Chromatin Architecture Regulatory Mechanisms in Pathogenesis of B-Cell Lymphomas

PROJECT 2: PROJECT SUMMARY Cell fate transitions and cancer phenotypes depend upon the establishment of specific transcriptional programs. The dynamic folding of chromatin acts as a key regulatory layer of gene expression and cell identity by allowing select chromatin regions to facilitate transcription factor (TF) action, spatiotemporal rewiring of enhancers with their target promoters, and transcription. We found that cell fate determining genes tend to reside within architectural topographies that we call CRE hubs, which consist of networks of interacting enhancers and promoters anchored by highly connected architectural hotspots. Thanks to the development of the Pore-C method, with our collaborators we have now discovered that CRE hubs can form larger assemblies called chromunities, through bridging elements that connect CRE hubs. However, the underlying mechanisms of formation and regulatory potential of CRE hubs and chromunities are not well understood. Based on our preliminary data generated through this P01 grant, we found that cohesin and CTCF play a critical role in assembling enhancer loops within hubs in germinal center B-cells, from which most B-cell lymphomas arise. We performed the first CRISPR screen of a CRE hub enhancer and identified the biochemical mechanisms that explain its ability to form in a cell context specific manner. We also discovered that loss of chromatin folding due to histone 1 (H1) mutations in lymphoma can release stem cell enhancers from silenced chromatin compartments, endowing B-cells with stem-like properties. Therefore we hypothesize that (i) physiological reprogramming of CRE hub and chromunity regulatory elements creates de novo coordination between sets of genes required to establish specific cell states and phenotypes during the humoral immune response, (ii) SVs occurring in DLBCL alter these CRE hub and chromunity structures or create new ones giving a selective advantage to malignant clones, and (iii) rewiring of epigenetic compartments induced by H1 mutations results in architectural and plasticity permitting formation of aberrant oncogenic CRE hubs. Finally, we predict that analysis of mechanisms supporting this process represents a therapeutic vulnerability in B cell lymphomas. Therefore, we will (i) identify CRE hubs and chromunities driving phenotypic transitions and cell fate decisions during the humoral immune response, (ii) explore how these structures mediate cell lineage identity, (iii) determine how aberrant genomic structures in lymphoma patients create oncogenic driver chromunities, and (iv) establish whether and how mutations in epigenetic modifiers drive lymphoma phenotype by forming malignant aberrant chromunities. This work will provide fundamentally novel insight into the role, mechanism of action and biological relevance of 3D CRE hubs, chromunities and their regulatory hotspot elements in controlling cell identity and driving malignant transformation, as well as explain how they evolve from oncogenic genetic or epigenetic lesions.