Differential Regulation of RNA Polymerase II Proximal-Pausing During Sexual Differentiation in Mice
|dc.contributor.author||Weston, Mitchell Kyle|
|dc.identifier.citation||Weston, M. K. (2015). Differential Regulation of RNA Polymerase II Proximal-Pausing During Sexual Differentiation in Mice (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/6084||en|
|dc.description.abstract||During animal development, correct spatial and temporal gene expression is necessary to ensure the organism develops appropriately. Errors in gene regulation during development can result in any number of problems, resulting in anything from tumour formation to embryonic lethality. While most studies have focused on gene regulation by looking at the initiation phase of transcription, recent evidence suggests that the elongation phase of transcription is also tightly regulated. RNA Polymerase II (Pol II) has been shown to pause around 30-50 base pairs downstream of the +1 transcription start site (TSS) following the release of Pol II from the preinitiation complex (PIC). The regulation of this is dependent on three protein complexes, namely Negative Elongation Factor (NELF), DRB Sensitivity Inducing Factor (DSIF) and Positive Transcription Elongation Factor b (P-TEFb). While other studies have shown that up to half of all developmental genes have stalled Pol II near the promoter region, our ChIP-seq studies have shown sex-specific patterns of Pol II pausing occurring in brain tissue from 13.5 days post coitum (dpc) mouse embryos. I hypothesise that the observations made from the ChIP-seq experiments could be explained by sexually differential expression of the NELF, DSIF and P-TEFb complexes. To examine this, I have used a combination of RT-qPCR and whole mount in situ hybridisation to characterise the expression patterns of the subunits of NELF, DSIF and P-TEFb during mouse embryonic development in two sexually dimorphic tissues, specifically the gonads and the brain, over a four day timespan from 12.5 to 15.5 dpc, a crucial developmental stage of sexual differentiation. RT-qPCR analysis revealed sexually dimorphic expression of all four subunits of NELF and the Supt4a subunit of DSIF at 13.5 dpc in the gonads, as well as increased expression of the CycT1 and CycT2 subunits of P-TEFb in the female gonad at 15.5 dpc. Additionally, all subunits of P-TEFb were expressed at higher levels in the female brain, while the Cobra1 and Whsc2 subunits of NELF and the Supt4a subunit of DSIF were all expressed at higher levels in the male brain at 14.5 dpc. Whole mount in situ hybridisation of gonads from both male and female embryos at 13.5 dpc revealed higher expression within the testis cords when compared to the surrounding interstitial tissue. Finally, the Cobra1 subunit of NELF and the Supt4a subunit of DSIF mRNAs were expressed in both the mesonephric tubules and mesonephric ducts in male tissues. Female gonads showed a more uniform expression pattern for each mRNA examined. These findings overall suggest that factors regulating Pol II pausing are expressed in a sex-dimorphic manner, possibly explaining the results observed from earlier ChIP-seq experiments and supporting the notion that Pol II pausing dynamics may be regulated by expression of the pausing regulators, NELF, DSIF and P-TEFb. While further experiments are required to further characterise this, these findings show that the release of stalled Pol II may influence sexual differentiation and presents a novel line of investigation, such as looking at if and how Pol II pausing affects the expression of specific genes related to sex differentiation.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Differential Regulation of RNA Polymerase II Proximal-Pausing During Sexual Differentiation in Mice|
|thesis.degree.discipline||Anatomy / Genetics|
|thesis.degree.name||Master of Science|
|thesis.degree.grantor||University of Otago|
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