an ensem-
ble of cells. As we learn from ongoing molecular and clinical studies in the precision medi-
cine approach, cell populations are highly heterogeneous which may impede our
understanding of tested processes. There is a need for novel technology to establish con-
nections between the molecular events in living cells, including epigenetics. Chapter 9
describes recent advances in optical microscopy and spectroscopy to capture epigenetic
events in living cells. It further provides practical guidance on optical instrumentations for
different applications and reviews recent advancements in sensing live-cell epigenetics.
Super-resolution microscopy and Förster resonance energy transfer (FRET) are presented
as methods for studying localization of target molecules and interaction. Fluorescence fluc-
tuation spectroscopy can be applied for quantity and stoichiometry measurements whereas
dynamics and kinetics can be assessed using fluorescence cross-correlation spectroscopy
(FCCS), fluorescence lifetime correlation spectroscopy (FLCS), and fluorescence recovery
after photobleaching (FRAP). Visualization of DNA methylation by utilizing the binding
specificity between methyl-CpG-binding domain (MBD) proteins and methylated DNA is
also summarized. The workflow for these techniques is complemented with advantages and
disadvantages in current applications.
The book not only constitutes a resource document with advanced methodology but also
delivers an extensive literature review. The last two chapters are review articles that present the
current knowledge in microRNAs (miRNAs) and epigenetics of human diseases including
autoimmune diseases. Chapter 10 extensively discusses the role of miRNAs in human diseases
and their potential as biomarkers of drug-induced toxicity. It further demonstrates a step-by-
step practical guide to identify miRNA species and test the role of miRNAs in clinically impor-
tant samples using miRNA-modulating agents. In Chap. 11, readers will learn about genetics
and epigenetics of multiple sclerosis, one of the most debilitating autoimmune disorders. The
chapter presents an overview of how results from exome sequencing, genome-wide associa-
tion studies, transcriptome, and epigenome mapping contribute to deciphering the patho-
physiology, progression, and different subtypes of the disease.
Finally, we are grateful to all the contributors for their tremendous efforts to prepare
the chapters and to share their knowledge in various aspects of epigenetics and gene expres-
sion studies in cancer, inflammatory and immune diseases. It was a great pleasure and
invaluable experience to work with each of them.
West Lafayette, IA, USA Barbara Stefanska
Liverpool, UK David J. MacEwan
Contents
Preface.......................................................... v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 High-Throughput Techniques for DNA Methylation Profiling ............ 1
Sophie Petropoulos, David Cheishvili, and Moshe Szyf
2 Reduced Representation Bisulfite Sequencing (RRBS) and Cell Sorting
Prior to DNA Methylation Analysis in Psychiatric Disorders .............. 17
Wilfred C. de Vega, Atif Hussain, and Patrick O. McGowan
3 Targeted DNA Methylation Analysis Methods......................... 33
David Cheishvili, Sophie Petropoulos, Steffan Christiansen, and Moshe Szyf
4 Analyzing Targeted Nucleosome Position and Occupancy
in Cancer, Obesity, and Diabetes................................... 51
Prasad P. Devarshi and Tara M. Henagan
5 Synthesis and Application of Cell-Permeable Metabolites
for Modulating Chromatin Modifications Regulated
by α-Ketoglutarate-Dependent Enzymes............................. 63
Hunter T. Balduf, Antonella Pepe, and Ann L. Kirchmaier
6 High-Throughput Screening of Small Molecule Transcriptional
Regulators in Embryonic Stem Cells Using qRT-PCR................... 81
Emily C. Dykhuizen, Leigh C. Carmody, and Nicola J. Tolliday
7 Methods for MicroRNA Profiling in Cancer .......................... 97
Sushuma Yarlagadda, Anusha Thota, Ruchi Bansal, Jason Kwon,
Murray Korc, and Janaiah Kota
8 Microbiota and Epigenetic Regulation of Inflammatory Mediators ......... 115
Marlene Remely, Heidrun Karlic, Irene Rebhan, Martina Greunz,
and Alexander G. Haslberger
9 Optical Microscopy and Spectroscopy for Epigenetic Modifications
in Single Living Cells ........................................... 135
Yi Cui and Joseph Irudayaraj
10 MicroRNAs in Therapy and Toxicity................................ 155
David J. MacEwan, Niraj M. Shah, and Daniel J. Antoine
11 Genetics and Epigenetics of Multiple Sclerosis......................... 169
Borut Peterlin, Ales Maver, Vidmar Lovro, and Luca Lovrečić
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
ix
Contributors
Daniel J. antoine • Department of Molecular and Clinical Pharmacology, Institute
of Translational Medicine, University of Liverpool, Liverpool, UK
