Human Molecular Genetics 2004 13(Review Issue 2):R297-R302; doi:10.1093/hmg/ddh230
Human Molecular Genetics, Vol. 13, Review Issue 2 © Oxford University Press 2004; all rights reserved
Applications of genomic microarrays to explore human chromosome structure and function
Nigel P. Carter* and
David Vetrie
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
Received June 25, 2004; Revised July 8, 2004; Accepted July 16, 2004
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ABSTRACT
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The combination of genomic microarrays with comparative genomic
hybridization and with chromatin immunoprecipitation is providing
an increasingly detailed view of the way in which the human
genome is organized and functions and how disorganization and
disfunction can lead to disease. These studies are enhanced
by the flexibility of array technology, allowing resolutions
from coverage of the whole genome using 200 kb cloned DNA
inserts to detailed analysis using PCR products or oligonucleotides
of 100 bp or less. In particular, the use of chromatin
immunoprecipitation is providing new insights into chromosome
structure and gene regulation and control through the analysis
of protein–DNA interactions.
The DNA (
2 m) which comprises the human cell nucleus is condensed and packaged by being wrapped around histone octamers in nucleosomes which themselves are organized into higher ordered structures. This is most clearly visualized as the highly condensed chromosomes visible at metaphase prior to cell division. During interphase, the chromatin remains organized into chromosomal domains where expressed regions become decondensed to facilitate transcription and silenced regions remain in a condensed form (1). This hints at the functional role of chromatin, which in recent years, we have come to realize is important in the regulation of gene expression in development and its maintenance in differentiation (2–4). Thus, chromosome structure and function are not simply determined by the linear sequence and the genes and regulatory elements for which it codes, but also by higher order chromatin organization. Disruptions in this organization often lead to disease.
With the publication of the finished sequence of the human genome we now have access not only to the linear sequence of DNA base pairs, but also to a bewildering diversity of mapped cloned DNA resources. These resources have been harnessed in the form of genomic DNA microarrays to allow widescale or detailed analysis of chromosome structure and function. Typically, the microarray comprises a substrate onto which a large number of genomic DNA sequences are individually synthesized or spotted. Current technology allows over 60 000 features (spots) on a standard microscope slide. The features are then utilized as probes allowing highly parallel analysis of the abundance of homologous sequences in the DNA sample hybridized to the array. This current restriction in the number of features limits the scope and resolution of the arrays. Probes are typically derived from cloned genomic DNA inserts, PCR amplicons or oligonucleotides designed directly from the sequence. While the whole genome can be covered with 30 000 large insert clones of over 100 kb in size (5), for higher resolution analysis of DNA–protein interaction or chromatin modifications, over three million sequences of 1 kb in size would be required. As this is not currently achievable, higher resolution arrays are generally restricted to smaller areas of the genome or to specific chromosomes (6,7).
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DNA MICROARRAYS AND COMPARATIVE GENOMIC HYBRIDIZATION
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In a typical experiment using a spotted clone array, a test
DNA and a reference DNA are labelled with different fluorochromes
and co-hybridized (usually in the presence of unlabelled DNA
to block shared repeat sequences) to the array (Fig.
1).
After hybridization and washing, the microarray is scanned and
the fluorescence intensity of each fluorochrome for each feature
quantified. In this way, for each feature, the relative fluorescence
intensity of the test to reference sample is determined and
differences between the abundance of complementary sequences
in the two hybridized DNAs quantified. This process is analogous
to comparative genomic hybridization (CGH) where differentially
labelled test and reference genomes are hybridized to metaphase
spreads and the relative genomic copy number determined along
each chromosome, thus indentifying aberration in chromosome
structure (
8). In CGH, the detection of gains and losses in
the genome are limited to a resolution of
5 Mb by the highly
condensed target DNA in the metaphase chromosomes (
9). Microarrays
are now replacing metaphase spreads as the target for hybridization
in these studies (
10,
11) so that in array-CGH, resolution is
now limited only by the size and density of the target sequences.
Typically, studies utilize microarrays comprising large insert
clones spaced at approximately one clone per Mb (
12), but higher
resolution arrays comprising overlapping clone sets for chromosomes
or chromosome regions (
13) and more recently for the whole genome
(
5) are being employed for more detailed and sensitive studies.
