• Unraveling the Roles of C. elegans Vasa Homologs, GLH Proteins, in Argonaute Pathway Specificity and Transcriptome Surveillance

  Dai, Siyuan (2023-05-25)

  Epigenetic regulation of gene expression empowers organisms to alter phenotypic information without affecting DNA sequence. Germline Argonaute proteins, complexed with their cognate small-RNAs, are essential for transcriptome surveillance and maintenance of heritable gene silencing. In C. elegans, PIWI Argonaute PRG-1 employs piRNAs to screen thousands of germline transcripts through microRNA-like base pairing. Upon target recognition, RNA-directed RNA polymerases (RdRPs) are recruited to generate abundant antisense small RNAs (22G-RNAs), which are subsequently loaded onto worm-specific Argonautes (WAGOs) to establish long-term epigenetic memory. Both small RNA amplification and Argonaute surveillance are thought to occur within perinuclear liquid-like condensates called nuage or P granules. The precise mechanism by which the nearly one million different perinuclear Argonaute/guide complexes engage their targets to mediate gene regulation remain unclear. In this dissertation, we explore the functions of a family of VASA homologs, Germ Line Helicases (GLHs) in diverse germline small-RNA pathways. Our genetic and biochemical investigations reveal that these perinuclear-localized DEAD-box proteins engage germline transcripts and promote piRNA- and RNA interference-mediated transgenerational silencing in C. elegans. We provide evidence that GLH proteins scaffold multiple Argonautes responsible for epigenetic silencing, competing with paralogs for direct binding to target mRNAs. Additionally, GLHs enhance the specificity of Argonuate pathways by preventing WAGO/22G-RNA misrouting. Through examining mutants without severe P-granule disruptions, we sought to separate GLH-1 scaffolding and enzymatic functions. We found that the GLH-1 ATPase cycle promotes RNA duplex unwinding and the biogenesis of WAGO-bound 22G-RNAs while many P-granule components remain properly localized. Moreover, we show that GLH-1 N-terminal domains containing both FG repeats and zinc fingers are crucial for binding Argonautes and RNA substrates. Taken together, our findings support a model in which Argonautes recruit GLH proteins for target mRNA binding and retention, enabling GLHs to regulate pathway-specific small-RNA signals and transgenerational inheritance.
• Integrating evolution and genomics to investigate social development in wolf-dog hybrids

  Li, Xue (2023-05-05)

  Domesticated dogs separated from wolves around ~5000-7000 generations ago, with major differences in early social development that have enabled them to survive, and thrive, in close proximity to humans. Due to their unique evolutionary history and accessibility, canines serve as a natural model system to study the genetic factors underlying behavior adaptation within and between subspecies. The wolf/dog system can not only advance the understanding of evolutionary processes, but also help to better understand the neurogenetic pathways involved in human psychiatric disorders. Although wolves and dogs split relatively recently in evolutionary time, they are genetically distinct populations with numerous differences across their genome. This population structure makes it impossible to confidently associate particular genomic variants with domestication-related traits by simply comparing dogs and wolves. In this dissertation, I identified genes and pathways associated with domestication-related behavior using an unusual admixed population of wolf-dog hybrids housed in sanctuaries across the United States. I developed methods and approaches to map behavioral phenotypes in wolf-dog hybrids, and explored the overlap with dog social behaviors, and human psychiatric conditions. I first characterized the population history of wolf-dog hybrids using techniques including exploratory principal component analysis, ancestry calling, and population differentiation test. I defined the behavioral phenotypes by dimensional reduction analysis of coded video data, and identified associations between genes and regulatory elements with those phenotypes using admixture mapping and association test. Finally, I investigated the functional and biological mechanisms underlying the associated regions by gene-set analysis. I discovered that regions associated with domestication-related behavioral differences are enriched for brain expressed genes, especially those enriched in early infancy. To further investigate the candidate regions associated with canine domestication, I leveraged a powerful new data resource comparing the genomes of 240 mammalian species. Using data from massively parallel reporter assay experiments in human cells, I confirmed that this resource can distinguish which bases have regulatory function. Overall, variants in highly constrained positions are more likely to alter cellular function. In addition, I showed that dogs with ancestry from a single breed, which have shorter lifespans than outbred dogs, are also more likely to carry variants in constrained positions, suggesting they impact fitness. In the wolf-dog hybrids, I cataloged candidate causal variants that differed between dogs and wolves and were highly constrained across mammals. Overall, this thesis demonstrates how new genomic tools and data resources can be leveraged to investigate exceptional evolutionary adaptations in other species that may offer insight into human diseases. By utilizing the wolf-dog hybrid population, we can re-trace the ancient genetic changes of domestication that led to divergence of canine social and developmental behaviors, and potentially uncover genetic pathways that contribute to social behavioral disorders such as autism spectrum disorders.
• Phosphoregulation of Cell Cycle Transcription Factors by Cyclin-Dependent Kinase

