Investigating transcriptional regulation through epigenetic DNA modifications and coactivator recruitment using SELEX

Abstract

Transcription factors (TFs) recognise specific DNA sequences to regulate gene expression, and their binding preferences can be dynamically modulated by epigenetic modifications and interactions with co-regulatory proteins. In this study, I systematically investigated two major modulators of TF-DNA binding: DNA methylation and transcriptional coactivators. Using adapted high-throughput SELEX (systematic evolution of ligands by exponential enrichment) approaches, I developed novel platforms to profile how these factors reshape the sequence specificity of human transcription factors in vitro. The first part of the thesis focuses on methyl-SELEX, a technique adapted to examine the influence of cytosine methylation on TF binding. By incorporating 5-methyl-dCTP at defined ratios during PCR amplification of SELEX DNA pools, I established a methylation gradient (0%, 33%, 67% and 100%) and analysed binding preferences across 272 human TFs. Combined with previous data, over 300 unique TFs were covered, with widespread methylation-dependent binding signals observed. TFs were classified according to their sensitivity to CpG and non-CpG methylation (mCG and mCH), and further grouped into methyl-responsive categories (mCG+/− and mCH+/−) based on dose-dependent effects. For example, TFs such as TEAD2 showed altered binding specificity in response to non-CpG methylation (mCH−), which suggested an underexplored regulatory code in non-CpG contexts. Using a similar approach, I also tested the impact of DNA N6-methyladenine (6mA) by mA-SELEX. No significant motif shifts were observed, likely due to limited incorporation of N6-methyl-dATP by standard polymerases, suggesting that future work should focus on optimising polymerase compatibility with 6mA. In the second part, I investigated interactions between TFs and transcriptional coactivators using coactivator-SELEX. Human recombinant Mediator and p300 were introduced into SELEX reactions to probe their effects on TF binding. Mediator-SELEX yielded limited enrichment, likely reflecting weak or transient protein-protein interactions, while p300-SELEX showed a modest increase in the enrichment of TF binding sequences. Together, this thesis introduces new experimental approaches to study how DNA modifications and protein cofactors influence TF-DNA interactions. These findings expand our understanding of transcriptional regulation and provide mechanistic insights into dynamic gene control in development and disease.