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German, Spanish Team Uses Genomics to Explore Nucleolar Organization

By Andrea Anderson

NEW YORK (GenomeWeb News) – German and Spanish researchers reported online today in PLoS Genetics that they have used genomics to start uncovering the three-dimensional characteristics of the nucleolus, a dense protein and nucleic acid structure in the cell's nucleus.

The team used high-throughput sequencing, microarray analyses, fluorescence, and single cell experiments to characterize the coding and non-coding sequences associated with the nucleolus. By combining data from the experiments, they were able to gain insights into how the nucleolus is organized in two human cell lines — and clues about how it performs its growing list of cellular functions.

"We think the study is important because it sheds light on the structural organization of the nucleolus," senior author Gernot Längst, a biochemistry researcher at the University of Regensburg in Germany, told GenomeWeb Daily News. Rather than being randomly put together, he added, the nucleolus appears to have a specific structure that reflects its cellular functions.

The nucleolus is best known for its role in producing ribosomal RNA. But, the researchers noted, recent studies suggest the structure has other functions as well. For instance, the nucleolus appears to contribute to processes such as stress regulation and senescence, lead author Attila Németh, a researcher in Längst's lab at the University of Regensburg, told GWDN. And specific proteins implicated in human disease appear to localize to the nucleolus.

In an effort to explore nucleolar organization and function in more detail, the team first isolated nucleoli from HeLa cervical cancer cells. They then used a combination of fluorescence in situ hybridization, array comparative genomic hybridization with the Nimblegen 385K WG tiling array, and Roche 454 sequencing to explore which regions of the genome are associated with the nucleolus.

By comparing different types of data, Längst explained, "we could really get a high confidence list" of chromosomal regions and DNA sequences associated with the nucleolus.

The researchers found, for example, that 97 chromosomal regions — or some four percent of the genome — are associated with nucleoli.

When they compared the more than 1,000 genes found in these nucleolus-associated regions with those associated with lamina structure surrounding the nucleus, they found that both the nucleolar and nuclear lamina were associated with more odor reception and defensin-related genes than the genome in general. But, the team noted, these genes were more common in the nucleolar than laminar domains.

Along with RNA genes they expected to find in the nucleolar vicinity, the team noted, DNA regions associated with the nucleolus also contained a slew of zinc finger genes and genes coding for proteins involved in tissue development, embryo implantation, and more.

Satellite repeats, which the researchers proposed might play a role in facilitating interactions between the nucleolus and associated chromatin regions, were also ten times more common in these regions than in the genome at large.

By comparing their results with previous maps of histone modifications in the genome, the researchers found that regions interacting with the nucleolus coincided with a rise in histone modifications typically present when genes are repressed and a decrease in modifications usually found in active regions.

Even so, Németh explained, the presence of expressed genes in these regions — including 5S RNA and tRNA genes — suggests the nucleolus is not only associated with genes sequestered for inactivation, but with functional genes as well.

"[T]he presence of the highly expressed classes of 5D RNA and tRNA genes in nucleolus-associated chromatin indicates that the perinucleolar region is not exclusively transcriptionally silent," the researchers explained.

In their subsequent experiments, the team did immuno-FISH in the HeLa cells and a second human cell line (an embryonic lung fibroblast line called IMR90) to confirm the localization of some regions found near the nucleolus, along with experiments in which they tested the effects of blocking the RNA polymerases II and III.

While the current study offers a snapshot of nucleolar architecture and function in the two cell types tested, those involved in the research say more work is needed to understand additional aspects of nucleolar organization, including nucleolar dynamics.

In the future, the researchers plan to explore the organization, regulation, and dynamics of the nucleolus at even greater resolution, Längst said, and hope to characterize nucleolar features at various stages of the cell cycle and during different stages of cellular differentiation. They also hope to learn more about whether nucleolar organization and function are evolutionarily conserved in different species.

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