  Conti, Michelle (2023-04-27)

  To prevent the development of cancer, cells must regulate the cell division cycle. Cell cycle events are coordinated by an oscillatory gene expression program, established by a conserved transcription factor (TF) network. Most TFs in the network are phosphorylated by cyclin-dependent kinases (CDKs), which regulate their activity. However, the physiological consequences of disrupting TF phosphorylation remain poorly understood. The budding yeast repressive TFs Yhp1 and Yox1 are degraded following multisite phosphorylation by CDK. Surprisingly, I discovered that blocking phosphorylation of Yhp1 and Yox1 increased fitness compared to wild type cells, despite decreased expression of several essential cell cycle genes. We found that cells expressing non-phosphorylatable Yhp1 and Yox1 accelerated the G1/S transition and delayed mitotic exit. This suggests that by lengthening mitosis mutant cells have more time to correct chromosome segregation errors, which confers a fitness advantage to cells. Although hundreds of CDK targets have been identified, it is challenging to determine which phosphosites within a domain are required to regulate protein function. The conserved S-phase TF Hcm1 is activated by CDK-dependent phosphorylation of eight sites in its transactivation domain (TAD). Like Yox1 and Yhp1, disruption of Hcm1 TAD phosphorylation impacts cellular fitness. I leveraged these fitness phenotypes to develop a high-throughput approach, Phosphosite Scanning, that determines the importance of each phosphosite within a multisite phosphorylated domain. I identified multiple combinations of phosphosites that can activate Hcm1 and found that specific phosphorylations are required for phosphorylation throughout the TAD. These results highlight the importance of precise TF phosphoregulation and demonstrate that disruption of phosphoregulatory networks can have unexpected consequences on cellular physiology.
• The Molecular Mechanism of RIPK1-Induced Cell Death And Its Impact On The Immune Response

  Park, Christa (2023-04-25)

  Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a critical adapter protein with pleiotropic functions that regulate cell survival and death. RIPK1 is essential for immune homeostasis and thus is closely controlled during development and inflammation. RIPK1 overexpression has been implicated in multiple inflammatory disorders such as multiple sclerosis, atherosclerosis, cardiovascular disease, obesity, psoriasis, and tumor growth. To study the effects of the overactivation of RIPK1, a system was established that drives its overexpression in mouse fibroblasts. Remarkably, the overexpression of RIPK1 resulted in the induction of both apoptosis and necroptosis. While apoptosis is known to be immunologically silent, necroptosis is highly inflammatory. Additional assays using chemical inhibitors and genetic knockout mice established that RIPK1 kinase activity promotes both types of cell death. Furthermore, RIPK1-induced apoptosis and necroptosis require caspase 8 and MLKL, respectively, and the absence of both caspase 8 and MLKL inhibits RIPK1-induced cell death. RIPK1 induction activates NF-κB/MAPK and increases cytokine/chemokine production driven by cell death. This system was further explored to elucidate the effects of RIPK1-induced cell death on immune effector cells, revealing that RIPK1-induced apoptosis and necroptosis can promote DC activation. Lastly, to study the role of RIPK1 in DCs and its contribution to intestinal homeostasis and injury, mice lacking RIPK1 in the DC population were characterized. Importantly, RIPK1 functions in DCs to support colon homeostasis, but also plays a detrimental role during DSS-induced colitis. Collectively, these data further provide novel insights into the multifaceted functions of RIPK1 in cell death and inflammation, highlighting its critical contributions to the immune response.
• Artificial Intelligence for the Diagnosis of Pediatric Appendicitis: A Systematic Review

  Chekmeyan, Mariam (2023-04-21)

  BACKGROUND: While acute appendicitis is the most frequent surgical emergency in children, its diagnosis remains complex. Artificial intelligence (AI) and machine learning (ML) tools have been employed to improve the accuracy of various diagnoses, including appendicitis. The purpose of this study was to systematically review the current body of evidence regarding the efficacy of AL and ML approaches for the diagnosis of acute pediatric appendicitis. METHODS: This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to identify articles from Pubmed, Scopus, and iEEE Xplore. Eligible articles included full text, English-language articles assessing the use of AI technologies for the diagnosis of acute pediatric appendicitis. Study quality of reporting was appraised using The Transparent Reporting of a multivariable prediction model of Individual Prognosis Or Diagnosis (TRIPOD) statement. RESULTS: A total of fourteen studies were included in the final analysis of which ten were published after 2019. Two studies originated in the United States while half were carried out in Europe. Artificial Neural Network and Random Forest AI methods were the most commonly used modeling approaches. Commonly used predictors were pain and laboratory blood findings. The average area under the curve that was reported among the fourteen studies was greater than 80%. CONCLUSIONS: AI and ML technologies have the potential to improve the accuracy of acute appendicitis diagnosis in pediatric patients. Further investigation is needed to identify barriers to adoption of these technologies and to assess their efficacy in real world usage once integrated into clinical workflows.
• The HHV-6B U20 Glycoprotein Inhibits NK Cell Responses By Binding ULBP1 And Blocking NKG2D Activation

  Weaver, Grant (2023-04-06)

  Many viruses impede host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Hosts will often counter this through NK cells that sense the absence of MHC-I and kill the infected cell. Human Herpesvirus 6B (HHV-6B) has also been shown to downregulate MHC-I but this does not result in NK-mediated elimination of the virus. Previous work has shown that HHV-6B downregulates three NK-activating stress ligands: MICB, ULBP1, and ULBP3. More recently, the U20 glycoprotein of HHV-6A was implicated in the downregulation of ULBP1 but the precise mechanism remains undetermined. We set out to better understand the role of HHV-6B U20 in modulating NK cell activity. We performed structural modeling studies that suggest that U20 is likely a viral nonclassical MHC (vMHC). These vMHCs are common tools used by herpesviruses to modulate host immune responses. Through in vitro studies with recombinant protein, we demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity (225nM). Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels (at the surface or in toto). Soluble U20 has the same effect and can also inhibit activation of ULBP1-stimulated primary NK cells. When taken together, these data suggest that U20 helps to downregulate NK cell activity by binding ULBP1, masking it from recognition by NKG2D at the surface of infected cells, resulting in a decrease in antiviral NK cell activation and killing.
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  Mechanism of Sliding Clamp Loading During DNA Replication and Repair in Atomic Detail

  Liu, Xingchen (2023-03-30)

  DNA replication is a fundamental process that is essential for all forms of life, and it is made efficient by ring-shaped sliding clamp proteins. One such protein is the eukaryotic sliding clamp, Proliferating Cellular Nuclear Antigen (PCNA), which not only facilitates replication but also coordinates multiple cellular pathways, such as DNA repair, cell cycle regulation, and apoptosis. The proper function of PCNA is critical for maintaining genome stability, making it a crucial factor in human health. The clamp loader complex is the primary regulator of sliding clamp activity. Replication Factor C (RFC), the eukaryotic clamp loader, is responsible for opening the closed PCNA and loading it onto DNA. However, the mechanism by which RFC accomplishes this task has been elusive for years. Our research has contributed to this field by revealing multiple cryo-electron microscopy (cryo-EM) structures of the RFC:PCNA complex that describe the steps involved in the clamp loading reaction. Specifically, we found that RFC opens PCNA with a 'crab-claw' motion, allowing it to preferentially bind to PCNA before DNA. Additionally, during replication, primer-template DNA, which is RFC's primary DNA substrate, directly binds to the central chamber of the complex. Our study also sheds light on the mechanism by which RFC performs its role in loading PCNA during DNA repair. When RFC binds to gapped or nicked DNA during DNA repair, it uses an additional DNA binding site, and both sites work together to melt the DNA strands with their 'separation pins.' This discovery provides the first structural insight into how RFC accomplishes its crucial functions in DNA replication and repair. Overall, our findings provide detailed atomic-level insights into how RFC efficiently loads PCNA onto different DNA substrates, advancing our understanding of this essential biological process.
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  Activation of the NLRP1 inflammasome in human keratinocytes by the dsDNA mimetic poly(dA:dT)