Array-CGH has been most widely applied for the analysis of gains
and losses in tumours (
14) but is increasingly becoming applied
to the analysis of patients with constitutional rearrangements
(
15–
21). We have specifically used array-CGH to identify
small microdeletions and microduplications in patients with
learning disability and dysmorphology who by conventional analysis
had normal balanced karyotypes (
15). In a study of 50 such patients,
we identified seven microdeletions and five microduplications
and showed that in most cases the microdeletions were
de novo and the microduplications inherited. Using the ability to flow
sort aberrant chromosomes, we have been able to extend these
analyses to patients with balanced translocations in a process
we have called array painting, such that the composition and
breakpoints of the rearrangement can be determined at high resolution
providing the opportunity to identify candidate disease genes
(
17). In array painting, the two derivative chromosomes in a
balanced translocation are separated by flow sorting, differentially
labelled and hybridized to a genomic clone array. Because only
the DNA comprising each rearranged chromosome is analysed, the
composition and breakpoints of the rearrangement can be directly
determined by ratio changes from high to low (and vice versa)
when the data are plotted according to chromosomal location.
Surprisingly, in a study of only 10 patients with cytogenetically
balanced translocations we found that six displayed unexpected
karyotype complexity involving additional translocations, microdeletion,
microduplication and inversion. Extending these studies to more
focussed, higher resolution arrays allows us to more accurately
define the specific breakpoints and the mechanisms involved
in chromosome rearrangement. The development of arrays which
are completely sequence-defined and repeat-free has allowed
this resolution to be increased to 15–20 kb (
22).
More recently we have demonstrated that it is possible to use
PCR products containing single coding exons of genes as a means
for defining copy number changes at a resolution of 150 bp
(P. Dhami and D. Vetrie, unpublished data, Fig.
2).
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Figure 2. Detection of copy number changes using arrays of exons of human genes. Fluorescently labelled human male and female genomic DNA were used in a competitive hybridization in an array-CGH experiment onto an array containing elements of individual exons (average size 296 bp) for five human genes (COL4A5, DMD, NF2, PLP, PMP22). The plot shows the male/female ratios obtained for the exons (each represented as a black dot). Each exon is plotted as a function of its position 5' to 3' for each gene. The number of exon elements assayed for each gene is showed below the gene name. Exons for the three X-linked genes (COL4A5, DMD, PLP) and the two autosomal genes (NF2 and PMP22) showed ratios centered around the theoretical values of 0.5 and 1, respectively (data courtesy of Pawandeep Dhami, unpublished data).
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ANALYSIS OF CHROMOSOME STRUCTURE AND FUNCTION USING ChIP-ON-CHIP
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While array-CGH has proved very useful for the analysis of gross
chromosome structural aberrations, array technology is now being
increasingly applied to the study of normal chromosome structure
and function. The functional role of chromatin organization
and modification and of associated proteins and complexes has
now become well established (
2–
4) and is involved in fundamental
processes such as transcription, recombination, replication
and DNA repair. DNA microarrays are being used to study these
processes by combination with methods which allow fractionation
of chromatin associated with function. Of these, one of the
most powerful approaches is the use of chromatin imunoprecipitation
(ChIP) where the chromatin associated with specific modifications
or with proteins or protein complexes can be enriched, then
quantified and mapped using microarrays. The use of chromatin
immunoprecipitation with arrays has been termed ChIP-on-chip
or ChIP-chip. This method has been well established for several
years in organisms where the genome is less complex such as
Saccharomyces cerevisiae and
Drosophila. However, its application
to the study of mammalian genomes is rapidly emerging with the
availability of arrays which allow analysis of relevant subfractions
of the genome (promoters, CpG islands, etc.) or entire tile
paths of chromosomes or chromosomal regions, the latter allowing
sequences of interest to be identified in an unbiased way.
The basic principles of ChIP-on-chip are shown in Figure 3. The first important step in the process is the crosslinking of protein to the DNA with formaldehyde. The crosslinked DNA is sheared and then immunoprecipitated with a specific antibody to the protein of interest. This process not only precipitates the protein but also the DNA crosslinked to it. The crosslinking is then reversed and the associated DNA extracted. In many studies, the yield of extracted DNA is very low and either multiple extractions must be pooled or an amplification step must be employed before hybridization of the extracted DNA to the microarray. Typically, the enriched extracted DNA is compared on the array with un-enriched DNA so that the pattern and extent of enrichment of the regions represented on the array can be determined. However, we have found that using advanced array technology, sensitivity and specificity can be improved to a level where pooling of samples or amplification of enriched DNA is no longer necessary (unpublished data, Fig. 4). Thus, issues related to biases obtained due to the amplification process can be avoided, thus adding to the biological significance of the data obtained from such assays.
