Development of novel high- throughput assays to measure compound binding to transcription factors in cells

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Examensarbete för masterexamen
Master's Thesis

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Transcription factors (TFs) are proteins possessing domains that recognizes and bind to specific DNA sequences. These proteins also possess domains that recruit various collaborator proteins and the transcriptional regulatory machinery, thus regulating the rate of transcription in nearby genes. Dysregulations of gene expression mediated by TFs is associated with numerous diseases, making them attractive targets for research. However, the understanding of the intricate mechanisms governing TF-DNA binding remains limited. This lack of knowledge complicates the study of TF-DNA binding, making it both labour-intensive and costly. To overcome these challenges, there is a need for high-throughput screening (HTS) methods that can efficiently identify TF-DNA interactions, characterize functional binding site motifs, and evaluate the impact of compounds on the TF-DNA binding. This thesis project thereby aimed to develop a novel cell-based assay to measure TF-DNA interactions in high throughput by leveraging the split-reporter system NanoBiT®. This system comprises two subunits: the Large BiT (LgBiT) and Small BiT (SmBiT), that upon proximity forms an active enzyme that produces luminescence. The concept involved tagging dead Cas9 (dCas9) with a LgBiT, tagging various TFs with SmBiTs, and providing single guide RNA (sgRNA), directing dCas9 to target DNA sites. The study focused on two disease related TFs: signal transducers and activators of transcription 3 (STAT3) and androgen receptor (AR). Two distinct cell models were used: HEK-293 for STAT3 and LNCaP for AR. Plasmids expressing dCas9 linked to N- or C-terminus of the LgBiT were designed and synthesized. Following transfections with these constructs, the work of generating stable pools using geneticin antibiotic selection was initiated. Subsequently, TF-SmBiT plasmids were designed and synthesized, along with a range of sgRNAs. These guides were designed to target promoter sequences of downstream genes associated with the TF. The established cell lines expressing dCas9-LgBiT were then transiently transfected with the TF-SmBiT plasmid and various sgRNAs, enabling subsequent measurement of the TF-DNA interactions. In this project, stable pools of HEK-293 cells expressing dCas9-LgBiT-N/C-terminus were established and validated through western blots. Despite several attempts, generation of LNCaP cell lines stably expressing these constructs was never achieved. After generating the assay reagents, the NanoBiT® assay was developed and tested. Specific interactions were not evident in the initial testing phase, when testing the first set of sgRNAs. By designing and testing more sgRNAs, the developed system holds potential for evaluating TF-DNA binding in a high-throughput manner, identifying transcription factor binding sites (TFBSs), and assessing the impact of compounds on TF-DNA interactions. Nonetheless, further validation of the assay is imperative to ensure its reliability and applicability in future studies.

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Transcription factor, High-throughput screening, Cell-based assay, Split-reporter system,, dCas9, sgRNA, STAT3, AR, HEK-293, LNCaP

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