Regulation of pol III transcription by mTOR

Ramsbottom, Ben Alan (2006) Regulation of pol III transcription by mTOR. PhD thesis, University of Glasgow.

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Abstract

RNA polymerase III (pol III) is dedicated to the transcription of genes involved in protein synthesis (5S rRNA and tRNA genes). Cell division is dependent on the rate of growth, which is dependent on the rate of protein synthesis. Therefore, the rate of pol III transcription plays a fundamental role in cellular growth and proliferation. Regulation is mediated via a number of different mechanisms that can alter the activities of transcription factors, which direct pol III transcription. Work in this project was directed at uncovering potential mechanisms for pol III regulation. A primary target was the mTOR pathway, as it coordinates nutrient availability with cell growth. Addition of the mTOR inhibitor rapamycin resulted in a decrease in the level of pol III transcripts. Furthermore, inhibition of the mTOR pathway resulted in a decrease in the promoter occupancy of pol III and TFIIIB. This occurred without any changes in the abundance of these two factors. Past studies have shown that mTOR regulates the expression of pol I-transcribed genes through the kinase S6K. Consistent with this, knockdown of S6K reduced the expression of pol I-transcribed genes. However, knockdown of S6K had no effect on the abundance of pol III transcripts. This highlights a difference in the regulation of pol I- and pol Ill-transcribed genes. mTOR may regulate gene expression through its direct recruitment to the transcriptional machinery, as raptor, a component of the mTOR complex, was found to co- immunoprecipitate with TFIIIC in this present study. Furthermore, chromatin immunoprecipitation revealed that mTOR is associated with pol Ill-transcribed genes. TSC2, Rheb and PKB, upstream components of the mTOR pathway, were all shown to regulate the expression of pol Ill-transcribed genes. TSC2, a tumour suppressor, was found to have a negative effect on the expression of pol Ill transcribed genes. Loss of TSC2 in a knockout cell line resulted in an increase in abundance of the 110kDa subunit of TFIIIC. This is consistent with previous studies where TFIIICl 10 was suggested to be the rate-limiting component of pol III transcription. However, work in the present study using a TFIIICl 10 inducible cell line was unable to substantiate these claims. Blocking the mTOR pathway also resulted in a decrease in the acetylation of histone H3 found on pol Ill-transcribed genes. Furthermore, addition of the drug trichostatin A (TSA), which promotes the acetylation of histones and other cellular proteins, increased the level of pol III transcripts. TSA also increased the promoter occupancy of pol III and TFIIIB, while at the same time increasing the acetylation of histones H3 and H4 on pol Ill-transcribed genes. In addition to being acetylated, histones are also methylated on pol Ill-transcribed gene. ChIP analysis has demonstrated the presence histone H3 lysine 4 methylation and histone H3 lysine 9 methylation on pol Ill-transcribed genes. Further work is needed to characterize the role of these covalent modifications.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-71049
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71049

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