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Ashworth, Nolan

University of British Columbia

Imprinting, an epigenetic phenomenon where only one allele of a gene is expressed depending on its parental origin, plays a significant role in mammalian development. The sub-proximal MMU6 imprinting domain, particularly, includes the paternally expressed gene Mest and the maternally expressed genes Copg2 and Klf14. In addition to the mature mRNA, Mest has a much longer isoform produced via alternative polyadenylation, termed MestXL. Mest is a candidate gene for the growth restriction Silver-Russell syndrome (SRS), since maternal uniparental disomy for the chromosomal region containing MEST (mUPD7) has been identified in 5-10% of SRS patients and because Mest knockout mice exhibit a growth restriction phenotype. The tissue-specific switch from Mest to MestXL formation seems to be linked to differential expression of the downstream gene Copg2. Copg2, which is adjacent and antisense to Mest/MestXL, is normally biallelically expressed. However, in tissues where MestXL is produced, such as the developing central nervous system, Copg2 shows preferential maternal expression, suggesting a potential regulatory role for MestXL in this process since it is exclusively expressed form the paternal allele. This thesis explores the expression and regulation of MestXL, which induces secondary imprinting of Copg2 within the central nervous system (CNS). Although it is known that MestXL is expressed in the CNS, the specific cell types, mechanisms behind its production and effect on paternal Copg2 expression remain unclear. I leveraged RNA-seq data and established an ESC-derived cortical neuron differentiation protocol to investigate MestXL expression. To study the sufficiency of the Mest mRNA polyA signal (PAS) in MestXL production in cortical neurons, I also developed a double-fluorescent Mest primary PAS transgenic ESC line. Finally, I constructed an endogenous MestXL reporter-truncation vector, for eventual integration at Copg2 and forward genetics studies of factors involved in MestXL expression and Copg2 imprinting in F1 ESCs. These tools and methodologies provide a foundation for understanding the regulation and function of the Mest locus, with broader implications for APA regulation and secondary imprinting.

Text

2024-11-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/89674

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/