Hunter t. BalDuf • Department of Biochemistry, and Purdue Center for Cancer
Research, Purdue University, West Lafayette, IN, USA
rucHi Bansal • Department of Medical and Molecular Genetics, Indiana University
School of Medicine (IUSM), Indianapolis, IN, USA
leigH c. carmoDy • Center for the Development of Therapeutics, Broad Institute
of Harvard and MIT, Cambridge, MA, USA
DaviD cHeisHvili • Department of Pharmacology and Therapeutics, McGill University
Medical School, Montreal, QC, Canada
steffan cHristiansen • Department of Biomedicine, Aarhus University, Aarhus, Denmark
yi cui • Department of Agricultural and Biological Engineering, Bindley Bioscience
Centre, Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA
PrasaD P. DevarsHi • Department of Nutrition Science, Purdue University, West Lafayette,
IN, USA
emily c. DykHuizen • Department of Medicinal Chemistry and Molecular Pharmacology,
Purdue University, West Lafayette, IN, USA
martina greunz • Department of Nutritional Sciences, University Vienna, Vienna, Austria
alexanDer g. HaslBerger • Department of Nutritional Sciences, University Vienna,
Vienna, Austria
tara m. Henagan • Department of Nutrition Science, Purdue University, West Lafayette,
IN, USA
atif Hussain • Department of Biological Sciences, Centre for Environmental Epigenetics and
Development, University of Toronto Scarborough, Toronto, ON, Canada
JosePH iruDayaraJ • Department of Agricultural and Biological Engineering, Bindley
Bioscience Centre, Purdue Center for Cancer Research, Purdue University, West
Lafayette, IN, USA
HeiDrun karlic • Ludwig Boltzmann Institute for Leukemia Research and Hematology,
Hanusch Hospital, Vienna, Austria
ann l. kircHmaier • Department of Biochemistry, Purdue University, West Lafayette, IN,
USA; Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA
murray korc • Department of Biochemistry and Molecular Biology, IUSM, Indianapolis,
IN, USA; The Melvin and Bren Simon Cancer Center, IUSM, Indianapolis, IN, USA;
Pancreatic Cancer Signature Center, Indiana University and Purdue University-
Indianapolis (IUPUI), Indianapolis, IN, USA
JanaiaH kota • Department of Medical and Molecular Genetics, Indiana University School
of Medicine (IUSM), Indianapolis, IN, USA; The Melvin and Bren Simon Cancer
Center, IUSM, Indianapolis, IN, USA; Pancreatic Cancer Signature Center, Indiana
University and Purdue University-Indianapolis (IUPUI), Indianapolis, IN, USA
xi
xii Contributors
Jason kwon • Department of Medical and Molecular Genetics, Indiana University School
of Medicine (IUSM), Indianapolis, IN, USA
luca lovrečić • Department of Obstetrics and Gynecology, Clinical Institute of Medical
Genetics, University Medical Center Ljubljana, Ljubljana, Slovenia
viDmar lovro • Department of Obstetrics and Gynecology, Clinical Institute of Medical
Genetics, University Medical Center Ljubljana, Ljubljana, Slovenia
DaviD J. macewan • Department of Molecular and Clinical Pharmacology, Institute
of Translational Medicine, University of Liverpool, Liverpool, UK
ales maver • Department of Obstetrics and Gynecology, Clinical Institute of Medical
Genetics, University Medical Center Ljubljana, Ljubljana, Slovenia
Patrick o. mcgowan • Department of Biological Sciences, Centre for Environmental
Epigenetics and Development, University of Toronto Scarborough, Toronto, ON, Canada;
Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada;
Department of Psychology, University of Toronto, Toronto, ON, Canada; Department of
Physiology, University of Toronto, Toronto, ON, Canada
antonella PePe • Purdue Center for Cancer Research, Purdue University, West Lafayette,
IN, USA
Borut Peterlin • Department of Obstetrics and Gynecology, Clinical Institute of Medical
Genetics, University Medical Center Ljubljana, Ljubljana, Slovenia
soPHie PetroPoulos • Department of Clinical Science, Intervention and Technology
(CLINTEC), Karolinska Institutet, Stockholm, Sweden
irene reBHan • Department of Nutritional Sciences, University Vienna, Vienna, Austria
marlene remely • Department of Nutritional Sciences, University Vienna, Vienna,
Austria
niraJ m. sHaH • Department of Molecular and Clinical Pharmacology, Institute of
Translational Medicine, University of Liverpool, Liverpool, UK
mosHe szyf • Department of Pharmacology and Therapeutics, McGill University Medical
School, Montreal, QC, Canada
anusHa tHota • Department of Medical and Molecular Genetics, Indiana University
School of Medicine (IUSM), Indianapolis, IN, USA
nicola J. tolliDay • Center for the Development of Therapeutics, Broad Institute of
Harvard and MIT, Cambridge, MA, USA
wilfreD c. De vega • Department of Biological Sciences, Centre for Environmental
Epigenetics and Development, University of Toronto Scarborough, Toronto, ON, Canada;
Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada
susHuma yarlagaDDa • Department of Medical and Molecular Genetics, Indiana
University School of Medicine (IUSM), Indianapolis, IN, USA