Investigating the role of mammalian SWI/SNF chromatin remodeling complexes in lymphocyte differentiation and function

Abstract

The dynamics of chromatin architecture and accessibility permit healthy cell type-specific biology, which when dysregulated, can lead to various human diseases. The mammalian SWI/SNF (mSWI/SNF) family of complexes are ATP-dependent, multi-subunit molecular machines that alter DNA-histone interactions to generate accessibility at key gene regulatory elements that control gene expression. Importantly, mutations in SWI/SNF subunit genes occur in over 20% of cancers emphasizing their protective functions in normal cell processes and their increasingly recognized and mechanistically interrogated roles in oncogenesis. The role for mSWI/SNF complexes in modulating chromatin accessibility in the setting of lymphocytes is relatively understudied due to technical limitations in experimental methodologies and complexities of maintaining primary cells in culture. These studies aim to uncover the functional contribution of mSWI/SNF localization, accessibility generation, and gene expression programs in healthy cellular processes including T cell exhaustion and B cell maturation, and within B cell malignancies. To understand the role of SWI/SNF in T cell activation and exhaustion, we used transient or chronic antigen-independent stimulation across early-activation and late-exhaustion timepoints in CD8+ human T lymphocytes. SWI/SNF occupancy, accessibility generation, and downstream gene expression, in the absence or presence of SWI/SNF genetic and pharmacologic modulators, were evaluated through integrative genomics-centered approaches, including CUT&Tag, ATAC-seq and RNA-seq. We determined that SWI/SNF chromatin targeting, and activity specifically modulates distinct and temporally controlled stages of T cell activation and exhaustion. Furthermore, chemical and genetic perturbation of the SWI/SNF complex enhance T cell persistence and CAR-T expansion. These studies uncovered the contributions of SWI/SNF in regulating T cell activation and exhaustion and highlight the potential to modify current immunotherapy therapies to increase CAR-T fitness. In addition to the chromatin architecture changes that accompany stimulation conditions in T cells, B cells undergo similar dynamic chromatin rearrangements during differentiation and maturation. Consequently, we investigated whether SWI/SNF localization directs chromatin remodeling to regulate cell-type specific RNA expression in differentiating B cell types. Pro-B, pre-B, immature B, mature B and other specialized B cells were isolated using cell-surface markers and genomic methodologies were performed to investigate SWI/SNF localization, accessibility generation, and resultant gene expression programs. These results uncovered that SWI/SNF localizes at transcription factor-specific enhancers to generate accessibility at critical lineage-specific B cell genes in cell-types across differentiation. A distinguishing feature of B cell malignancies is the dysregulation of proper B cell development. The BCL7 subunit, a dedicated and evolutionarily conserved component of the SWI/SNF family, is disproportionately mutated in B cell malignancies. Importantly, the functional dissection of BCL7 in biochemical integrity, genomic targeting, chromatin remodeling activity in cells and effect on downstream gene expression has yet to be fully characterized. In these studies, the role of BCL7 in SWI/SNF biochemical activity was explored using CRISPR/Cas9-engineered human cell lines and purification of BCL7A-null, -wild-type and -mutant complexes. BCL7 structural domains and disease variants were functionally characterized through SWI/SNF complex incorporation and integrity. Lastly, the role of BCL7 in SWI/SNF localization, chromatin accessibility and gene expression were examined in B cell lymphoma cell lines. Taken together, these findings provide new mechanisms underlying the functional consequence of BCL7-loss in B cell lymphomas. Collectively, these studies uncover SWI/SNF chromatin remodeling complex activities in lymphocytes during basic immune cell processes and in human disease, and highlight the distinct contributions of mutated subunits in the setting of B cell malignancies.