Study of Condensin

Name of Research Sponsor: Dr. David Engelke

Dates of Involvement: n/a

Abstract

The 274 tRNA genes in Saccharomyces cerevisiae are scattered throughout the linear genome, but are clustered together in the nucleolus in 3-demensional space. This spatial arrangement of tRNA genes contributes to tRNA gene-mediated (tgm) silencing of RNA polymerase II transcription adjacent to tRNA genes. Condensin is thought to keep the tRNA genes clustered together, regardless of the presence of other organizational factors- such as microtubules. I will research how condensin binds to the tRNA transcription complex. Moreover, I will study the possibility of condensin-condensin interactions as the mechanism for tRNA gene localization. I will do these things through in vitro reconstitution of the tRNA transcription complex. I will perform electrophoretic mobility shift assay (EMSA) shifts and electron microscopy to obtain results that I will then interpret. It has been suggested that condensin binds to TFIIIC, a transcription factor that binds to the promoter regions of tRNA genes. Also, it has been suggested that the clustering of tRNA genes is done through condensin-condensin interactions. All of this will act to broaden the understanding of the mechanism behind the spatial arrangement of eukaryotic genomes. This may have consequences in cancer studies. There is evidence that repetitive elements in the genome, such as Alu elements which are descendents of tRNA gene-like elements, have consequences in certain types of cancers.

Background

The DNA of living cells needs to be compacted to fit neatly in the nucleus, while maintaining space for necessary processes of transcription. Very little is known about how the genome is condensed and organized within the nucleus. For yeast, there are two models for spatial organization. The first being that there are condensed chromosome territories; the second, that there is extensive looping. Although it is accepted that localization is not static, current data suggests that the truth is a combination of the two models. The organization of the genome plays important roles in gene interaction and the activation and repression of gene expression.

This is significant because retrotransposons integrate at silenced loci because there is a selective advantage. Ty retrotransposons have a close association with tRNA genes. Ty elements or remnants of Ty elements flank on one or both sides >60% of tRNA genes. This has links to cancer research. It has been thought that increasing numbers of tRNA genes could help to deregulate cell growth. More accepted though is the thought that chromosomal translocations and other abnormalities that lead to cancers are influenced by the spatial proximity of the loci involved. Therefore, this research not only extends the understanding of eukaryotic cell organization and gene expression, but also has significance in its mammalian relatives. tRNA genes are considerably conserved elements of the genome. In this lab, Saccharomyces cerevisiae, a species of yeast, are studied. No animals or human subjects are used. The lab does have collaborators that may or may not do such testing.

The 274 tRNA genes in Saccharomyces cerevisiae are scattered throughout the linear genome, but are clustered together at the nucleolus in 3-demensional space throughout the cell cycle. This spatial arrangement of tRNA genes is necessary for tRNA gene-mediated (tgm) silencing of RNA polymerase II transcription adjacent to tRNA genes. This is a novel silencing method that is different from other methods of known silencing. The consequences of this phenomenon are largely unknown; however, in the yeast genome, pol II genes are underrepresented adjacent to tRNA genes, suggesting a potential physiological relevance.

For tgm silencing to occur, the full tRNA transcription complex- pol III and its two DNA-binding transcription factors, TFIIIB and TFIIIC- must be assembled at the tRNA gene. Whereas deletions in MAF1, RIS1 and MOD5 release tgm silencing while keeping localization in the nucleolus, mutations in other genes that cause a the dispersion of tRNA genes from the nucleolus also release tgm silencing. Most importantly, mutations in any of the subunits of condensin release tgm silencing. The effect of condensin on clustering of tRNA genes will be discussed here.

It is known that microtubules keep the tRNA gene clusters positioned at the nucleolus. It is thought that condensin keeps the clusters of tRNA genes together, however, regardless of the condition of microtubules. Microtubule disruption caused the clusters of tRNA genes to delocalize from the nucleolus, but they remained intact clusters (Haesuler et al, 2008). This suggests that some other factor is holding the tRNA genes together.

Condensin has been shown to be critical in the clustering of tRNA genes. Moreover, condensin has been found to have low level associations with the assembled tRNA gene complex (Haesuler et al, 2008). It also associates with TFIIIC and TFIIIB when the transcription factors are not bound to DNA, suggesting that condensin recognizes these transcription factors independently. Condensin remains associated with tRNA genes throughout the cell cycle as well, which is expected because the tRNA genes remain clustered throughout the cell cycle. Additionally, data from several groups show that condensin-condensin interactions are used for nucleolar compaction. This mechanism is likely to be similar to the one used for the clustering of tRNA genes. It is, however, unknown how condensin binds to the tRNA gene complex.

Mircoarray data show that condensin and TFIIIC binding sites are co-localized throughout the genome. Condensin binds specifically to TFIIIC binding sites- a process that is likely directed by protein-protein contacts. I will be looking into how condensin binds. Specifically, I will be reconstituting tRNA gene-condensin complexes in vitro.

Expected Results

Starting with radiolabebled DNA containing a tRNA gene, I will add in purified TFIIIC, TFIIIB, and pol III to reconstruct the tRNA gene complexes. These complexes will be made with stoichiometric amounts of TFIIIC alone, TFIIIB alone, TFIIIC and TFIIIB, and TFIIIC and TFIIIB and pol III. Condensin will be titrated into these complexes in an attempt to bind condensin to the complex. Moreover, complexes will be made with a tRNA gene and combinations of only condensin, condensin and TFIIIB, condensin and TFIIIC, condensin and pol III, condensin and TFIIIB and pol III, etc. Whenever condensin is added, titrated amounts of nonspecific, nonlabeled, competitor DNA will also be present. EMSA shifts will be used to determine binding.

Additionally, I will be testing condensin dependent looping. It is known that tRNA genes interact with high frequency with other tRNA genes. DNA fragments with two tRNA genes will be created. tRNA complexes will be built at the same time on tRNA gene. After fixing the cells with glutaraldehyde, the two complexes will be observed by electron microscopy. If looping does occur, the process will be repeated with increasing numbers of tRNA genes present. Because tRNA genes preferentially localize with other tRNA genes of the same family, it would be interesting to test the difference in creating fragments with tRNA genes in the same family to the fragments with tRNA genes in different families. Because tRNA family has little to nothing to do with tRNA gene complexes and condensing binding, as far as we known, it should not show a difference.

The PI has done many experiments similar in nature to the ones listed above and expects few difficulties. The hypothesis is that condensin binds to part of the tRNA gene complex and through condensin-condensin interactions causes the clustering of tRNA genes.

My Contributions

Although I wrote the above for the application for the UROP Biomedical Summer Fellowship, this project has not been started.

Sources

Haeusler, R., Pratt-Hyatt, M., Good, P., Gipson, T., & Engelke, D. (2008). Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes. Genes Development, 22(16), 2204.