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Figure 4. ChIP-chip analysis using a genomic tiling path array. Fluorescently labelled unamplified ChIP DNA and genomic DNA were used in a competitive hybridization onto a PCR product-based array constituting a near complete tile path (400 bp resolution) of a 256 kb region of human chromosome at 1p32 containing the TAL1 locus. The plot shows the fold enrichment obtained for each PCR product (represented as joined-up red dots) in the ChIP DNA for histone H3 Lys4 trimethylation across the genomic region represented on the array (data courtesy of Pawandeep Dhami, unpublished data). H3 Lys4 trimethylation is associated with actively transcribed genes. The organization of genes across the region is shown at the bottom of the figure (green); the direction of transcription is denoted by the arrows under the blocks representing the genomic region encompassed by each gene.
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Modifications to the histone proteins which make up nucleosomes
are tightly linked to the way in which the chromatin is packaged
and in the regulation of gene expression. Modifications to the
histone tails include acetylation, methylation, phosphorylation
and ubiquitination. Microarrays have been used to map the distribution
of such modifications using specific antibodies with chromatin
immunoprecipitation. For example, Kondo
et al. (
23) used
separate specific antibodies against acetylated and methylated
histone H3 lysine 9 to immunoprecipitate acetylated and methylated
histones from a cancer cell line, which were then hybridized
to a human CpG island microarray. Using this approach, they
were able to identify novel targets of gene silencing in cancer,
since methylation at H3 lysine 9 is a central mechanism involved
in gene repression and silencing (
4). We have also visualized
histone modifications across tiled genomic regions providing
clues of the chromatin state and transcriptional regulation
of loci of interest (P. Dhami and D. Vetrie, unpublished data,
Fig.
4). Such analyses provide new insights into the histone
code hypothesis (
24) and the relationship between DNA elements
which control gene expression and chromatin structure.
Microarray analysis using ChIP-on-chip is particularly suited to the study of direct interaction of proteins and protein complexes with DNA. There are numerous examples of this in the study of the transcription-factor–DNA interactions in the human genome. Good early examples include detailed analysis of the E2F transcription-factor family which plays a critical role in cell cycle progression. Weinmann et al. (25) used chromatin immunoprecipitation with an antibody against E2F4 in combination with a human CpG island microarray to identify 68 unique targets, 25% of which recruit E2F by a mechanism other than through the known consensus binding sequence. Ren et al. (26) also investigated the co-ordinated regulation of genes by both E2F1 and E2F4 using ChIP in combination with an array containing the promoters of 1500 genes. Transcription-factor–DNA interactions have also been assayed using high resolution tile paths of human chromosomal regions (27) and entire chromosome tile paths (28–30), thus providing detailed information on the spatial organization of transcription-factor binding sites with respect to genes and other genome features.
The ChIP-on-chip approach has also been applied to the study of other functional chromosomal elements such as neocentromeres. Neocentromeres are regions of rearranged chromosomes which carry out the function of the normal centromere in providing mitotic stability in the aberrant chromosome. Alonso et al. (31) used immunoprecipitation with an antibody to centromere protein A (CENP-A) with an overlapping BAC clone array of chromosome 13q31.3–13q33.1 to identify the precise location of three neocentromeres which had been previously localized by fluorescence in situ hybridization to 13q32. They showed that each of the neocentromeres was located at distinct locations and that there was no underlying sequence motif that could be found predisposing to neocentromere formation. Their data support the view that neocentromere formation is a sequence-independent epigenetic mechanism
While high resolution arrays are most suited for the analysis of protein–DNA interactions due to the range over which binding occurs, current technology limits the scope of these arrays such that the whole genome cannot be scanned. Where this is required, it is currently necessary to use large insert clone arrays spanning the whole genome. We have used both 1 Mb resolution and chromosome tiling arrays to map the distribution of proteins associated with cellular senescence. We applied chromatin immunoprecipitated with an antibody against phosphorylated histone H2AX, a marker of DNA damage which associates with DNA damage and DNA checkpoint proteins, and showed that H2AX was specifically enriched at human chromosome telomeres (32). We proposed that telomere-initiated senescence reflects a DNA damage checkpoint response that is activated by direct contribution from dysfunctional telomeres.
