Research areas: Microbiology, RNA Biology, Biochemistry, Molecular Biology and Genetics
Research in the lab aims to understand how cells control expression of genes during normal growth, in response to stress, and in cancer. There are two main research areas in the lab.
1. Activation and re-initiation of transcription by RNA Polymerase II
In eukaryotic cells, RNA Polymerase II (RNAP II) is responsible for transcribing all protein-coding genes as well as a number of non-coding RNA genes. When a cell requires production of a specific protein or gene product, its gene is “turned on” by the process of transcriptional activation. This involves a step-wise assembly of a number of transcription factors and related complexes on the gene’s promoter, which enables the recruitment of RNAP II. RNAP II then initiates transcription of the gene.
The details of transcriptional activation have been studied for decades and are quite well-understood at many levels. However, not much is known about what happens to the promoter-bound transcription factors and complexes after the first molecule of RNAP II has initiated transcription. Research in the lab addresses this by studying how transcriptional activators, general transcription factors, and other promoter-bound complexes are regulated to make sure that further rounds of transcription can take place (i.e. re-initiation of transcription), and to ensure that the cell can shut down transcription of genes when their gene products are no longer needed.
2. Regulation of gene expression by SUMO post-translational modifications
Post-translational modifications (PTMs) play important, often essential, roles in practically every cellular process. A relatively newly discovered PTM is protein sumoylation, which involves attachment of the SUMO peptide to specific lysine side chains on target proteins. Sumoylation is an essential and widespread modification that targets hundreds of proteins involved in many cellular processes in all eukaryotes. The effects of protein modification by SUMO vary depending on the target protein, but include changes to localization, stability, and activity of the target.
The lab is interested in sumoylation because several proteins involved in gene expression are known targets of SUMO modification. In particular, numerous transcription and splicing factors are sumoylated, but in most cases, it is not known why they are modified, nor how sumoylation affects their function. Research in the lab aims to characterize how transcription and splicing are regulated by sumoylation of specific proteins, as well as entire protein complexes, involved in each process.
Interestingly, the number of proteins that are sumoylated in cells increases dramatically during exposure to stress, like heat shock. Furthermore, many of the sumoylation enzymes are over-expressed in tumours, presumably resulting in high levels of sumoylated proteins in cancer cells. Therefore, another of the goals of the lab is to determine how changes to sumoylation levels during stress and in cancer affect gene expression. For example, does increased sumoylation due to heat shock facilitate increased transcription specifically of heat shock genes?
Approaches and Techniques Used in the Lab
Both budding yeast and human cell culture systems are used with a combination of molecular biology, biochemistry and yeast genetics approaches. Examples of commonly used procedures include chromatin immunoprecipitation, western blotting, protein immunoprecipitation, and quantitative RT-PCR.
Ng CH , Akhter A , Yurko N , Burgener JM , Rosonina E , Manley JL. (2015). Sumoylation controls the timing of Tup1-mediated transcriptional deactivation. Nature Communications. 6: 6610.
Rosonina E , *Yurko N , Li W , Hoque M , Tian B , Manley JL. (2014). Threonine-4 of the budding yeast RNAP II CTD couples transcription with Htz1-mediated chromatin remodeling. Proceedings of the National Academy of Sciences 111(33): 11924-31
Rosonina, E., Duncan, S.M., and Manley, J.L. (2012) Sumoylation of transcription factor Gcn4 facilitates its Srb10-mediated clearance from promoters in yeast. Genes Dev. 26: 350-355.
Garcia, A., Rosonina, E., Manley, J.L., Calvo, O. (2010) Sub1 globally regulates RNA polymerase II CTD phosphorylation. Mol. Cell. Biol. 30: 5180-5193.
Rosonina, E., Duncan, S.M., and Manley, J.L. (2010) SUMO functions in constitutive transcription and during activation of inducible genes in yeast. Genes Dev. 24: 1242-1252.