  Zhou, Jeffrey Y. (2023-03-29)

  The accrual of cytosolic DNA leads to transcription of type I IFNs, proteolytic maturation of the IL-1 family of cytokines and pyroptotic cell death. Caspase-1 cleaves pro-IL-1b to generate mature bioactive cytokine and gasdermin D which facilitates IL-1 release and pyroptotic cell death. Absent in melanoma-2 (AIM2) is a sensor of dsDNA leading to caspase-1 activation, although in human monocytes, cGAS-STING acting upstream of NLRP3 mediate the dsDNA activated inflammasome response. In healthy human keratinocytes, AIM2 is not expressed yet caspase-1 is activated by the synthetic dsDNA mimetic poly(dA:dT). Here, we show that this response is not mediated by either AIM2 or the cGAS-STING-NLRP3 pathway and is instead dependent on NLRP1. Poly(dA:dT) is unique in its ability to activate NLRP1, as conventional linear dsDNAs fail to elicit NLRP1 activation. DsRNA was recently shown to activate NLRP1 and prior work has shown that poly(dA:dT) is transcribed into an RNA intermediate that stimulates the RNA sensor RIG-I. However, poly(dA:dT) dependent RNA-intermediates are insufficient to activate NLRP1. Instead, poly(dA:dT) results in oxidative nucleic acid damage and cellular stress, events which activate MAP3 kinases including ZAKa that converge on p38 to activate NLRP1. Collectively, this work defines a new activator of NLRP1, broadening our understanding of sensors that recognize poly(dA:dT), and advances understanding of the immunostimulatory potential of this potent adjuvant.
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  Mechanistic Role of the Sliding Clamp in Genome Stability and Disease

  Magrino, Joseph (2023-03-27)

  DNA replication is an essential task to all life. To ensure precise genome duplication, cells utilize a network of factors that copy, surveil, and repair DNA. The coordination of all these factors heavily relies on the homotrimeric sliding clamp protein, Proliferating Cell Nuclear Antigen (PCNA). Like other sliding clamps, PCNA slides along DNA and acts as a molecular tether that increases the processivity of various DNA-acting enzymes. In addition, PCNA plays a much more multifaceted role in coordinating the concerted efforts of dozens of proteins involved in DNA replication, DNA repair, chromatin remodeling, cell cycle, and apoptosis. Proper interactions with PCNA are necessary for these enzymes to perform their functions. Yet, how PCNA controls its vast network remains unclear. How does partner binding affinity, PCNA levels, and lifetime on DNA influence PCNA’s ability to coordinate various DNA metabolic events? To understand the biophysical principles behind these questions, I investigated how two disease-associated substitutions in PCNA impact partner binding, stability, and its loading onto DNA. Furthermore, I made C. elegans strains that harbored these substitutions to understand their effects at the organismal level. Finally, I identified an electrostatic patch within PCNA that plays a role in partner binding affinity. My work collectively identifies residues that are critical for PCNA function. More broadly, my work provides insight into the evolution of PCNA and how substitutions impact both genome stability and human health.
• The Interplay Between Stress and the Drosophila piRNA Pathway

  Ho, Samantha (2023-03-22)

