Complex Genome Regulation Characterized by Atypical Signatures in DNA Methylation

In mammals, DNA methylation is essential for development, and helps determine cell identity in over 200 specialized cell types. Dynamic CpG methylation during cell differentiation is correlated with changes in gene regulation and expression, with loss of methylation typically reflecting enhancer or gene activation. In this work, I identify and characterize unusual signatures in the human methylome that reveal unique modes of cell type-specific gene regulation. Most CpGs in the genome are fully methylated or unmethylated, implying a binary relationship with gene expression. In the first part of this thesis, I discover regions of intermediate DNA methylation (IM) in the genome that exhibit variable methylation between alleles or cells in a population of uniform cell type. Thousands of IM regions are identified in multiple cell types, and are conserved across tissues, individuals, and species. The IM state is associated with intermediate levels of gene expression and exon usage, indicating a potential mechanism of quantitative gene regulation. I then conduct an in-depth examination of the enigmatic human placenta epigenome, which has unusually low levels of DNA methylation similar to those observed in tumors. Integration of whole-epigenome and transcriptome data reveals that different histone modifications distinguish active and repressed genes in the absence of DNA methylation. This implies the existence of DNA methylation-independent regulatory mechanisms unique to the placenta genome. These studies demonstrate the utility of high-throughput epigenomic data analysis in understanding the role of DNA methylation in development. Additionally, they support a relationship between epigenetic modification and gene regulation that is more nuanced and pliable than previously thought

Complex Genome Regulation Characterized by Atypical Signatures in DNA Methylation

In mammals, DNA methylation is essential for development, and helps determine cell identity in over 200 specialized cell types. Dynamic CpG methylation during cell differentiation is correlated with changes in gene regulation and expression, with loss of methylation typically reflecting enhancer or gene activation. In this work, I identify and characterize unusual signatures in the human methylome that reveal unique modes of cell type-specific gene regulation. Most CpGs in the genome are fully methylated or unmethylated, implying a binary relationship with gene expression. In the first part of this thesis, I discover regions of intermediate DNA methylation (IM) in the genome that exhibit variable methylation between alleles or cells in a population of uniform cell type. Thousands of IM regions are identified in multiple cell types, and are conserved across tissues, individuals, and species. The IM state is associated with intermediate levels of gene expression and exon usage, indicating a potential mechanism of quantitative gene regulation. I then conduct an in-depth examination of the enigmatic human placenta epigenome, which has unusually low levels of DNA methylation similar to those observed in tumors. Integration of whole-epigenome and transcriptome data reveals that different histone modifications distinguish active and repressed genes in the absence of DNA methylation. This implies the existence of DNA methylation-independent regulatory mechanisms unique to the placenta genome. These studies demonstrate the utility of high-throughput epigenomic data analysis in understanding the role of DNA methylation in development. Additionally, they support a relationship between epigenetic modification and gene regulation that is more nuanced and pliable than previously thought

Degron mediated BRM/SMARCA2 depletion uncovers novel combination partners for treatment of BRG1/SMARCA4-mutant cancers.

Recent studies have highlighted that cancer cells with a loss of the SWI/SNF complex catalytic subunit BRG1 are dependent on the remaining ATPase, BRM, making it an attractive target for cancer therapy. However, an understanding of the extent of target inhibition required to arrest cell growth, necessary to develop an appropriate therapeutic strategy, remains unknown. Here, we utilize tunable depletion of endogenous BRM using the SMASh degron, and interestingly observe that BRG1-mutant lung cancer cells require near complete depletion of BRM to robustly inhibit growth both in vitro and in vivo. Therefore, to identify pathways that synergize with partial BRM depletion and afford a deeper response, we performed a genome-wide CRISPR screen and discovered a combinatorial effect between BRM depletion and the knockout of various genes of the oxidative phosphorylation pathway and the anti-apoptotic gene MCL1. Together these studies provide an important framework to elucidate the requirements of BRM inhibition in the BRG1-mutant state with implications on the feasibility of targeting BRM alone, as well as reveal novel insights into pathways that can be exploited in combination toward deeper anti-tumor responses

CRISPR screening identifies T cell-intrinsic regulators of CD3-bispecific antibody responses.

CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics

Exquisite Sensitivity to Dual BRG1/BRM ATPase Inhibitors Reveals Broad SWI/SNF Dependencies in Acute Myeloid Leukemia

Various subunits of mammalian SWI/SNF chromatin remodeling complexes display loss-of- function mutations characteristic of tumor suppressors in different cancers, but an additional role for SWI/SNF supporting cell survival in distinct cancer contexts is emerging. In particular, dependence on the catalytic subunit BRG1/SMARCA4 has been observed in acute myeloid leukemia (AML), yet the feasibility of direct therapeutic targeting of SWI/SNF catalytic activity in leukemia remains unknown. Here, we evaluated the activity of BRG1/BRM ATPase inhibitors across a genetically diverse panel of cancer cell lines and observed that hematopoietic cancer cell lines were among the most sensitive compared to other lineages. This result was striking in comparison to data from pooled short hairpin RNA screens, which showed that only a subset of leukemia cell lines display sensitivity to BRG1 knockdown. We demonstrate that combined genetic knockdown of BRG1 and BRM is required to recapitulate the effects of dual inhibitors, suggesting that SWI/SNF dependency in human leukemia extends beyond a predominantly BRG1-driven mechanism. Through gene expression and chromatin accessibility studies, we show that the dual inhibitors act at genomic loci associated with oncogenic transcription factors, and observe a downregulation of leukemic pathway genes including MYC, a well-established target of BRG1 activity in AML. Overall, small molecule inhibition of BRG1/BRM induced common transcriptional responses across leukemia models resulting in a spectrum of cellular phenotypes. Our studies reveal the breadth of SWI/SNF dependency and support targeting SWI/SNF catalytic function as a potential therapeutic strategy in AML

The discovery of SWI/SNF chromatin remodeling activity as a novel and targetable dependency in uveal melanoma

Uveal melanoma is a rare and aggressive cancer that originates in the uveal tissue of the eye. Currently, there are no approved targeted therapies for this cancer, and very few effective treatments are available. While activating mutations in the G protein alpha subunits, GNAQ and GNA11, are key genetic drivers of the disease, other targetable molecular players are only partially understood. Through new analysis of unbiased, functional genomics screens and comprehensive validation studies in a panel of uveal melanoma cell lines, we find evidence that the SWI/SNF complex is essential in uveal melanoma. The mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) are often mutated in cancers and described as tumor suppressors, yet context specific roles for these complexes in the maintenance of certain cancers are beginning to emerge. We determined that the catalytic activity of SWI/SNF is critical, and further translated these findings with our recently described small molecule inhibitors of BRM and BRG1, the closely related catalytic subunits of the SWI/SNF complexes. Finally, we describe a functional relationship between the SWI/SNF complex and the melanocyte lineage specific transcription factor MITF, suggesting that SWI/SNF cooperates with MITF to drive a lineage specific transcriptional program essential for uveal melanoma cell survival. These studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path to the treatment of this cancer

Functional DNA methylation differences between tissues, cell types, and across individuals discovered using the M&M algorithm.

DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities

Functional DNA methylation differences between tissues, cell types, and across individuals discovered using the M&M algorithm.

DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities