3D Chromatin architectural dynamics at ultra-high resolution

Project Summary/Abstract This proposal will investigate the dynamic 3D folding of the genome at ultra-high resolution across length and time scales as it relates to gene activity. Studies in Aim 1 will investigate how newly discovered “microcompartments” between active cis-regulatory elements (CREs) form as cells exit mitosis and enter G1 phase. Because microcompartments cannot be resolved with conventional Hi-C or Micro-C methods, we will apply ultra-high resolution Region Capture Micro-C (RCMC). Using acute degradation technology, we will investigate the role of a battery of key proteins in microcompartment formation. Studies at the mitosis-to-G1 transition will be complemented using a dynamic cell transition system that considers CRE dynamics during transcriptional repression. Genome-wide insights will be obtained using newly developed machine learning- based imputation. Aim 2 is motivated by the hypothesis that similar to transcriptionally active microcompartments, intricate fine scale chromatin organization exists within heterochromatin. We will apply RCMC to representative regions of constitutive and facultative heterochromatin. These studies will be complemented by experiments perturbing key heterochromatic regulators. Furthermore, machine learning based imputation will be applied to obtain genome-wide insights, followed by validation experiments, aimed at uncovering the fine-scale microstructure of the repressive chromatin compartments. Importantly, both aims will be enhanced by mechanistic 3D polymer modeling with the goal of developing a polymer model from first principles that can explain the data, to make experimentally testable predictions, and to estimate key parameters which are otherwise not experimentally observable. In Aim 3 we will validate and extend our findings using super-resolution chromosome tracing experiments as an orthogonal and sequencing independent method. This will address if microcompartments form through simultaneous multi-way interactions, will reveal cellular or allelic heterogeneity in chromosomal folding, and deepen our understanding of the relationship between 3D genome folding and transcription.