  piRNAs are required to silence transposons in the germline to maintain genome stability and transmission of an intact genome to the next generation. In Drosophila, biogenesis of these small RNAs occurs in three distinct compartments: the nucleus, perinuclear nuage, and the outer mitochondria membrane. My thesis focuses on how genomic instability via transposon-initiated DNA damage and heat stress impact piRNA pathway organization and function. We show that activation of Chk2, a Checkpoint kinase required for DNA damage signaling, disrupts nuage composition and this complicates piRNA mutant phenotypes. Stripping away DNA damage signaling in piRNA mutants provided new insight into how the nuclear piRNA proteins organize the cytoplasmic nuage. Additionally, we found that localization of key components to nuage is dispensable for piRNA production and transposon silencing. piRNA pathway proteins are not only susceptible to genomic instability, but also sensitive to heat shock. Rhino is a core component of the nuclear piRNA pathway and displays drastic localization changes upon heat shock that recovers with time. piRNAs have been proposed to help localize Rhino and heat stress provided a unique platform to test this model. We show that Rhino recovery after heat shock does not require Piwi, the sole nuclear protein bound to piRNAs, and this points to piRNAs having a less significant role in Rhino localization. Taken together, we show how different types of stress can modulate the piRNA pathway in unexpected manners.
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  Canonical Wnt Signaling Maintains Human Mesenchymal Progenitor Cell Multipotency During Adult Adipose Tissue Development

  Yang Loureiro, Zinger (2023-03-21)

  Human adipose tissue development and repair requires mesenchymal progenitor cells capable of self-renewal and differentiation into adipocytes. These progenitors are within adipose tissue throughout life, and how a progenitor pool is sustained is not well understood. As adipocytes have exceedingly slow turnover rate in vivo, we used a human adult primary adipose tissue-derived progenitor model to study adipogenesis at scale. Initially, single-cell transcriptome profiling of progenitors during early adipogenic induction identified common-origin developmental trajectories toward two fates. One fate is toward adipocyte terminal differentiation, and the other is toward a non-differentiated state characterized by up-regulation of extracellular matrix and canonical Wnt target genes. This latter cell population, herein referred to as Structural Wnt Adipose Tissue resident (SWAT) cells, retains features of progenitors, including the capability to proliferate and to differentiate towards multiple mesenchymal fates, but SWAT cells have a distinct transcriptional profile compared to progenitor cells. We conducted an in-depth investigation of Wnt signaling in SWAT cells. Canonical Wnt signaling reporter assay revealed Wnt activity was rapidly and transiently up-regulated upon adipogenic induction, and perturbation of Wnt activity alters the proportion of differentiated adipocytes. In additional to experimental validations, this research includes meta-analyses of published human adult adipose tissue single-nuclei transcriptome profiles, providing in vivo clinical evidence for expression of Wnt target genes and the transcriptomic signature of the maintained adipose progenitors. Our study identifies canonical Wnt signaling as a critical mechanism for mesenchymal progenitor maintenance and demonstrates its essential role in human adult adipose tissue homeostasis.
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  Structure and Assembly of a Thermophilic Bacteriophage

  Agnello, Emily (2023-03-20)

  Bacteriophages (phages) are ubiquitously abundant bacterial viruses and the most numerous biological entities on Earth. In order to fully understand the phage life cycle, we must understand how phage particles self-assemble. Further, an understanding of phage structure and assembly permits the application of phages to human health. Phage assembly is largely governed by two parallel pathways: capsid assembly and tail assembly. The capsid houses the genome, while the tail is the channel through which the genome travels to infect its host, making both components essential for successful infections. In this dissertation, I investigate the assembly pathways of the hyperthermophilic phage P74-26 as a model for understanding long-tailed phages (~85% of all phages). Primarily, I combine cryoEM structures of the P74-26 tail tube with an in vitro system for studying assembly kinetics to propose the first molecular model for the assembly of long-tailed phage tail tubes. Additionally, the tail is attached to a Tail Tip Complex (TTC) which recognizes the surface of the host. I present a cryo-EM structure of the P74-26 TTC, identifying the protein components for the first time. Finally, I explore the self-assembly of the capsid protein with the potential for establishing an in vitro system for studying capsid assembly and present proof-of-concept studies for using the particles as functional nanoparticle therapeutics. Together, this work explores principles of phage assembly and thermostability in tailed bacteriophages and how we can take advantage of these principles for future development of therapeutic delivery tools and nanoparticle applications.
• Molecular Mechanisms of Amino Acid Sensing Upstream of mTORC1

  Egri, Shawn B. (2023-03-10)

