Table Of ContentMethods in
Molecular Biology 1094
Juan C. Stockert
Jesús Espada
Alfonso Blázquez-Castro Editors
Functional
Analysis of DNA
and Chromatin
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
Functional Analysis
of DNA and Chromatin
Edited by
Juan C. Stockert
Department of Biology, Faculty of Sciences, Autonomous University of Madrid,
Cantoblanco, Madrid, Spain
Jesús Espada
Departamento de Biología, Facultad de Ciencias,
Universidad Autónoma de Madrid, Madrid, Spain
Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM, Madrid, Spain
Alfonso Blázquez-Castro
Department of Biology, Faculty of Sciences, Autonomous University of Madrid,
Cantoblanco, Madrid, Spain
Editors
Juan C. Stockert Jesús Espada
Department of Biology Departamento de Biología
Faculty of Sciences Facultad de Ciencias
Autonomous University of Madrid Universidad Autónoma de Madrid
Cantoblanco, Madrid, Spain Madrid, Spain
Instituto de Investigaciones Biomédicas
Alfonso Blázquez-Castro
“Alberto Sols” CSIC-UAM
Department of Biology
Madrid , S pain
Faculty of Sciences
Autonomous University of Madrid
Cantoblanco, Madrid, Spain
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-705-1 ISBN 978-1-62703-706-8 (eBook)
DOI 10.1007/978-1-62703-706-8
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Pref ace
In eukaryotic cells, the nucleus is a highly dynamic compartment in which the functional
profi le of the chromatin fi ber is fl exibly maintained through each DNA replication round in
order to obtain the most adequate output of molecular activity required in each particular
situation. In multicellular organisms, each cell type has a specifi c transcriptional readout of
the DNA molecule as a result of a complex network of genetic and epigenetic regulatory
mechanisms that depend on autonomous as well as nonautonomous informational cues.
This dynamic view of the cell nucleus drastically differs from that transmitted by
cytologist in the late nineteenth to mid-twentieth century and is a direct consequence of
the progressive implementation of adequate and increasingly precise experimental tools.
The development of new and more robust tools in cell and molecular biology has
undoubtedly led to exciting and impressive advances in our current understanding of the
structure and function of the genome. In this context, the present volume of MiMB aims
to provide an overview of standard and more recent methods for the functional analysis
of DNA and chromatin.
Chapters in this volume are arranged as a continuum, dealing as much as possible with
DNA and chromatin analysis displayed in an increasing range of organization and complex-
ity. They include DNA-dye binding modes, chromatin staining, nuclear dispersion assays,
DNA labeling in vivo, sister chromatid exchanges, FISH, DNA and chromatin imaging by
fl uorescence, electron and atomic force microscopy, detection of apoptotic DNA, cytosine
methylation and hydroxy-methylation, DNA thermophoresis, improved methods for his-
tones, chromatin immunoprecipitation, and analysis of rDNA genes and chromatin-
associated RNA.
In Chapter 1 , the helical and hydrogen-bonding parameters for binding to DNA minor
groove are deduced from DNA models. A simplifi ed geometrical stencil can be applied on
computer-drawn molecules to predict minor groove binding and recognition of AT and
GC sequences. The process of drug binding to DNA and chromatin can alter their struc-
ture. The methodology described in Chapter 2 is designed to evaluate changes in the DNA
structure as a result of its interaction with a drug. On account of differences in the migra-
tion pattern as a direct consequence of structural changes due to drug binding, a simple
method to study such interactions based on a microchip electrophoresis setup is
presented.
Giemsa is one of the most widely used biological stains for the nuclear compartment,
inducing a purple color of chromatin DNA, which contrasts with the blue-stained RNA-
containing nucleoli and cytoplasm. Chapter 3 describes standard Giemsa staining of chro-
mosomes after specifi c treatments that cause chromatin disorganization (banded or
harlequin chromosomes).
Chapter 4 describes a handy technique to analyze DNA damage that combines features
from both the COMET assay and FISH. On the other hand, the alkaline variation of nuclear
dispersion assay allows a great fl exibility and availability to study genotoxicity at the single
cell level. Chapter 5 presents in detail the origins and experimental setup of this technique.
v
vi Preface
Due to their particular chemical structure and spatial arrangement, DNA and chroma-
tin present a strong polarization interaction with visible light. Thus it is possible to evaluate
changes in the genetic material (e.g., repetitive sequences or protein interactions) by mea-
suring the change in the polarization characteristics of DNA/chromatin. Chapter 6
describes a simple technique to take advantage of this polarizing property of DNA in
extended chromatin fi bres at the single-cell level.
