AN INVESTIGATION OF THE MECHANISMS OF GROUCHO MEDIATED TRANSCRIPTIONAL REPRESSION

Groucho (Gro) is the Drosophila member of an evolutionarily conserved family of proteins that act as co-repressors for a wide range of transcription factors and includes four orthologs in humans, known as Transducin-like enhancer of split proteins (TLE1-4). This family of co-repressors play crucial roles in many developmental pathways (including Notch, Wingless and BMP/Dpp), and have been implicated in the pathogenesis of some human cancers. Despite the important roles that Gro proteins play during development and in adult life, there is not an established model that describes the mechanism underpinning Gro-mediated transcriptional repression. Recent studies that used Gro ChIP-seq data from Drosophila cells and embryos indicated that Gro could mediate transcriptional repression by promoting Pol II promoter-proximal pausing. In this work, I took three different approaches to test this model, and gain understanding of Gro-mediated repression. For the first approach, genetic assays exploiting the GAL4/UAS system were used to drive the tissue-specific RNAi downregulation of gro and genes involved in Pol II pausing in the developing wing and eye. These assays revealed that gro interacts genetically with the pausing factors Trl, Nelf, bin3, and Larp7, indicating that they work together during Drosophila development, and provide the first in vivo evidence that Gro promotes Pol II pausing. Additionally, genetic assays in which the levels of Gro were decreased or increased in a Notch-dependent Drosophila tumour model showed the essential role that Gro has during normal growth and tumorigenesis during eye development. Secondly, I performed a bioinformatic analysis of several published ChIP-seq datasets in Drosophila Kc167 to study the genome-wide overlap between Gro peaks and the binding sites of factors involved in Pol II pausing (including GAF, Nelf-E, Cdk9, and Myc), as well as Pol II peaks. It was also explored the co-localization between Gro and Psq, which together with GAF is known to bind to GAGA motifs, as well as with factors that associate with the two different Psq isoforms, containing and lacking the BTB domain. This analysis revealed a large overlap of Gro-bound regions with the binding sites of GAF and Nelf-E, which are two factors key in the establishment of the poised Pol II. A large overlap was also observed between Gro peaks and the binding sites of Myc, and Cdk9, which are involved in the release of the paused Pol II. This indicates that Gro may block the positive action of these factors on transcription and provides new hints on the possible mechanism by which Gro promotes Pol II pausing. Gro also showed different degrees of co-localization with the two Psq ChIP-seq datasets analysed, as well as with their associated factors. A very high ChIP-seq signal was observed at Gro-bound regions for Psq total (antibody against all Psq isoforms), ISWI, and CBP. A weaker signal was observed for BTB-containing Psq, and Pc, a very weak signal for the architectural proteins Su(Hw), CP190 and Mod(mdg4). Thus, these results suggest a preferent co-localization of Gro with non-BTB Psq and their associated proteins (ISWI, CBP, and Pc). For the third approach, I performed two unbiased high-throughput screens to find novel proteins that interact with Gro. Specifically, I used the full-length Drosophila Gro protein as bait in the yeast 2-hybrid screen, and in a TurboID-based proximity labelling assay in Drosophila Kc167 cells. However, these approaches resulted in unfruitful finding novel interacting partners. Taking together all the evidence from this work I proposed a model of Gro-mediated repression, in which Gro “competes” with activators to regulate transcription.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2021

Author dc:creator

• Martinez Quiles, Maria Lorena

Contributors dc:contributor

• Jennings, Barbara

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/zr1x-4935

OAI identifier oai:identifier

tle:d0a75911-3fa4-497a-bb99-db742251d447:d6bd9758-527a-46cd-bfe2-c433766e8fca:1