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MAPPING HIGHER-ORDER CHROMATIN STRUCTURES
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Bruce
H. Howard, MD, Head, Human Genetics
Section Valya
Russanova, PhD, Staff Scientist Yaohui
Chen, PhD, Research Fellow Andrei
Tchernov, PhD, Visiting Fellow Nicholas
Sammons, BS, Postbaccalaureate Fellow |
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Our
investigations focus on links between epigenome structure, development, and
aging. We work from the basic hypothesis that remodeling of chromatin
structures is fundamental to the reprogramming of gene expression. We examine
such remodeling in the contexts of embryonic stem cell differentiation or the
transformation of monocytes to macrophages or dendritic cells. At the same
time, we analyze changes in epigenome structure in relation to the much
slower processes of postnatal development and aging. Human skin fibroblasts
provide a model system for the latter processes. A long-term goal is to
understand how errors in epigenome stability or remodeling contribute to
developmental defects, cancer, and age-related diseases. We have developed
new functional genomics techniques to facilitate progress in these areas. Genome
sampling, bioinformatics, and high-throughput approaches to map higher-order
chromatin domains Russanova, Howard In
previous studies, we detected examples of differentiation- and cell
senescence–linked chromatin remodeling at several loci by using a
genome sampling–chromatin immunoprecipitation (gsChIP) technique. We
applied the same approach further to reveal age-related changes in epigenome
structure in the human chromosome 4p16.1 and 4q35.2 regions. Our primary goal
over the past year was to characterize these changes in greater detail. To
this end, we developed and implemented efficient chromatin mapping
approaches. Rapid
primer design was the initial element required to set in place an integrated
high-throughput methodology. Of the large number of computer-based primer
design algorithms that are available, we chose Primer3 for interfacing with
custom software. A key to the strategy is code that performs the following
basic operation loop: (1) recover a short template sequence for primer
design; (2) write an input file for Primer3; (3) call a Bourne shell script
that launches Primer3 and returns a success/fail value; (4) interpret the
output from Primer3; and (5) write an output file line containing the primer
ID and target position, primer pair sequences, and the 96-well positions into
which they are to be placed. The program continues until the requisite number
of primer pairs has been designed or the end of the region to be mapped is
reached. Output from the above linking software, primerMap, is formatted to
serve directly as order information for a commercial biotechnology source.
Importantly, primers are returned in a 96-well format and normalized to
constant concentrations, allowing simple dilution and direct use in a
robot-based PCR set-up. The
second major task was to find a high-throughput approach to validate primers
in order to perform PCR assays that would remain in the linear range and to
generate an output form that could be analyzed quickly by pattern recognition
on a 96-well scale. We identified an ion-pair reverse-phase HPLC based on a
poly(styrene-divinylbenzene) matrix that would serve this purpose. The
sensitivity of the system is enhanced by post-fractionation staining with SYBR
gold. Appropriate code generates 96-well virtual gel images and thus permits
rapid data interpretation. The
third task in the integrated approach is a rapid graphics summary of the ChIP
results. The solution is based on the SVG graphics language. The code
represents acetylation levels, or differences in acetylation levels,
according to arbitrarily chosen color scales (red-gray-blue and
red-yellow-green for high versus low and increasing versus decreasing
acetylation levels, respectively). In the former scale, gray corresponds to
average epigenome histone H4 acetylation as determined by both
chromatin-associated DNA recovery and multiple gsChIP comparisons. A
summary of mapping around the 4p16.1 site reveals diminishing H4 acetylation
over an interval spanning fetal development through early childhood. The
region of remodeling extends over more than 700 kilobase pairs. Two pairs of
genes are situated near or overlapping the edges of this region. Of interest
is the MIST gene (LOC166522), given that the most striking remodeling,
over 11-fold, spans the 5ยด part of the gene and extends through the promoter
region. MIST/Clnk and related proteins are adaptors involved in signal
transduction, mostly studied in hematopoietic lineages. Novel
regions of chromatin structure are likely initiated by combinations of
sequence-specific DNA binding factors. To seed the mapping of such regions,
we developed a set of custom search algorithms that allow comparisons of
mouse and human genomes for the presence of conserved binding sites. In
excess of 100,000 sites per genome can be analyzed, followed by more than 10
billion blast comparisons, to yield potential binding sites of interest. The
sites can be prioritized according to proximity to CpG islands, genes, and so
forth. The in silico predictions can then be rapidly tested by
chromatin immunoprecipitation experiments coupled with the high-throughput
approaches noted above. Russanova VR, Hirai TH, Howard BH. Semi-random sampling to
detect differentiation- and age-related epigenome remodeling. J Gerontol A
Biol Sci Med Sci, in press. Russanova VR, Hirai TH, Tchernov AV, Howard BH. Mapping
development- and age-related chromatin remodeling by a high throughput
ChIP-HPLC approach. J Gerontol A Biol Sci Med Sci, in press. Histone
H3 methylation marks the murine H19-Igf2–imprinted region Tchernov, Howard; in
collaboration with Pfeifer, Stewart The
murine H19-Igf2–imprinted region is characterized by maternal
expression of the H19 locus versus paternal expression of the Igf2 gene.
A differentially methylated region (DMR) is located upstream from the H19
locus. In maternal cells, the DMR is unmethylated, binds to the transcription
factor CTCF, and functions as an insulator to prevent activation of the Igf2
promoter by enhancers located downstream from H19. Conversely, in paternal
cells, the DMR is methylated and repressed with respect to transcriptional
and insulator activities. Histone H3 methylation at lysine position 9 (K9)
appears to precede DNA methylation as a mark for transcriptional silencing in
a number of systems. Experiments by members of this group and collaborators
revealed a high level of histone H3-K9 methylation near the H19-associated
DMR and promoter region in paternal but not maternal cells. In contrast,
H3-K9 methylation was detected at control regions for the Igf2 locus
in paternal cells only. Of note, bisulfite mapping of DNA CpG methylation
sites confirmed that H3-K9 and DNA methylation occurs with opposite
parental bias upstream from the Igf2 gene. Moreover, the differential
H3-K9 methylation defined an upstream region distinct from that marked by DNA
methylation. This work reveals considerably greater complexity in the
relative histone and DNA methylation patterns than previously appreciated. COLLABORATORS Keiko Ozato, PhD, Laboratory of
Molecular Growth Regulation, NICHD, Karl Pfeifer, PhD, Laboratory of Mammalian
Genes and Development, NICHD, Colin Stewart, PhD, Cancer and
Developmental Biology Laboratory, NCI, Frederick, MD
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