Animal and cell-based models of human disease offer simplified biological systems for studying the basis of more complex pathologies under well-controlled conditions. An ever-expanding suite of genomic and transcriptomic tools allows us to thoroughly characterize these models, highlighting disease-driving molecular features and exposing novel therapeutic targets. Here, we integrate diverse DNA- and RNA-sequencing strategies to describe the gene-regulatory chromatin landscape of models for hepatoblastoma and retrovirally-infected CD4+ T-cells. We first developed a conditional hepatoblastoma mouse model using doxycycline-inducible YAP1 overexpression and constitutive β-cateninDelN90. We found that YAP1 withdrawal alone is sufficient to trigger tumor regression and substantially increase survival. We reasoned that a thorough chromatin profile of this tumor model during YAP1 withdrawal could reveal YAP1-driven mechanisms of hepatoblastoma tumorigenesis. Our integrated approach revealed 31 novel YAP1-targeted cis-regulatory element-gene pairs. Subsequent validation confirmed that regulation of Jun-Dimerization Protein 2, among others, is both YAP1-dependent and functionally consequential for the hepatoblastoma phenotype in human cells and in hepatic malignancies. To expand our efforts to apply multi-omics technologies to disease models, we next engineered a fluorophore-containing murine leukemia virus (MLV-GFP) stably integrated into Jurkat CD4+ T-cells to report on defective transcriptional silencing by the retroelement-silencing complex, HUSH. A CRISPR knockout screen identified DHX29 as essential for HUSH-mediated silencing of newly-integrated retroviruses. Profiling genomic and transcriptomic features of MLV-GFP Jurkat cells after HUSH and DHX29 knockout revealed their epistatic roles in silencing, and revealed a suite of loci targeted by HUSH. Finally, we used site-specific proteomics and chromatin profiling to identify HUSH-associated factors at the newly integrated proviral reporter.

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we show that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can efficiently edit mouse genomic loci and revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

Thyroid cancer is the most prevalent endocrine malignancy in the United States with greater than 53,000 new cases in 2020. There is a significant gender disparity in disease incidence as well, with women developing thyroid cancer three times more often than men; however, the underlying cause of this disparity is poorly understood. Using RNA-sequencing, we profiled the immune landscape of papillary thyroid cancer (PTC) and identified a significant inverse correlation between androgen receptor (AR) levels and the immune checkpoint molecule PD-L1. The expression of PD-L1 was then measured in an androgen responsive-thyroid cancer cell line. Dihydrotestosterone (DHT) treatment resulted in significant reduction in surface PD-L1 expression in a time and dose-dependent manner. To determine if androgen-mediated PD-L1 downregulation was AR-dependent, we treated cells with flutamide, a selective AR antagonist, and prior to DHT treatment to pharmacologically inhibit AR-induced signaling. This resulted in a > 90% restoration of cell surface PD-L1 expression, suggesting a potential role for AR activity in PD-L1 regulation. Investigation into the AR binding sites showed AR activation impacts NF-kB signaling by increasing IkBalpha and by possibly preventing NF-kB translocation into the nucleus, reducing PD-L1 promoter activation. This study provides evidence of sex-hormone mediated regulation of immune checkpoint molecules in vitro with potential ramification for immunotherapies.