  In order to initiate cellular growth, a cell must weigh its nutrient availability against its anabolic needs. A critical pathway responsible for this process is the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 is a serine threonine protein kinase complex that will phosphorylate its downstream substrates in order to promote anabolic reactions. mTORC1 activity is dependent upon the Rag GTPase heterodimer. In the presence of amino acids, the Rags will activate mTORC1 by promoting its translocation to the lysosomal surface. In contrast, amino acid withdrawal results in mTORC1 inactivation. In order to ensure faithful mTORC1 signaling, the nucleotide loading state of the Rag GTPase is tightly regulated. In this thesis, we combine biochemical and biophysical approaches to investigate the molecular mechanism of how the nucleotide loading state of the Rag GTPase subunits are regulated. First, we characterized a conserved interdomain hydrogen bond within the Rag GTPases that is responsible for maintaining the GDP-bound state of the subunits. Elimination of this hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in dysregulated mTORC1 signaling. Second, we utilize cryo-EM to describe the molecular mechanism of how GATOR1, a potent negative regulator of the mTORC1 pathway, modulates the nucleotide loading state of the Rag subunits in response to nutrient deprivation. These results reveal the molecular details of how the Rag GTPases are regulated in response to changes in amino acid availability, and furthermore how disruptions to those mechanisms can lead to dysregulation of the mTORC1 signaling pathway.
• Impact of stress on the piRNA Pathway

  Rice, Nicholas P (2023-03-10)

  Barbara Mcclintock proposed the genomic stress hypothesis, which states that transposable element activation is an adaptive response to generate new variation. Transposons are ubiquitous selfish genetic elements that compose a large fraction of eukaryotic genomes. In Drosophila melanogaster’s germline, transposons are regulated by small RNAs called Piwi Interacting RNAs (piRNAs). piRNAs originate from clusters of repeats, whose expression is driven by a germline-specific protein Rhino, and cleavage products produced by Argonaute proteins concentrated in nuage, a phase-separated granule. The “genomic stress” model must operate in the germline, and the effect of stress on the piRNA pathway is not well understood. In my thesis, I investigated how two types of stress, temperature and DNA damage via transposon mobilization, affect the piRNA pathway. We show heat shock rapidly and reversibly disrupts the nuclear biogenesis factors required for piRNA production and the collapse of cluster transcription. This loss and recovery of Rhino and its associated factors allowed us to show that cluster assembly is independent of piRNAs. Mutations in the piRNA pathway upregulate transposons and activate the DNA damage response complicating any single piRNA mutant phenotype. We thus created double mutants with a kinase required for DNA damage signaling (Chk2) and demonstrated that Chk2 signaling alters nuage composition. Stripping away damage signaling revealed that components of the nuclear piRNA pathway are required to link nuage properly to clusters. Together we show that the piRNA pathway responds to stress in different ways and that this differential response is an additional tool for investigating the piRNA pathway.
• Exploring the Phenomenon of MNNG Dose-Dependent Death Polypharmacology

  Fontana, Rachel (2023-02-23)

  Regulated cell death (RCD) is composed of several pathways that control cell fate. While each pathway is mechanistically distinct, these pathways have been shown to interact. Most of these interactions tend to be antagonistic, such that activation of one pathway blocks the subsequent activation of another pathway. This highlights that death pathways tend to be mutually exclusive. Thus, combining two cytotoxic drugs that activate different death pathways could result in less cell death than predicted, hampering therapeutic efficacy. As such, it is necessary to characterize which death pathways are activated by clinically relevant drugs, particularly for drug combination studies. However, studies of death pathway engagement are complicated by the fact that many drugs are capable of activating multiple RCD pathways. In order to improve annotations of RCD pathway activation by specific stimuli, we need to learn what features dictate which death pathway is activated. To study this phenomenon, we focused on characterizing RCD execution after treatment with methylnitronitrosoguanidine (MNNG), a DNA alkylating agent. MNNG is the canonical activator of parthanatos, an inflammatory form of RCD dependent on PARP-1 hyper-activation. We found that MNNG exhibits dose-dependent changes in death features, such as death onset time and death rate, indicative of a death mechanism change. As such we hypothesized that MNNG can induce multiple RCD pathways in a dose-dependent fashion. We found that this dose-dependent change in death features was generalizable to multiple cell lines. Moreover, we established that the phenotype was not due to PARP-trapping effects. Importantly, we uncovered that MNNG does induce a death mechanism switch. We found that MNNG is capable of inducing either parthanatos or apoptosis, depending on the dose. We also found evidence that the two death pathways induced by MNNG were mutually exclusive. And lastly, we established that the death mechanism switch was not due to altered mismatch repair (MMR). The information from this study could help to shed light on clinical outcomes from drug combination trials, specifically combinations with DNA damaging agents and PARP inhibitors.
• STING Mediated Autoinflammation in Endothelial Cells Initiates Autoimmune Interstitial Lung Disease

