Write-up of research and development interests/focus, past and present goals:
R & D interests:
My lab is interested in unraveling the epigenetic mechanisms underpinning the phenomenon of gene silencing in a genetically tractable single cell eukaryote, fission yeast, Schizosaccharomyces pombe. An underlying feature in these phenomena is the ability of various alternative epigenetic states of chromatin, exhibiting differing levels of gene activity, to be propagated as units of inheritance or Mendelian epialleles during multiple generations of mitosis and meiosis. We have investigated the role of different pathways in ensuring this faithful inheritance.
In one line of ongoing study the role of DNA replication machinery in propagating the epigenetic state of chromatin has been shown by the demonstration of interaction of the catalytic subunit of DNA pola with the heterochromatin protein Swi6/HP1, suggesting a coupling of DNA replication with assembly of heterochromatin. Further work has revealed that both Pola and Pold, the main enzymes involved in semi-conservative DNA replication in all eukaryotes, interact both with Swi6 and Clr4. These interactions may help in duplicating the heterochromatin code during the chromatin replication coincidentally with DNA replication. Because of the conservation of the components of heterochromatin and DNA replication machineries, the process of replication of heterochromatin may also be evolutionarily conserved.
Our earlier work had shown the role of DNA repair gene Rhp6/Rad6 in mating type silencing in fission yeast. Rhp6, being involved in polyubiquitylation of target proteins, subsequent work led to identification of a Uhp1, a 22kd protein of unknown function, as the target/mediator of silencing. Recent studies have revealed that this protein indeed has several interacting partners: histone H2B, ubiquitin and Clr4. Furthermore, it has an associated activity causing a lowering of histone H3-K4 dimethylation, which remains to be identified. It also contains an FMN-binding pocket and CUE domain found to be conserved among proteins interacting with ubiquitin in vitro and in vivo. Current work is focused on understanding how Uhp1 is involved in mating type and centromere silencing by modulating the effectors involved in histone H3 K9 and K4 methylation.
Our recent work has revealed a surprising role of Anaphase Promoting Complex (APC) subunits Cut4 and Cut9 in interaction with and recruitment of Swi6/HP1 and Clr4/Suv39h in a mutually cooperative manner to the heterochromatin loci, like mating type, centromere and rDNA. APC polyubiquitylates Cdc13 to cause its degradation by the proteasome thus facilitating mitotic exit. Similarly, APC also ubiquitylates Cut2 to release Cut1, which degrades the cohesin complex, thus facilitating sister chromatid separation during metaphase-to-anaphase transition. In conjunction with earlier observations that Swi6 recruits the cohesion complex to centromeric and mating type heterochromatin, we speculate that a well times recruitment of APC by Swi6 may help to orchestrate the timely degradation of Cdc13 and cohesin. Interestingly, like RNAi mutants, APC mutants also exhibit bidirectional transcription from the dg-dh repeats of centromere 1. Future work will investigate the possible role of heterohromatin in cell cycle progression through interaction with cell cycle regulatory components and the interaction of APC with RNAi machinery.
We have made highly interesting finding of genetic interaction between components of RNAi and heterochromatin protein. Studies are in progress to check whether there is direct physical interaction between RNAi and heterochromatin components. Future studies will address the mechanism in greater molecular detail.
In an application oriented project, we are developing S. pombe as an expression system for producing proteins at a lower cost and increased productivity. A patent and been filed in the US and PCT-designated countries for a new promoter that provides maximum expression level of several proteins within 3 hours of a temperature shift from 36oC to 25oC. We are also working with new promoters to achieve commercial scale expression of proteins that are important for therapeutic and industrial applications.