3D chromatin architectural dynamics at ultra-high resolution

The National Human Genome Research Institute awarded The Children's Hospital of Philadelphia $863,248 on February 1, 2026, under the Human Genome Research program (CFDA 93.172) to investigate three-dimensional chromatin architectural dynamics at ultra-high resolution as it relates to genome folding and gene activity.

The research examines how microcompartments between active cis-regulatory elements form as cells exit mitosis and enter G1 phase, using ultra-high resolution region capture micro-C (RCMC) and acute degradation technology to interrogate the role of key proteins in microcompartment formation. A second aim applies RCMC to constitutive and facultative heterochromatin regions to uncover fine-scale chromatin microstructure within repressive compartments. Both aims employ machine learning-based imputation to derive genome-wide insights and incorporate mechanistic three-dimensional polymer modeling to explain experimental data and make testable predictions. The third aim validates findings using super-resolution chromosome tracing as an orthogonal method. The award runs through January 31, 2030, with place of performance in Pennsylvania.

Massachusetts Institute of Technology received a sub-award (R01HG014500-FP00047817_SUB01_01) effective April 29, 2026. Dr. Hansen's lab at MIT performs all computational analysis of RCMC and Micro-C data including alignment, loop and feature calling, machine learning-based imputation of genome-wide maps, and leads three-dimensional polymer modeling and imaging-based chromosome tracing via Hi-M. Dr. Blobel's lab at CHOP leads cell culture, cell line engineering, and RCMC experiments, with work redistributed between sites as project development warrants.

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.