  Gao, Kevin MingJie (2023-02-16)

  Pediatric patients with constitutively active gain-of-function (GOF) mutations in the cytosolic double-stranded-DNA sensing adaptor STING develop an autoinflammatory syndrome known as STING associated vasculopathy with onset in infancy (SAVI). Despite persistent circulating lymphopenia, SAVI patients suffer from interstitial lung disease (ILD) with lymphocyte predominant bronchus associated lymphoid tissue (BALT). Mice expressing SAVI mutations (STING V154M [VM]) recapitulate lymphopenia concomitant with ILD. We find that although lymphopenia results from central developmental defects attributable to immune intrinsic STING GOF, lymphocytes accumulate in lung tissue and are critical for lung pathology and mortality. However, lethally irradiated VM recipients fully reconstituted with wild type (WT) immune cells still develop ILD, indicating that STING GOF in hematopoietic cells is not required for lung disease. Additionally, WT B cells that develop in a VM host produce auto-antibodies against lung and show a repertoire dependent activation in lung tissue. This indicates that STING GOF in radioresistant stromal cells initiates lung autoimmunity. We identified lung endothelial cells as radioresistant cells that express STING. Transcriptional analysis of VM endothelial cells revealed significant up-regulation of chemokine, cytokine, and antigen presentation genes. Conditional expression of the STING GOF mutation in endothelial cells was sufficient to initiate recruitment of lymphocytes to lung tissue. Together, our data show that VM-expressing radioresistant cells, particularly endothelial cells, initiate lymphocyte driven interstitial lung disease in VM mice and provide insights for treatment of SAVI patients, with implications for ILD associated with other connective-tissue-disorders.
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  The Genomics of Canine Behavior and its Comparative Relevance to Human Neuropsychiatric Conditions

  Morrill, Kathleen (2023-02-08)

  Mounting genomic evidence suggests that biological contributions to psychiatric disorder susceptibility are genetically complex, environmentally mediated, and highly comorbid. By paralleling human initiatives, I propose that comparative genomics in the domestic dog offers informational and translational utility to investigations of such complex conditions. Dogs present problematic behaviors with ostensible similarity to human disorders, such as separation anxiety and compulsive behaviors. As behavior played a major role in dog evolution, canine genomes may be enriched for common genetic variants underlying many behaviors. Breeds represent a distillation of diversity in appearance and behavior, but there has been limited success in linking the latter to genes using only pedigreed dogs. The genomes of “mutts” present a natural experiment in genome-wide admixture, which I leverage to map high-dimensional phenotypes across thousands of pet dogs. While I find that most behavioral differences are heritable, especially functional behaviors characteristic of major lineages, I also show that breed alone is an unreliable and confounded predictor of dog behavior. By intersecting genomic studies in people and dogs, this work supplies a roadmap for discerning and ranking cross-species relevance for a wide array of canine and human phenotypes, including psychiatric conditions.
• The Role of Chromatin Architecture in Motor Neurons in Maturation and Amyotrophic Lateral Sclerosis

  Uyan, Ozgun (2023-02-03)

