Research Test-Bed Unit: PROJECT SUMMARY The overall goal of the U54 Northwestern University Center for Chromatin Nanoimaging in Cancer (NU-CCNIC) is to develop and deploy a multi-scale chromatin nanoimaging platform together with molecular analyses and computational modeling to characterize chromatin structure and transcriptional patterns associated with the cancer stem cell (CSC) and chemoresistance phenotype. The immediate application of the proposed studies will be ovarian cancer, a malignancy of unmet need. It has been speculated that CSCs represent the reservoir from which recurrent, chemotherapy-resistant tumors arise. The key biological question addressed by this Center is whether reprogramming of the transcriptome through epigenetic and chromatin organization-regulated mechanisms leads to significant transcriptional plasticity, which is critical for CSCs to withstand and survive stressors in the tumor environment, driving tumor initiation and progression. As part of NU-CCNIC, the Research Test-Bed Unit (RTB) will provide source materials for the nanoimaging technologies developed by the Technology Development Unit (TECH). These resources include cells and tissues at various transition points between stem cell and non-stem cell phenotypes and between chemotherapy-sensitive and resistant states. The studies conducted by the RTB will test the applicability of the Nanoscale Chromatin Imaging and Analysis (nano- ChIA) platform and provide feedback to optimize its use. In addition, the RTB will perform state-of-the-art computational genomic analyses of CSCs and chemotherapy-resistant cells, including single-cell mRNA sequencing and genome mapping (e.g., Hi-C, ATAC-sequencing and ChIP-sequencing). The specific objectives of this unit are: 1) To identify CSC-specific epigenomic features and 3D chromatin packing conformations by integrating genome-wide maps of chromatin accessibility, contact frequency and gene expression networks with high resolution nano-scale chromatin imaging features. 2) To identify whether the transition to a chemotherapy-resistant state promotes stemness-like chromatin packing and conformation. Locus specific epigenetic manipulations will be coupled with high resolution imaging technologies to investigate resistant-state specific 3D chromatin packing and its relation to the CSC state. 3) To discover how global epigenome manipulations induced by small enzymatic inhibitors block stemness and chemo-resistance through alterations of chromatin packing. In all, the integrated interrogation of cancer through chromatin nanoimaging methods (TECH) and genome-wide mapping (RTB) will discover how transcriptional plasticity of CSCs and chemo-resistant cancer cells is regulated. The project is at the forefront of the field by using highly innovative molecular and nanometer-scale chromatin imaging technologies to better understand the relationship between higher level chromatin structure and key drivers of stemness and chemo-resistance in...