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SARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associated with mild or moderate symptoms were already robust and included severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity was marked by upregulation of classical antiviral pathways, including those regulating IRF1 and IRF7, whereas in moderate disease, these classical antiviral signals diminished, suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.

Patient‐Derived Organoids (PDO) and Xenografts (PDX) are the current gold standards for patient‐derived models of cancer (PDMC). Nevertheless, how patient tumor cells evolve in these models and the impact on drug response remains unclear. Herein, the transcriptomic and chromatin accessibility landscapes of matched colorectal cancer (CRC) PDO, PDX, PDO‐derived PDX (PDOX), and original patient tumors (PT) are compared. Two major remodeling axes are discovered. The first axis delineates PDMC from PT, and the second axis distinguishes PDX and PDO. PDOX are more similar to PDX than PDO, indicating the growth environment is a driving force for chromatin adaptation. Transcription factors (TF) that differentially bind to open chromatins between matched PDO and PDOX are identified. Among them, KLF14 and EGR2 footprints are enriched in PDOX relative to matched PDO, and silencing of KLF14 or EGR2 promoted tumor growth. Furthermore, EPHA4, a shared downstream target gene of KLF14 and EGR2, altered tumor sensitivity to MEK inhibitor treatment. Altogether, patient‐derived CRC cells undergo both common and distinct chromatin remodeling in PDO and PDX/PDOX, driven largely by their respective microenvironments, which results in differences in growth and drug sensitivity and needs to be taken into consideration when interpreting their ability to predict clinical outcome. Patient‐derived CRC cells undergo two‐axes chromatin remodeling in PDO and PDX/PDOX, largely driven by their respective microenvironments. Transcription factors and genes affected by this remodeling significantly impact tumor growth and drug sensitivity. Therefore, the chromatin adaptation of different PDMC may interfere with their ability to predict therapeutic outcomes.

Advancements in technologies for sequencing the genome and the computational methodologies to process and extract information from the data have rapidly accelerated the understanding of the genetic basis for many diseases. The practice of precision medicine enables more precise targeting of disease, better appreciation of individual patient needs, and a higher resolution understanding of the genetic basis underlying drug responses by translating insights from genomics to clinical practice. Looking to the future, modulation of the epigenome as a therapeutic intervention promises major potential to revolutionize the approach to managing complex diseases like cancer and inflammatory or metabolic disorders. The major goals of the work presented in this dissertation are to leverage genomic data to improve our understanding of 1) immunity – particularly how dysfunctional immune cells respond differently to infectious diseases like COVID-19; 2) disease prognosis – whereby epigenetic biomarkers reveal how immune responses are primed within the first few days of infection and underlie the severity of symptoms; and 3) pharmacogenomics – in order to develop a molecular framework for the evaluation of selective glucocorticoid receptor modulators.To address the first goal, we investigated differences in innate immunity associated with mortality in COVID-19 ICU patients. Prior to decompensation, abundances of non-classical monocytes were significantly lower, and we profiled these cells following stimulation with TLR agonists using single-cell RNA-seq. I identified a transcriptional pattern of tolerance against TLR activation in the monocytes from deceased patients that explained the absence of a robust innate immune response in the agonist conditions. We concluded that secondary infections may occur more frequently in these patients, thus increasing the risk of mortality, and tested nucleic acid-scavenging MnO nanoparticles as a potential therapy to neutralize the hyperinflammatory environment and reverse the tolerance phenotype.To address the second goal, we identified biomarkers that differentiated mild from moderate Covid-19 prior to IgG seroconversion, when antibodies begin to be produced, using single-cell ATAC-seq. The IgG-negative window lasts for only the first few days following infection. Thus, our goal was to improve the understanding of COVID-19 immunity – that is, how immune responses were differentially primed and associated with disease severity – as well as to develop translational molecular targets with prognostic and therapeutic potential. The chromatin landscape was indeed remodeled significantly prior to seroconversion. Furthermore, classical monocytes had the highest enrichment of regions with differential accessibility, and I characterized prognostic biomarkers that included ~1000 domains of regulatory chromatin and differences in TF activity associated with monocyte maturation that underlie disease severity.To address the third goal, we conducted the largest comparative study of genomic responses to 10 GR ligands, measuring differential gene expression and regulatory element activity using RNA-seq and STARR-seq. 1 in 5 Americans have used short-term classical glucocorticoid therapies, such as dexamethasone, and repeated or excessive use carries a significant risk of adverse side effects. Safer ligands, proposed to selectively modulate GR activity by impairing trans-activation of some target genes, have consistently underperformed in clinical trials. I explained why by demonstrating that these ligands activate the same gene expression response as dexamethasone, just to different degrees in strength. Indeed, the ligand-specific activities at ~50,000 enhancers were dominantly explained by dexamethasone effects (R2 = 0.82). I reported the genes in the most ligand-dependent mode of these responses to be used for future genetic screens. Finally, I simulated enhancer activities using a two-component Gaussian mixture model to represent activation of a subset of dexamethasone-responsive enhancers by a hypothetical highly selective ligand, the response to which was significantly different from any of the ligands we tested (p = 0.03).

SARS-CoV-2 infection triggers highly variable host responses and causes varying degrees of illness in humans. We sought to harness the peripheral blood mononuclear cell (PBMC) response over the course of illness to provide insight into COVID-19 physiology. We analyzed PBMCs from subjects with variable symptom severity at different stages of clinical illness before and after IgG seroconversion to SARS-CoV-2. Prior to seroconversion, PBMC transcriptomes did not distinguish symptom severity. In contrast, changes in chromatin accessibility were associated with symptom severity. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif occupancy for individual PBMC cell types. The most extensive remodeling occurred in CD14+ monocytes where sub-populations with distinct chromatin accessibility profiles were associated with disease severity. Our findings indicate that pre-seroconversion chromatin remodeling in certain innate immune populations is associated with divergence in symptom severity, and the identified transcription factors, regulatory elements, and downstream pathways provide potential prognostic markers for COVID-19 subjects.

Synthetic glucocorticoids (GCs), which induce the transcription factor activity of the glucocorticoid receptor (GR), are frequently prescribed anti-inflammatory therapeutics that have been in use for over 70 years. Despite their broad immunosuppressive utility, sustained use of GCs is often intolerable due to the prevalence of adverse side effects. A longstanding goal has been to make synthetic GCs safer by developing selective GR ligands that have similar anti-inflammatory activity but without the burden of side effects. To evaluate the ability of synthetic GCs to target specific subsets of the GC response, we completed a genome-wide comparative analysis of changes in gene expression and gene regulatory element activity in response to ten ligands with various evidence of dissociated adverse side effects. We measured the gene expression response using mRNA-seq and the gene regulatory element response using genome-wide STARR-seq. Effects associated with each ligand were highly correlated with and linearly related to the response to dexamethasone, a strong, non-selective GR agonist used as a positive control for this study. Furthermore, 93% of the variation in regulatory element activity responses could be explained by the efficacy of each ligand alone. We also found limited evidence of differential enrichment of chromatin context-specific markers of regulatory activity with each ligand. Based on those findings, we developed a simulation framework to evaluate selectivity of GR ligands. We conclude that the ligands we tested elicit attenuated molecular responses according to their respective efficacies, and do not selectively target subsets of the molecular GC response.

