Understanding chromosomes: (left to right) Shiv Grewal, NCI; Thomas Ried, NCI; session chair John Niederhuber, NCI director; Gary Felsenfeld, NIDDK; and Francis Collins, NHGRI

Francis Collins–
The Genetics of Common Disease

Efforts to uncover the genetics of common disease are "rocketing forward" thanks to a revolution in genomic tools and technologies and the emergence of new, multidisciplinary collaborations, said NHGRI Director Francis Collins.

A huge leap forward was the recent creation of a map of common human genetic variation, called the HapMap. The HapMap confirmed that variations in DNA sequence—single nucleotide polymorphisms (SNPs)—travel in neighborhoods, called haplotypes. More important, the map paved the way for genome-wide association studies (GWAS), a new approach that involves rapidly scanning SNPs across the genomes of many affected and nonaffected people to find genetic variations associated with a particular disease.

An impressive array of NIH-led initiatives, including the Genetic Association Information Network and the Genes, Environment and Health Initiative, have been launched to tap into this powerful resource. Data generated by these and other NIH-supported GWAS are already being deposited in the Database of Genotype & Phenotype (dbGAP), a powerful database developed by NCBI for use by the scientific community, Collins said.

Collins has applied the new tools to his own research endeavors. Working in close collaboration with two other international teams of epidemiologists, geneticists, and bioinformaticians, Collins’ group in April 2007 published results of a genome-wide survey of genetic risk factors involved in type 2 diabetes—a disease long considered "the geneticist’s nightmare."

The studies identified at least four new variants associated with increased risk of diabetes. The variants lie in an intergenic region of chromosome 11 and near IGF2BP2, CDKAL1, and CDKN2A/B, a cell-cycle gene also implicated in myocardial infarction. The research also confirmed several variants previously associated with type 2 diabetes.

Thomas Ried–Cancer:
A Disease of the Chromosome

It was just in 1956 that the correct number of human chromosomes was established. Since that time, the role of chromosomal abnormalities in blood cancers has been definitively recognized with the identification of the Philadelphia chromosome in chronic myeloid leukemia and other translocations in lymphomas, Thomas Ried observed. He then addressed the complexities of chromosomes and solid tumors.

Chromosomes in epithelial cancers are defined by catastrophic mitoses and centrosome amplifications that lead to cells with abnormal chromosome numbers, complex karyotypes, and ongoing chromosome instability, noted Ried, head of the Cancer Genomics Section, Genetics Branch, NCI.

Studies to determine the cytogenetic makeup of solid tumors, however, have been slower in coming, requiring advances in visualization techniques, such as comparative genomic hybridization and spectral karyotyping.

These are the tools that Ried’s group has used to study cervical cancer, identifying chromosomes that are altered in invasive tumors and following progression from early- to late-stage cancer.

Studies of invasive cervical carcinomas have revealed that the long arm of chromosome 3 is frequently amplified, suggesting a "fundamental genomic insult" and a driving event for disease progression, Ried said. A Pap test can identify morphologically altered cells indicative of cervical cancer, but, he noted, it doesn’t predict invasive propensity. Recognition of the role that amplification of chromosome 3 plays in progression from low- to high-grade lesions may lead to using cytogenetics in the staging and prognosis of cervical cancer.

Aneuploidies, in general, are a defining feature of carcinomas, Ried said, and there is a continuous selection for and maintenance of aneuplodies that are not fatal to cells, but rather confer "stability on a different plateau."

These aneuplodies are tumor-specific and acquired early in tumorigenesis, suggesting they may be useful in diagnosis. Ried’s laboratory is currently investigating mouse models of hematological malignancies such as Burkitt’s lymphoma and of mammary gland adenocarcinomas.

In addition to studying mouse models, Ried is particularly interested in looking at the entire transcriptional landscape of cells with aneuploidies, addressing what happens to genes on an extra chromosome. By generating artificial trisomies in human cell lines using microcell-mediated chromosome transfer, Ried’s group found that, in general, there is an increase in the average message levels of genes on the affected chromosome. The researchers also noted that the expression of genes on other chromosomes was affected as well, indicating that aneuploidies not only target a few specific genes for increases in transcription, but also lead to a "massive and complex deregulation of [the] cancer transcriptome."

Gary Felsenfeld–
Chromatin Boundaries

His lab’s work on DNA insulators, observed Gary Felsenfeld, has shed light on the nature of chromosomes and the role of chromatin boundaries in gene expression.

To maintain specific patterns of gene expression, organisms have evolved a variety of ways to "establish boundaries between regions with different properties," observed Felsenfeld, chief of the Physical Chemistry Section, Laboratory of Molecular Biology, NIDDK. As a result of thoroughly characterizing the chicken b-globin locus, Felsenfeld’s lab identified a specific DNA sequence that is capable of blocking enhancer-promoter interactions. DNA insulators, as these sequences are termed, are found in different places in the genome and help form boundaries by blocking inappropriate interactions within the nucleus.

Felsenfeld’s group identified a single protein in vertebrates, CTCF, for its ability to bind the chicken b-globin insulator and confer insulation. Recent studies of CTCF have begun to elucidate its genome-wide locations as well as the role of CTCF sites in mice that mediate the formation of DNA loops and higher-order structures.

In addition to their enhancer-blocking role, DNA insulators can act as barriers to block the spread of condensed chromatin. By systematically analyzing the presence or absence of various modified histones throughout the chicken b-globin locus, Felsenfeld’s laboratory discovered that nucleosomes at the insulator are highly marked with modifications frequently associated with open chromatin.

Further studies established that a heterodimer—USF1 and USF2—binds the insulator and recruits the histone H4R3-specific methyltransferase PRMT1 and, subsequently, a barrage of "positive" histone modifications to maintain a local environment of open chromatin.

Nucleosome stability can also play a role in regulation of chromatin-coupled mechanisms, Felsenfeld’s group has recently discovered. In addition to regulation of histones by modification, histone variants such as H3.3 and H2A.Z are incorporated into nucleosomes and can mark specific sites in the genome.

In particular, H3.3 is concentrated at regulatory sites and is displaced through nucleosome-disrupting activities such as transcription.

By studying the salt-dissociation properties of nucleosomes containing different combinations of these variants, Felsenfeld’s group has been able to determine a hierarchy of nucleosome stability. Nucleosomes with both H3.3 and H2A.Z are highly unstable, Felsenfeld said. These nucleosomes are predominantly found on the promoters of transcriptionally active genes and over the coding regions of genes transcribed at high levels, suggesting that these histone variants can serve as an epigenetic signal in the genome.

Shiv Grewal–Heterochromatin:
A Versatile Platform of the Genome

Continuing Felsenfeld’s theme of context within chromosomes, Shiv Grewal focused on the organization within chromosomes of so-called "junk" DNA—which, he observed, not only constitutes a significant fraction of eukaryotic genomes, but also contributes to regulating cellular processes and genomic evolution and stability.

Senior investigator and head of the Chromosome Biology Section, Laboratory of Biochemistry and Molecular Biology, NCI, Grewal has paid special attention to heterochromatin.

Within the fission yeast Schizosaccharomyces pombe, there are distinct peaks of heterochromatin, marked by the presence of a specific histone H3 modification on lysine 9 (H3K9), which recruits heterochromatin protein 1. These peaks are found at centromeres, telomeres, regions of ribosomal DNA, and the mating-type locus.

Grewal’s studies of these regions has revealed a role for small RNAs in the formation of heterochromatin: In a self-reinforcing loop mechanism, repeats in the DNA sequence in these regions are transcribed and resulting RNAs are processed into small RNAs that lead to the recruitment of H3K9 methyltrans-ferases and the spreading of heterochromatic marks.

More recent work has demonstrated that heterochromatin can recruit diverse regulatory proteins involved in histone modification, transcriptional silencing, RNAi, chromosomal architecture, and chromatid cohesion. Specifically at the mating-type region, heterochromatin is necessary for the spreading of the RNA-induced initiation of transcriptional gene-silencing complex and also inhibits RNA polymerase II from localization to these sites.

Grewal’s group recently identified a multiprotein effector complex, called SHREC, that coats all major heterochromatin domains and contains the histone deacetylases CLR3. This complex is recruited to heterochromatin in both RNAi-dependent and -independent manners.

Grewal’s work has contributed to challenging the dogma that heterochromatin is a static, rigid structure. Rather, Grewal and his team are discovering heterochromatin changes throughout the cell cycle. Their ongoing studies on the dynamic regulation of heterochromatin have provided new insights into the mechanisms of heterochromatin assembly, Grewal said.

The establishment and maintenance of these important heterochromatic sites, he said, are conducted through a carefully orchestrated dance of histone modifications and protein binding.

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