  Amyotrophic lateral sclerosis (ALS) is a progressive and lethal neurodegenerative disorder that is caused by the selective degeneration of upper and lower motor neurons (MNs) in the central nervous system. The causes of ALS are poorly understood and likely to be heterogenous; a compelling approach to understand ALS has been to investigate the 10% of cases with a positive familial history. More than 40 genes have been associated with familial cases. The most common ALS causing gene, C9orf72, has a unique hexanucleotide repeat expansion (HRE) located in the first intron. The hallmark of the C9orf72 pathology includes RNA foci accumulation and translation, and aggregates of repeat-associated dipeptides (DPR) in the nucleus and cell body. In recent years, using 3C-based chromosome conformation technologies, chromatin folding of various cell types, such as fibroblasts, embryonic stem cells (ESC) and neurons, have revealed crucial information to understand 3D folding mechanisms and architectural structures in the nucleus. I performed Hi-C 2.0 and RNA-seq to investigate the three-dimensional genome architectures and transcriptome profiles in three different cell types generated from healthy and ALS individuals with C9orf72 mutation through reprogramming fibroblasts into induced pluripotent stem cells (IPSCs), IPSC differentiation into MNs, and maturation of MNs. Firstly, I analyzed healthy cell types to understand how reprogramming, neural differentiation, and long-term maturation alter genome folding. Secondly, I investigated whether chromatin folding is affected in fibroblasts, IPSCs and MNs of ALS patients with C9orf72 HRE mutation by using the same strategy. This work demonstrated that MNs require long term maturation to establish proper transcriptome and genome folding. Moreover, C9orf72 HRE mutation does not cause any alteration in fibroblast and IPSC lines, however, partial alterations in large scale genome folding were observed in motor neurons.
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  Investigating the role of mutational interdependencies on viral protein function and the evolution of drug resistance

  Samant, Neha S. (2023-01-24)

  Interaction of mutations is ubiquitous in understanding protein fitness landscapes. Fitness landscapes are critical in understanding protein evolution and drug resistance. I aim to elucidate functional consequences of mutations in viral proteins. Retroviral proteases cleave highly diverse substrates, for example, HIV-1 protease (PR) cleaves dramatically different cleavage sites, making it a challenging and interesting system to investigate epistasis. Epistasis also plays an important role in shaping the emergence and evolution of drug resistance, for example in Oseltamivir resistance in Influenza A virus (IAV). To systematically investigate interaction of mutations in important proteins of RNA viruses, we used a mutational scanning approach, EMPIRIC, to investigate the fitness landscape of cleavage sites of HIV-1 PR. We observed that the cleavage sites had higher preferences for hydrophobic and aromatic amino acids. We also observed that negatively charged amino acids are preferred at positions distal to the scissile bond, where these positions are not involved in binding in the PR active site. Studying the fitness landscapes revealed that biophysical features and context-dependencies both mediate cutting of the cleavage sites. However, in-depth analysis of long-range and short-range contextuality would provide further insights on functional determinants of PR cleavage. I also explored the interaction of mutations in the neuraminidase (NA) of influenza A virus in response to inhibitor oseltamivir and identified positive epistasis between drug resistant mutation and a permissive mutation. Our data revealed the potential of epistasis in the evolution of drug resistance in circulating viruses. In summary, these studies provide a framework to examine evolutionary constraints and biochemical mechanisms of viral proteins that can contribute to the evolution of drug resistance.
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  A Transcriptional Cofactor Regulatory Network in the C. Elegans Intestine

  Horowitz, Brent (2023-01-23)

  Transcription is fundamental to growth, development, and homeostasis of all known organisms. It is imperative that genes are expressed correctly, both in time and in specific tissues. Proper spatiotemporal gene expression is controlled by a combination of regulatory factors: DNA binding transcription factors and transcriptional cofactors and chromatin modifiers. Chromatin modifiers are responsible for rearranging DNA structure to affect local transcription. Other transcriptional cofactors work by binding transcription factors and regulating RNA polymerase activity to regulate transcription. While transcription factors have established roles in activating and repressing specific genes, chromatin modifiers and cofactors also act specifically. In multicellular organisms each tissue has its own unique transcriptional program (gene regulatory network) driven by combinations of transcriptional regulators governing the expression of genes necessary for development and homeostasis. To characterize the transcriptional cofactor gene regulatory network, I performed a screen in the Caenorhabditis elegans intestine. Here, the effects of chromatin modifiers and cofactors on nineteen promoters in the C. elegans intestine are assessed using RNAi knockdowns and a set of transcriptional fluorescent reporters. I find no cofactors are universally required to activate all nineteen transcriptional reporters, but certain cofactor complexes seem to work cooperatively to activate or repress the transcription from specific promoters. I also found that transcription from one promoter, that of acdh-1, uses separate transcription factors but common cofactors depending on the biological perturbation activating its transcription. This study demonstrates that cofactors do not act identically at all promoters, similar to the activities of transcription factors.

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