Metabolic DNA labeling in vivo using nucleoside analogues is a current method to
assess cell proliferation in cell cultures and tissues. It is also successfully used to localize
adult stem cell niches. Chapter 7 describes a standard protocol for BrdU labeling of DNA
in mouse skin and the hair follicle, as well as a simple procedure using Fiji image processing
software to accurately calculate the ratio of label retained in long-term experiments with
respect to the total chromatin content in tissues. Likewise, BrdU is widely used to show
sister chromatid differentiation (SCD) in metaphase chromosomes. Chapter 8 describes
several procedures to produce SCD, using Hoechst 33258 or acridine orange dyes, either
directly or after Giemsa staining, and discusses some possible mechanisms for SCD.
Chromatin packaging plays a signifi cant role in regulating gene transcription. Analysis
of the higher-order packing states of chromatin can provide essential clues for the detection
of functional changes in the nuclear compartment. These changes are involved in processes
associated with or preceding alterations in transcriptional activities. Chapter 9 describes the
application of methods for detecting chromatin remodeling by using image analysis.
Chapter 1 0 reviews in detail a number of techniques currently at the vanguard of FISH
methods for using in plant genetics, whole-cell hybridization in bacterial populations, clini-
cal diagnosis, comparative genomic hybridization, etc. Structural and histochemical meth-
ods are very useful for the characterization of the XY body (the structure formed by the XY
pair during meiotic prophase) in mammals. At present these methods are used for the
detection of abnormalities leading to human infertility, and they are thoroughly described
in Chapter 11 . The replication banding pattern of human chromosomes analyzed by the
combined use of fl uorescence and atomic force microscopy is described in Chapter 12 . This
combined technique is useful to analyze the relations between chromosome structure and
banding pattern.
Degradation of apoptotic cells is critical for the completion of apoptosis and its detec-
tion is extremely useful in cancer research and autoimmune diseases where clearance of
dying cells plays a central role. Chapter 13 presents a simple and selective fl uorescence
technique for visualization of phagocytic cells participating in waste management. The tar-
geted delivery of fl uorescently labeled, DNA-modifying proteins into the nuclei of living
cells permits studies on chromatin/DNA function and damage. A simple approach for
specifi c intranuclear transportation of vaccinia topoisomerase, based on its cationization, is
described in Chapter 1 4 .
The combination of psoralen photo-crosslinking and electron microscopy has been
exploited to reveal the fi ne structure of DNA replication intermediates. Chapter 1 5 describes
how replication structures are stabilized in vivo by psoralen photo-crosslinking prior to
extraction and enrichment procedures, allowing their visualization at the transmission elec-
tron microscope. Replication interference by specifi c DNA sequences has a key role in the
etiology of different human diseases, but studies in human cells have been hampered by
poorly defi ned replication origins on genomic DNA. Chapter 1 6 describes a new SV40-
based episome system to isolate replication intermediates in vivo with high yield and purity.
A major epigenetic mark in mammalian genomes is the methylation at C-5 position of
cytosine in CpG dinucleotides, implicated in the regulation of embryo development and
Preface vii
the maintenance of adult tissue. Standard methods for analysis of DNA methylation (bisulfi te
sequencing, methylation-specifi c PCR, and 5mC immunodetection) are described in
Chapter 17 , using the mouse skin and the hair follicle as biological models. In this sense,
Chapter 18 presents an effi cient experimental approach for rapid biochemical assessment of
DNA methylation through a DNA-stretching methodology.
The discovery of 5-hydroxymethylcytosine (5hmC) as an abundant base in mammalian
genomes has excited the fi eld of epigenetics and stimulated an intense research activity
aimed at decoding its biological signifi cance. Chapter 19 presents an exhaustive review on
the techniques currently available to study 5hmC. This approach has culminated in the
recent publication of two complementary techniques for quantitative, base level-resolution
mapping of 5hmC, the fi rst true mammalian hydroxymethylomes. In this sense, Chapter 2 0
describes an immunoprecipitation approach for distinguishing between 5mC and 5hmC.
