Using single molecule footprinting to study chromatin regulation in cardiac differentiation and diseases

About This Grant

Project Summary/Abstract Gene regulatory mechanisms are a key driver of the dynamic changes from multipotent progenitor cells into progressively more differentiated cell types during organ development, including cardiac development, and tightly regulated in time and space. Mutations that affect gene regulation often lead to congenital defects or increase risk for complex diseases. However, in most cases, the molecular mechanisms causing these effects remain unknown. This knowledge gap exists because current genomics methods cannot fully capture the coordinated and dynamic gene regulatory processes, including resolving the interactions between transcription factors (TFs), cis-regulatory elements (CREs), DNA methylation, and chromatin. In this proposal we will focus on the gene regulatory mechanisms of cardiomyocyte differentiation and cardiac disease. We will use single molecule footprinting (SMF) which combines methyltransferase-based measurement of chromatin organization with native long-read sequencing, to assess chromatin accessibility, nucleosome positioning, TF binding, and DNA methylation simultaneously on single chromatin fibers from human cells. We will collect both SMF data and single-cell multiome data from induced pluripotent stem cells at multiple time points as they differentiate into cardiomyocytes. Using multimodal data captured from haplotype-resolved individual DNA molecules, we will explore how genetic perturbation of CREs and TFs - including those observed in congenital and complex cardiac diseases - affect chromatin organization and transcription. In this study we aim to develop better models of the gene regulatory mechanisms that drive cardiomyocyte differentiation, providing direct insights into developmental cardiac defects; we aim to establish a novel paradigm for the genome-wide study of dynamic and coordinated gene regulatory processes.