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DNA METHYLATION
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The methylation of DNA in most CpG dinucleotides is important
in gene regulation in that methylation is an inhibitor of gene
expression (
33–
36). In promoter regions associated with
CpG islands, the methylated DNA is bound by proteins such as
MeCP2 which in turn recruits histone deacetylase. The combination
of methylated DNA and deacetylated histones generates a transcriptionally
inactive chromatin state and gene silencing. The pattern of
methylation and histone deacetylation changes during development
and differentiation as different genes are switched on and off.
DNA methylation is also intimately involved in genomic imprinting
(
37–
39). Errors in DNA methylation or in the enzymes associated
with methylation lead to disease such as Cranofacial and Rett
Syndromes (
40), Beckwith–Weidemann and Prader Willi/Angleman
syndromes via errors in imprinting (
41). This also appears to
be very important in cancer where the silencing of tumour suppressor
genes is thought to be causative in many sporadic tumours (
34).
As DNA methylation is so intimately involved in gene regulation,
it is inevitable that the list of diseases where altered methylation
patterns lead to altered gene expression and so to the disease
state is likely to grow. DNA microarrays are now being employed
to assess methylation patterns and several approaches have emerged.
One approach uses methylation-sensitive restriction enzymes
and DNA size selection (
42) or specific PCR amplification to
differentially amplify methylated and unmethylated loci (
43,
44).
These products are then hybridized to an array where stronger
hybridization indicates hypermethylation (
43). Using this approach,
it has been possible to correlate hypermethylation with hormone
status in breast tumours (
45) and progression-free survival
in ovarian cancer (
46). In an alternative approach, Gitan
et al. (
47) utilized bisulphite modification of DNA to convert unmethylated
cytosines to uracil which after PCR is converted to thymine.
Thus unmethylated sites can be distinguished from methylated
sites by the conversion of cytosine to thymine after the bisulphite
treatment. The conversion was detected by hybridization of the
bisulphite-treated DNA to oligonucleotide arrays designed to
be sensitive to the base change. Using this method, this group
mapped methylated CpG sites within a human oestrogen receptor
gene CpG island. Adorjan
et al. (
48) were able to use this
type of approach to distinguish between different types of leukaemia
and between carcinomas and their corresponding normal tissues.
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REPLICATION TIMING
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It has been long known that DNA replication progresses in an
ordered process where different parts of the genome replicate
at different times through S phase. Regions of the genome being
actively transcribed replicate early in S phase where as heterochromatin
and regions of gene inactivity replicate late. We have developed
a microarray-based method for assessing replication timing using
DNA microarrays (
49), which extends studies in yeast (
50) and
Drosophila (
51) to the human genome. The basis of the method
is the separation, by flow cytometry, of nuclei in S phase from
nuclei in G1 phase of the cell cycle from an asynchronously
dividing cell population (Fig.
5). The S phase fraction
contains nuclei at all stages of replication including nuclei
just entering S phase through to nuclei at the end of S phase.
Loci which replicate early will have doubled their DNA content
in most S phase nuclei, whereas loci which replicate late will
remain at a single copy in the majority of nuclei. Thus the
average DNA content of a locus in all of the cells in S phase
will be directly related to the time through S phase at which
the locus replicates. Thus the earliest locus to replicate will
have twice the DNA content of the last locus to replicate. By
hybridizing the differentially labelled S phase fraction against
a reference where no replication has taken place (the G1 fraction),
the relative ratio will theoretically vary by locus from 2 : 1
(early replication) to 1 : 1 (late replication). Using
this approach we have measured the replication timing across
the whole human genome using our array with a resolution of
one large insert clone per 1 Mb as well as at higher resolution
on an array of chromosome 22q containing overlapping clones
(
49). Our results confirmed the correlation of early replication
with high gene density, high GC content, high Alu repeat content
and low SINE repeat content, and confirmed the correlation of
the replication timing with the transcriptional state of the
DNA by comparison with gene expression microarray analysis.
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CONCLUSIONS
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The application of microarrays to the study of chromosome structure
and function is still in its infancy. The power of microarrays
to quantify hybridization at high resolution over large areas
of the genome is perfectly matched to methods that are able
to separate the genomic DNA into functional fractions. As the
technology develops, we can expect microarrays which are more
sensitive, of higher resolution and that are more widely available
and with this, increasing contributions to our understanding
of chromosome structure and function.
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ACKNOWLEDGEMENT
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N.P.C. and D.V. are supported by the Wellcome Trust.
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FOOTNOTES
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* To whom correspondence should be addressed. Tel: +44 1223494860; Fax: +44 1223494919; Email:
npc{at}sanger.ac.uk
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ABSTRACT
DNA MICROARRAYS AND COMPARATIVE...