Thermophoresis (Soret effect) is a physical phenomenon that manifests as different
molecular diffusion patterns under a temperature gradient. Chapter 2 1 describes a method
to analyze DNA interactions with histones and histone-binding proteins by making use of
microscale thermophoresis to measure the DNA diffusion time. Chapter 22 provides a very
useful introduction to the mass spectroscopic analysis of histone posttranslational modifi ca-
tions (PTMs, i.e., methylation, acetylation, etc.). Chapter 2 3 describes three new protocols
for histone isolation avoiding conventional steps that affect PTMs, and the cumbersome
use of reverse-phase high-performance liquid chromatography.
Chromatin immunoprecipitation (ChIP), a basic experimental approach to study chroma-
tin dynamics and crosslinking ChIP variant (XChIP), is described in Chapter 2 4 . The ChIP-
chop technique described in Chapter 2 5 allows the determination and analysis of epigenetically
modifi ed chromatin, especially of genomic regions containing a high density of repeated
sequences, like clusters of rDNA gen repeats that span several interspersed CpG islands.
Chapter 26 presents a methodology that has already been successfully implemented in
the yeast Saccharomyces cerevisiae to assess single-copy gene loci. This selective isolation
protocol enables, not only the analysis at the genetic level but also the study of epigenetic
modifi cations and DNA–protein interactions. The technique holds the potential for chro-
matin isolation in other species.
In Chapter 2 7 a technique to detect chromatin-associated RNA (caRNA) is described.
This is a new class of RNA that stably associates with actively transcripting chromatin and
helps to maintain the chromatin arrangement to be accessible to transcription. Finally
Chapter 28 presents an informatic tool that allows the analysis and comparison of caRNA
sequences. This tool is based on the Galaxy toolbox, a computer-based platform for the
large-scale interactive analysis of genomes.
We believe that this topic selection will be tempting and useful for most researchers
investigating the cellular and molecular biology of the nucleus. Our aim has been to pro-
vide these professionals with an actual, relevant, rigorous range of descriptions and tech-
niques that will help to expand our current understanding of the complex but fascinating
genetic and epigenetic processes that regulate eukaryotic cell function and structure. We
hope to have achieved this goal with the present volume of Methods in Molecular Biology.
Finally, we acknowledge the encouragement and lasting advice and assistance of John M.
Walker (Series Editor) and David Casey (Humana Press, Springer).
Madrid , Spain Juan C . Stockert
Madrid , Spain Jesús Espada
Madrid , Spain Alfonso B lázquez-Castro
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
1 Predictive Binding Geometry of Ligands to DNA Minor Groove:
Isohelicity and Hydrogen-Bonding Pattern. . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Juan C. Stockert
2 Using Microchip Gel Electrophoresis to Probe DNA–Drug
Binding Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Nan Shi and Victor M. Ugaz
3 Identifying Different Types of Chromatin Using Giemsa Staining . . . . . . . . . . 2 5
Juan C. Stockert, Alfonso Blázquez-Castro, and Richard W. Horobin
4 Analysis of DNA Damage and Repair by Comet Fluorescence
In Situ Hybridization (Comet-FISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9
Michael Glei and Wiebke Schlörmann
5 Alkaline Nuclear Dispersion Assays for the Determination
of DNA Damage at the Single Cell Level. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Piero Sestili and Carmela Fimognari
6 Polarization Microscopy of Extended Chromatin Fibers . . . . . . . . . . . . . . . . . 7 1
Maria Luiza S. Mello and Benedicto de Campos Vidal
7 DNA Labeling In Vivo: Quantification of Epidermal Stem Cell
Chromatin Content in Whole Mouse Hair Follicles Using Fiji Image
Processing Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 9
Elisa Carrasco, María I. Calvo, and Jesús Espada
8 A Historical Overview of Bromo-Substituted DNA
and Sister Chromatid Differentiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Roberto Mezzanotte and Mariella Nieddu
9 Image Analysis of Chromatin Remodelling . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 9
Benedicto de Campos Vidal, Marina B. Felisbino, and Maria Luiza S. Mello
10 FISH Methods in Cytogenetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 09
Miguel Pita, Juan Orellana, Paloma Martínez-Rodríguez,
Ángel Martínez-R amírez, Begoña Fernández-Calvín, and José L. Bella
11 Ultrastructural and Immunofluorescent Methods for the Study
of the XY Body as a Biomarker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 37
Roberta B. Sciurano and Alberto J. Solari
ix
