Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.780553
Title: Investigating the transcriptional role of FoxP in decision making in Drosophila melanogaster
Author: Hak, Laura Chan Wah
ISNI:       0000 0004 7966 1943
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Abstract:
Transcription factors play crucial roles in the expression of genes and thus control the cellular abundance of proteins, which in turn define cell fates, developmental processes, cellular states and ultimately behaviour. One family of transcription factors of special interest in molecular and developmental neurobiology, cognitive science, linguistics and evolutionary anthropology is the evolutionarily conserved forkhead box P (FoxP) proteins, which have been implicated in vocal communication, motor learning, cognitive development, and decision-making. To fully understand the role of FoxP family members in these processes, it is important to analyse how they impact cellular functions through the regulation of gene expression. At the molecular level, the study of transcription factors involves identifying their target genes and specific binding patterns, analysing the effects of transcription factor-DNA interactions on gene expression and understanding gene regulatory networks. The Drosophila FoxP gene gives rise to 2 isoforms, of seemingly differential expression. Elucidating the binding patterns, the putative targets and the gene regulatory networks of each isoform will give an insight into why the isoforms are differentially expressed and how the different isoforms influence different behaviours, such as decision-making, differentially. In order to analyse the putative targets of FoxP in the Drosophila brain, targeted DNA adenine methyltransferase identification (targeted DamID or TaDa), coupled with next generation sequencing was carried out. The binding profiles were consistent with FoxP regulating gene expression at the level of transcription by acting on silencers and promoter regions of its target genes. Consensus FoxP binding sites were determined by analysing for enrichment of common motifs in FoxP-bound regions. Targeted DamID identified about 600 targets for each isoform, with less than 50% overlapping genes. Most of the putative targets of both isoforms play crucial roles in the nervous system, in agreement with the known or suspected roles of FoxP. These targets were confirmed with a complementary method, chromatin immunoprecipitation (ChIP) in vitro in neuronal cells and in vivo in Drosophila brains. Among the targets of the FoxPC isoform is the gene encoding the voltage-gated potassium channel Shal, confirming biophysical evidence that FoxP sets neuronal integration and behavioural decision times by controlling the abundance of Shal in a small population of FoxP-positive, decision-critical neurons. One of the major challenges of transcription factor studies is that binding sites are common in the genome and binding per se does not necessarily have a functional effect. The functional consequences of the transcription factor-DNA binding were tested by luciferase reporter assays and translating ribosome affinity purification (TRAP). Like its mammalian orthologues, Drosophila FoxP, acts as a transcriptional repressor, decreasing mRNA levels of the most highly-bound genes. Since most transcription factors work in a cooperative manner to regulate gene expression, potential FoxPC cofactors were identified to allow a more in-depth understanding of gene regulation, especially in the decision-making process. Yeast two-hybrid and coimmunoprecipitation assays identified three transcription factors, glial cells missing (gcm), Sp1-like factor for pairing sensitive-silencing (Spps) and Dichaete (D) as potential interaction partners. ChIP and luciferase assays confirmed that FoxPC cooperates with these transcription factors to regulate its target genes. Together, these results offer not only a global view of gene expression control by FoxP but also a molecular explanation for the decision phenotype of FoxP mutant flies.
Supervisor: Miesenboeck, Gero ; Glitsch, Maike Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.780553  DOI: Not available
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