Ansari - Research

The research in this lab is directed towards understanding the regulation of transcription in eukaryotes. The major emphasis is on two aspects of transcription. The first aspect deals with the promoter-terminator interaction during transcription. The prevailing view of transcription of eukaryotic class II genes is that RNA polymerase II transcribes a linear template with spatially separate promoter and terminator regions. The “Chromosome Conformation Capture” (3C) technology was used to study promoter-terminator interaction in budding yeast, Saccharomyces cerevisiae. The CCC results clearly demonstrated that promoter and terminator regions of a gene interact during transcription such that RNAP II transcribes a looped rather than a linear DNA template as shown in the Fig 1. The looped configuration required Ssu72 and Pta1, the 3’end processing factors also required for transcription termination. The future research will focus on elucidating the effect of promoter-terminator interaction on transcription reinitiation. Attempts will be made to determine if juxtaposition of promoter and terminator results in enhanced transcriptional efficiency due to facilitated reinitiation by RNAP II. The role of chromatin structure in the process will be investigated.


RESEARCH INTERESTS
The research in this lab is directed towards understanding the regulation of transcription in eukaryotes. The major emphasis is on two aspects of transcription. The first aspect deals with the promoter-terminator interaction during transcription. The prevailing view of transcription of eukaryotic class II genes is that RNA polymerase II transcribes a linear template with spatially separate promoter and terminator regions. The “Chromosome Conformation Capture” (3C) technology was used to study promoter-terminator interaction in budding yeast, Saccharomyces cerevisiae. The CCC results clearly demonstrated that promoter and terminator regions of a gene interact during transcription such that RNAP II transcribes a looped rather than a linear DNA template as shown in the Fig 1. The looped configuration required Ssu72 and Pta1, the 3’end processing factors also required for transcription termination. The future research will focus on elucidating the effect of promoter-terminator interaction on transcription reinitiation. Attempts will be made to determine if juxtaposition of promoter and terminator results in enhanced transcriptional efficiency due to facilitated reinitiation by RNAP II. The role of chromatin structure in the process will be investigated.
The second aspect of my research deals with the role of chromatin structure and the cofactors involved in transcriptional regulation. Nucleosomes, the repeating units of chromatin, are often the target of modification by cofactors in response to environmental signals. The cofactors modify nucleosomes both covalently and non-covalently (Fig 2). To elucidate cooperative interaction of cofactors in effecting transcriptional regulation by signaling molecules is one of the primary focuses of my lab. To address the issue I will be using a novel method for biochemical isolation of selected chromatin fragments from yeast chromosomes. This method was developed while I was studying silencing in yeast. Briefly, site-specific recombination will be employed in vivo to produce DNA rings from the locus of interest and differential centrifugation used to separate excised chromatin rings from chromosomes and other cellular debris. The rings will be purified further by affinity chromatography on a lac-repressor column. The purified chromatin preserves the salient structural features of its native state.

			The main advantage of the approach is that specific chromosomal sequences can be isolated directly from chromosomes in high yield. Recombination is rapid and inducible, thereby permitting experiments which analyze chromatin at defined stages in development or the cell cycle as well as under specialized growth conditions. Another major advantage of the approach is that the purified material is amenable to further in vitro functional analysis, for example, to study transcription, DNA replication and recombination. I will be using this approach to investigate the transcription of URA3 gene in response to changes in uracil concentration in the growth medium. Ppr1 is the factor that regulates transcription URA3. Preliminary evidences in my lab have shown that Ppr1 is both an activator and a repressor of URA3 transcription. Whether, Ppr1 activates or repress URA3 depends on the concentration of uracil in the medium. Furthermore, Ppr1 interacts with Tup1, the global transcription repressor that recruits histone deacetylases. The role of Ppr1 in bringing about transcriptional regulation of URA3 in response to uracil as the environmental signal will be investigated using affinity purified chromatin rings.


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