The role of dTopors in nuclear structure and meiotic chromosome segregation in Drosophila melanogaster

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)

Andrea Maria Binder (Creator)

Institution

The University of North Carolina at Greensboro (UNCG )

Web Site: http://library.uncg.edu/

Advisor

John Tomkiel Dean

Abstract: Ubiquitination and sumoylation are post-translational modifications that control a variety of cellular processes. Topors, a protein that can act as both an E3 ubiquitin and SUMO-1 ligase, is critical for regulation of gene expression, DNA repair and cell division. The Drosophila homolog, dTopors, is required for establishing proper nuclear structure and chromosome segregation in male meiosis. The relationship between ubiquitination and/or sumoylation in these functions, however, is unknown. Using CRISPR-Cas9, I created separation-of-function mutants to specifically alter the ubiquitin ligase activity and used these to investigate the role of ubiquitination in these processes. In combination with mass spectrometry, I also utilized these mutants to identify potential targets of dTopors’ ubiquitination and sumoylation in testis. Furthermore, to determine which E2 ligase acts with dTopors in the germline, I performed an RNAi knockdown screen of each E2 ligase and assayed for nondisjunction and nuclear defects. Positives were then tested for enhancement of a dtopors hypomorphic allele. I found that the nuclear structure was more sensitive to lack of dTopors’ ubiquitination than was chromosome segregation, suggesting that there may be different targets of dTopors ubiquitination important for each phenotype. Mass spectrometry identified over 800 peptides that were differentially ubiquitinated in the presence or absence of dTopors’ ubiquitination. Notably these included chromatin proteins, Lamin Dm0 and numerous components of the ubiquitination pathway. Similarly, differentially sumoylated targets were identified including proteins involved in chromatin structure and transcription. Effete (UbcD1) and Bruce (BIR repeat containing ubiquitin-conjugating enzyme) were identified as potential E2 ligase partners of dTopors. Expanding our knowledge of targets and E2 ligase partners are critical steps towards understanding the relationship between ubiquitination/sumoylation and germline nuclear structure and chromosome segregation.

The role of dTopors in nuclear structure and meiotic chromosome segregation in Drosophila melanogaster
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Created on 8/1/2022
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Additional Information

Publication

Dissertation

Language: English

Date: 2022

Keywords

Drosophila, dTopors, Lamina, Meiosis, Spermatocytes, Ubiquitination

Subjects

Fruit-flies

Germ cells

Meiosis

Small ubiquitin-related modifiers

Sumoylation

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