From Transcription Factors to Transcriptional Pausing in Development and Disease: Insights from Drosophila Model

Date of Award

12-12-2024

Degree Name

Ph.D. in Biology

Department

Department of Biology

Advisor/Chair

Amit Singh

Abstract

The Drosophila model is an extremely useful tool to study development and disease. It offers several advantages with its short lifespan, high number of genetically identical offsprings, repertoire of genetic tools and conservation in developmental pathways and processes. With high genetic conservation and ease of screening phenotypes, the Drosophila eye offers an excellent model for studying development and disease. In this study, we have used the Drosophila eye to study the regulation achieved by a transcription factor, and transcriptional pausing factor in eye development with implications in human craniofacial development. In addition, we also assayed the impact of various proteins of SARs-CoV2 using the Drosophila eye model. In the first study, we focused on the role of a dorsal selector gene - defective proventriculus, that encodes a K-50 homeodomain containing transcription factor. Axial patterning is required to establish antero-posterior, dorso-ventral and proximo-distal axes and is crucial for the formation of a 3D organ. In the eye, dorso-ventral axis is the first to form. Previously, we characterized dve as a dorsal selector gene based on (1) its expression in the dorsal head vertex region of the eye disc, (2) its gain-of-function phenotype showing complete eye suppression and (3) its loss-of-function phenotype showing dorsal eye enlargements, as in other dorsal eye genes. We also reported that differences in Dve expression impact the placement of eyes on the head of an organism resulting in diversity across animal groups. The human ortholog of Dve is SATB1 and it is highly conserved. Misexpression of SATB1 in the developing eye results in eye suppression and ectopic Wg expression, as in dve gain-of-function. SATB1 is highly expressed in cancers and metastasis. As part of this thesis, I have performed structure-function analysis of Dve protein to dissect its diverse roles in development and growth. We identified N-terminal+ULD+Hox1 as the minimal domain sufficient to mimic full spectrum of Dve function during eye development. Misexpression of N-terminal+ULD+Hox1 in the entire developing eye results in complete eye suppression and ectopic induction of Wg. Furthermore, C-terminal of Dve represses Dve function during eye development. In the second study, we identified a novel role of Motif-1 binding protein during eye development. During development, cell death is one crucial component in determining the shape and size of the organ. It is also important for organ sculpting and removing damaged and superfluous cells. Apoptosis and autophagy and two important forms of genetically distinct cell death mechanisms that occur during development. M1BP is a transcriptional pausing factor and its downregulation causes complete eye suppression. M1BP is thus required to suppress Wg expression and promote eye fate during development. In this study, we have shown that M1BP is required to suppress Wg and JNK pathways and dependent cell death during development. Downregulation of M1BP in the developing eye results in induction of both apoptosis and autophagy in a JNK dependent manner. Furthermore, blocking either cell death shows a weak rescue while blocking both cell death modes showed significant rescue of eye suppression. This study uncovers a novel regulatory mechanism by M1BP mediated transcriptional pausing that is crucial for regulating cellular homeostasis. In the third study, we also looked at the impact of SARS-CoV2 proteins in cells and tissues using the Drosophila eye model. SARS-CoV-2 virus causes COVID-19 resulting in symptoms like inflammation, severe acute respiratory syndrome (SARS), and cognitive impairments like brain fog. With its recent history, the impacts of SARS-CoV2 on cells and the long-term effects of COVID-19 are not known. Upon misexpression of different SARS-CoV-2 proteins we observed that Nonstructural protein 3 (Nsp3) causes eye degeneration and dark necrotic spots. Nsp3 misexpression leads to reactive oxygen species (ROS) production and ectopic induction of cell death markers like TUNEL, Dcp1, and atg8a-mCh. Additionally, we observed progressive age dependent worsening of the phenotype in adult flies. When misexpressed in an Alzheimer’s disease transgenic fly model (GMR>Aβ42), Nsp3 further worsens the neurodegenerative rough eye phenotype due to increased cell death.

Keywords

defective proventriculus, structure-function, dve, M1BP, transcriptional pausing, eye development, drosophila, growth, development, cell death

Rights Statement

Copyright © 2024, author.

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