Understanding the binding of multiple transcription factors by base-pair-resolution chromatin accessibility

Abstract A fundamental challenge in studying cell function and disease is understanding how cis-regulatory regions control gene expression. Sequences of these regions specify the binding sites of multiple transcription factors, and understanding regulatory function requires the understanding of how multiple transcription factors bind simultaneously. This is particularly important for understanding the impact of genetic variants lying within regulatory regions. We recently discovered that regulatory variants in a cell-type specific silencing element in an intron of gene HK1 cause congenital hyperinsulinism. We found over 26 variants within 50 bp, identifying three transcription factor binding sites. To understand this locus, we need to understand how these transcription factors simultaneously bind the same DNA molecule. Technologies that measure transcription factor binding or chromatin accessibility generally lack the capacity to measure simultaneous binding of transcription factors. Here, we employ the capacity of nanopore sequencing to directly detect DNA modifications to understand base-pair-level chromatin accessibility. We use exogenous bacterial DNA methyltransferases to mark accessible DNA: Eco-GII to mark 6mA, providing an average accessibility resolution of <2 bp; and M.CviPI to mark 5mGC, providing an average accessibility resolution of 25 bp. We detect these modifications using nanopore sequencing to build maps of chromatin accessibility. We report on our progress, establishing methodologies and data pipelines. We calibrate against wellunderstood chromatin accessibility surrounding CTCF binding sites and investigate the co-operative binding of multiple transcription factors at our HK1 disease locus. Biography Ailsa MacCalman received her PhD in Medical Studies from the University of Exeter in 2023. During her PhD with Professor Jon Mill, Ailsa investigated the genomics of human fetal pancreas development. She investigated the epigenetic and transcriptomic changes regulating pancreatic development and their implications in pancreatic disease. Following her PhD, Ailsa began her postdoctoral research with Dr. Nick Owens, who is supported by a Wellcome Career Development award, investigating the cis-regulatory code. She uses sequence changes in rare and common pancreatic diseases as a model to gain insight into genomic regulation.