Patient-Derived Organoids (PDO) and Xenografts (PDX) are the current gold standards for patient derived models of cancer (PDMC). Nevertheless, how patient tumor cells evolve in these models and the impact on drug response remains unclear. Herein, we compared the transcriptomic and chromatin accessibility landscapes of six matched sets of colorectal cancer (CRC) PDO, PDX, PDO-derived PDX (PDOX), and original patient tumors (PT) and discovered two major remodeling axes. The first axis delineates PDX and PDO from PT, and the second axis distinguishes PDX and PDO. PDOX were more similar to PDX than they were to PDO, indicating that the growth environment is a driving force for chromatin adaptation. Using bivariate genomic footprinting analysis, we identified transcription factors (TF) that differentially bind to open chromatins between matched PDO and PDOX. Among them, KLF14 and EGR2 footprints were enriched in all six PDOX relative to matched PDO, and silencing of KLF14 or EGR2 promoted tumor growth. Furthermore, EPHA4, a shared downstream target gene of KLF14 and EGR2, altered tumor sensitivity to MEK inhibitor treatment. Altogether, patient-derived CRC cells undergo both common and distinct chromatin remodeling in PDO and PDX/PDOX, driven largely by their respective microenvironments, which results in differences in growth and drug sensitivity and needs to be taken into consideration when interpreting their ability to predict clinical outcome.

ABSTRACT Sexual dimorphisms in immune responses contribute to coronavirus disease 2019 (COVID-19) outcomes, yet the mechanisms governing this disparity remain incompletely understood. We carried out sex-balanced sampling of peripheral blood mononuclear cells from confirmed COVID-19 inpatients and outpatients, uninfected close contacts, and healthy controls for 36-color flow cytometry and single cell RNA-sequencing. Our results revealed a pronounced reduction of circulating mucosal associated invariant T (MAIT) cells in infected females. Integration of published COVID-19 airway tissue datasets implicate that this reduction represented a major wave of MAIT cell extravasation during early infection in females. Moreover, female MAIT cells possessed an immunologically active gene signature, whereas male counterparts were pro-apoptotic. Collectively, our findings uncover a female-specific protective MAIT profile, potentially shedding light on reduced COVID-19 susceptibility in females. Competing Interest Statement MTM reports grants on biomarker diagnostics from the Defense Advanced Research Projects Agency (DARPA), National Institutes of Health (NIH), Sanofi, and the Department of Veterans Affairs. TWB reports grants from DARPA and is a consultant for Predigen; MTM, TWB, ELT, GSG, and CWW report patents pending on Molecular Methods to Diagnose and Treat Respiratory Infections. ELT reports grants on biomarker diagnostics from DARPA, the NIH/Antibacterial Resistance Leadership Group (ARLG) ; an ownership stake in Predigen; GSG reports an ownership stake in Predigen; CWW reports grants on biomarker diagnostics from DARPA, NIH/ARLG, Predigen, and Sanofi; and has received consultancy fees from bioMerieux, Roche, Biofire, Giner, and Biomeme.

The Puroindoline genes (Pina and Pinb) together comprise the wheat (Triticum aestivum L.) Hardness locus (Ha) located on chromosome 5D and control grain texture. While hard wheats contain a mutation in either Pina or Pinb, there is no puroindoline allelic diversity among soft hexaploid wheats as all carry the Pina-D1a/Pinb-D1a alleles. However, Pina and Pinb allelic variation exists within synthetic hexaploid wheats created using novel D genome donors. Here we tested the effects of four Aegilops tauschii-derived Ha locus haplotypes (Pina-D1c/Pinb-D1h, Pina-D1e/Pinb-D1i, Pina-D1a/Pinb-D1i, and Pina-D1j/Pinb-D1i) found in synthetic wheats by crossing them into the soft white spring wheats ‘Alpowa’ and ‘Vanna’. The effect of each Ha haplotype on grain hardness was measured by analyzing backcross or F2–derived lines. All novel Ha loci increased grain hardness while still conditioning soft wheat texture independent of durum or Ae. tauschii synthetic wheat parent line. The Pina-D1c/Pinb-D1h haplotype was found to increase grain hardness relative to the wild-type Ha locus by an average of 6.5 units, Pina-D1e/Pinb-D1i by 5.6 units, Pina-D1a/Pinb-D1i by 12.6 units, and Pina-D1j/Pinb-D1i by 3.8 units. None of the novel Ha locus haplotypes significantly affected Pina or Pinb transcript or protein expression levels. The results indicate that Ae. tauschii derived novel Ha loci could be useful in modifying soft wheat grain texture and end product quality.

Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world’s population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock.