Delineating Mechanisms of Signal-Induced RNA Polymerase II-Transcription

Signal-induced transcriptional programs regulate critical biological processes through the precise spatio-temporal activation of inducible gene programs. Understanding the dynamics by which RNA Polymerase II precisely induces transcription to activate these programs is important for dissecting the basis for their role in cell fate responses and disease progression. Here, we utilize high-resolution genomic approaches coupled with temporal signal induction to characterize how individual transcription steps contribute to the gene expression cycle in signal-induced transcription activation. Our first story (chapter 2) utilizes acute depletion approaches to reveal that the KAP1 protein is a positive regulator of transcription of immediate early genes, a class of signal-induced genes that regulate diverse biological processes including cancer and development. Mechanistically, KAP1 negatively regulates elongation rate at the early stages of transcription, which allows for proper kinetic progression through the transcription cycle by bolstering new initiation and full activation of gene expression. Overall, this study is the first report to link KAP1 "repressive" control of transcription to a positive role in gene activation and has implications for transcription-induced cell fate responses. Our second story (chapter 3) centers on ligand-induced transcription activation of the HIV-1 provirus. Decades of research has shown that HIV-1 transcription is primarily activated through pause release and transcription elongation by the HIV-1 transactivator Tat. Here, we use a novel Tat depletion approach to show that Tat function in pause release is the catalyst that promotes sustained RNA Polymerase II recruitment to the HIV-1 promoter. This recruitment of RNA Polymerase II is the mechanism that robustly induces "logarithmic" HIV-1 expression and viral replication. These data reveals a new significance for Tat function in transcription initiation and explains how Tat can sustain extended levels of HIV-1 transcription for proviral fate. Overall, the two studies have provided important mechanistic insights for understanding transcription dynamics in gene expression programs that have implications for diverse biological phenomena and pathogenesis

Chemical Investigations in Complex Alkaloid Synthesis

The concise and efficient synthesis of complex biologically active natural products often requires the elaboration of innovative chemical design paired with the development of modern chemical methods to push the forefronts of chemical space to further biochemical investigations. Complex alkaloids have been of interest to synthetic chemists for decades where their intriguing chemical structures have led to the development of new reactions as well as provided biologically relevant compounds for potential therapeutic use. The growing Myrioneuron and Sarpagine- related alkaloid classes are only a small fraction of the larger realm of complex alkaloid space, however, chemical investigations into these families will hopefully provide insight to the synthetic community as a whole through new chemical methods, synthetic design, and expand further biochemical understandings. Thus, Chapter 1 will serve to introduce the Myrioneuron alkaloids, beginning by detailing their breadth of biological activity, followed by their proposed biosynthetic origins. Lastly, the reported synthetic approaches to this family will be described, starting with early efforts towards simpler congeners, leading to recent, elegant approaches to the more elaborate family members. Next, Chapter 2 will describe the evolution of our synthetic approach to the complex tetracyclic Myrioneuron alkaloid myrioneurinol, a strategy leveraging the hidden symmetry within the molecule to assemble the core scaffold. To begin this chapter, I will discuss our strategic plan towards the complex myrioneuron scaffold, then the early efforts to assemble the required symmetrical intermediate, an in-depth investigation into the key desymmetrizing double reductive amination, efficient elaboration of the scaffold, issues surrounding a challenging hydrogenation and our topologically controlled solution, and finally, the completion of myrioneurinol. Also described will be the development of a formal asymmetric synthesis, the first asymmetric entry towards this complex Myrioneuron alkaloid. Chapter 3 will serve to introduce a similarly complex, yet structurally distinct, subset of indole alkaloids, the Sarpagine-related alkaloids. This chapter will cover this broad family's chemical diversity and wide range of biological activity, their biosynthetic origins, as well as discuss selected synthetic studies within the sarpagine-type alkaloids. Finally, in Chapter 4, our efforts to develop a concise asymmetric approach to the unusual Sarpagine-type alkaloid rauvomine B will be described. First, I will introduce the strategic approach to the distinct sarpagine alkaloid, the challenges of achieving the preparation of the core methyl-containing scaffold will be discussed, including adjustments to the synthetic design. Lastly, the formation of the signature cyclopropane and associated quaternary carbon center via a key late- stage Rh-catalyzed cyclopropanation will be detailed, allowing for completion of this alkaloid target

Dynamic Shuttling of AGO2 to the Nucleus Relieves MicroRNA Repression

The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC). This machinery mediates post-transcriptional silencing of mRNA targets by sequence-specific hybridization with microRNAs (miRNAs) in the cytoplasm. Localization to subcellular compartments is critical to understanding miRNA action, and our lab and others have shown that miRISC complexes are also present and active in mammalian nuclei. miRISC has several proposed nuclear functions, yet the biological significance and regulatory mechanisms of action of nuclear miRISC in mammalian cancer cells remain poorly understood. We observed that three different model systems that mimic colon tumor microenvironments: (1) 2D colorectal cancer cell, HCT116, cultures grown beyond a monolayer to high cell density, (2) 3D HCT116 tumor spheroid cultures grown in collagen matrix, and (3) primary tissue samples from malignant colon tumor and normal adjacent colon tissue, show significant nuclear enrichment of core RISC effector protein, AGO2. We tested the consequences of nuclear AGO2 localization on global microRNA regulation using a multipronged transcriptomics approach. We combined AGO2-eCLIP-sequencing, small-RNA-sequencing, and whole transcriptome sequencing to identify candidate gene targets bound by AGO2 that may be regulated by the most abundant microRNA families. We observed that the majority of AGO2-bound cytoplasmic 3'UTR miRISC targets were significantly upregulated, potentially de-repressed, when AGO2 is nuclear-enriched. Using an AGO2 construct with a nuclear localization sequence (NLS-AGO2-GFP), we were able to phenocopy the de-repression of cytoplasmic candidate genes and observe a correlation with a higher rate of migration. Our findings reveal dynamic relocalization of AGO2 to the nucleus in colon cancer culture systems and primary colon tumor tissue, which may impact our understanding of spatial regulation of microRNA action and reveal therapeutic targets that contribute to colon tumor progression. Another study from my thesis research provides a systemic evaluation of the effects of depleting microRNA biogenesis enzyme, DROSHA, and microRNA effector proteins, AGO1-4 and TNRC6A-C, on microRNA expression. Our findings help to define the boundaries of RNAi in a widely used cell line, HCT116, by refining a subset of abundant microRNAs that are DROSHA-dependent, associated with Argonaute proteins, and most likely to carry out robust gene regulation

Enhancing Nanoparticle Drug Delivery to Brain Tumors with Focused Ultrasound

Glioblastoma mulitforme (GBM) is regarded as an incurable and universally fatal disease, characterized by its physical inaccessibility to most therapies and ultimately serve as a source for tumor recurrence which leads to the patient's demise. Furthermore, the prognosis for GBM has not improved significantly over the last 20 years. To achieve meaningful advances in the treatment of GBM, novel strategies must be devised to effectively treat these invasive glioma cells, therein halting the progression of the disease. Our laboratory's research centers on a nanoparticle biologic with the ability to selectively kill cancer cells despite uptake into normal healthy cells. This novel biologic therapy is based on plasma‐derived low‐density lipoprotein (LDL) reconstituted with the natural omega‐3 fatty acid, docosahexaenoic acid (DHA) (herein referred to as LDL‐DHA). However, therapeutic delivery to the brain is infamous due to the BBB that regulates the homeostasis of the brain by cell tight junctions with limited passage to certain molecules and nutrients. To bypass the BBB, Focused Ultrasound (FUS) combined with microbubbles are used to mechanically open the BBB to allow transient and targeted delivery of the brain. FUS in combination with LDL-DHA for delivery in normal rat brains showed safety of FUS and no evidence of cytotoxicity, respectively. However, delivery of the LDL particle by FUS to the brain has not been done in a tumor bearing animal model as well as the evaluation of LDL-DHA toxicity, uptake, and mechanisms in normal and malignant brain cell lines. In this dissertation, we focused on the development of a GBM tumor-bearing mouse model, delivery of LDL particles using FUS to the tumor, and assessment of particle cellular uptake and cytotoxicity by LDL-DHA in normal and malignant murine brain cells lines. In the following chapters we discuss accumulation of LDL particles in the surrounding mouse tumor by FUS, assessed mechanisms of particle uptake in normal and malignant brain cells, and showed the selective toxicity of LDL-DHA to GBM cell lines

An Ultra-pH-Sensitive Logic Decodes Cancer Imaging and Immunotherapy

Pages 186-213 are misnumbered as pages 219-246.Binary classification of cancer is a challenge in clinical diagnosis due to continuously varying cancer-associated signals in both time and space. To distinguish cancer from healthy tissue it's necessary to assess binary relationships between signals at the molecular scale. However, the naked eye cannot discriminate differences at that resolution. Further, contrast enhancement with responsive small molecules shows minute changes between cancer and healthy tissues. Therefore, our group previously developed an ultra-pH-sensitive (UPS) nanoparticle platform to discretely amplify differences of acidotic thresholds. Predicated on molecular cooperativity, a divergent phase transition behavior from nanoparticle to polymer enables an irreversible capture-and-integration in the tumor microenvironment for robust tumor margin visualization. Based on previous observations on applications of the UPS platform, here I employ a formal first-order logic to quantify ultra-pH-sensitivity in cancer diagnosis. Based on this framework, I demonstrate novel applications of UPS nanoparticles in lymph node metastasis imaging. I show that UPS nanoparticles accumulate in microscopic cancer foci inside of lymph nodes, enabling discrimination between metastatic and benign lymph nodes. Further, I determine contexts wherein near-neutral pKa UPS nanoparticles lose pH-reporting fidelity in vivo. Both UPS and PC7A nanoparticles (pKa 6.9) have a short circulation half-life and poor tumor biodistribution. To overcome this, I employ cybernetic control by co-formulating two UPS polymers into a single nanoparticle. Attenuation of micelle disassembly, through strengthened non-covalent interactions, stabilizes UPS nanoparticles for improved pharmacokinetics in blood circulation. I demonstrate two applications of these long-circulating, hybrid nanoparticles. First, I engineer HyUPS nanoparticles for improved lymph node delivery of UPS6.9 polymers through co-assembly with UPS5.3. Reporting dual pH-thresholds enables accumulation in metastatic lymph nodes. Second, I develop HySTING nanoparticles for improved accumulation of PC7A in tumors. HySTING retains the unique biological activity of PC7A, yet accumulates greater in tumors after intravenous injection. HySTING enables tumor growth inhibition in mouse models of cancer by synergizing with synthetic cyclic dinucleotide payloads. Overall, a formal logic reveals robust discrimination of cancer from normal tissue by UPS nanoparticles. However, this logic fails in certain contexts. Restoring high fidelity logic through engineering control enables ON-target cancer imaging and immunotherapy

Design, Development, and Interrogation of Salmonella Typimurium Minimal Effector Networks During Infection

Pages 136-174 are misnumbered as pages 137-175.Salmonella enterica serovar Typhimurium utilize a Type Three Section System (T3SS) to deliver virulent "effector" proteins directly into the host cell cytoplasm. Some of these effectors have been biochemically characterized, but many of them can be deleted individually with little reduction in bacterial pathogenicity. Thus, determining the interrelationship between effector gene networks, host substrates, and pathogen virulence has been challenging. Using targeted mutagenesis and phenotypically informed genome reassembly, we engineered a minimal SPI-2 T3SS effector gene network that supports Salmonella enterica serovar Typhimurium (STm) intracellular replication in vitro and disease pathogenesis in a murine model of Typhoid-fever. Genetic analysis of the host immune response to this minimal effector gene network reveals a sophisticated interplay between host immunity and bacterial disease progression. Further analysis reveals the specific immune responses during infection at a dynamically novel resolution. The interplay between SPI-2 effector proteins and immune cell targets reveals an advanced complexity of host-pathogen interactions, illustrating the cooperation necessary to drive tissue tropism during bacterial pathogenesis and a powerful tool for interrogating effector functions in vivo which has further implications for Salmonella pathogenesis in the host

The Role of Metabolic Inflexibility in Heart Failure

Heart failure (HF) is a syndrome defined by the inability of the heart to supply adequate nutrients and oxygen to peripheral tissues. One of the most common causes of HF is chronic pressure overload due to hypertension, which leads to hypertrophic remodeling, accompanied by metabolic alterations. These changes are initially compensatory but lead to worse outcomes in the long term, making them targets for therapeutic intervention. When hypertension and diabetes are present simultaneously, a type of HF with preserved ejection fraction (HFpEF) ensues. HFpEF is distinct from HF with reduced ejection fraction (HFrEF) and diabetes in clinical presentation and physiology, necessitating alternative treatments. Understanding metabolic alterations which contribute to the development and progression of HFpEF can aid us greatly in creating these treatments. This study aimed to replicate the cellular metabolic profile in diabetic heart to 1) determine if energy deficiency plays a causative role in HFpEF progression, and 2) test the necessity of glucose oxidation for left ventricular hypertrophy development and progression to HF. Since pyruvate dehydrogenase (PDH) kinase 4 (PDK4) is the predominant PDK found in the heart and PDK4 expression in the heart increases with high circulating fat levels, I employed a genetic approach to modulate its expression in cardiomyocytes specifically. This animal model allows us to control the activity level of PDH by its phosphorylation as seen in diabetic patients while bypassing complications of altering metabolism systemically. Deletion of PDK4 in cardiomyocytes of adult mice prior to a high-fat diet prevented PDH inactivation in response to increased free fatty acids, allowing continued use of glucose for energy production by the TCA cycle. The impact on HFpEF development was unclear as the control group failed to fully develop the phenotype. In a separate experiment, constitutive overexpression of PDK4 gene was used to persistently inhibit PDH before surveying cellular metabolism and its effect on cardiac function at baseline as well as in response to increased cardiac work due to pressure afterload. This revealed that cardiac energetic deficiency did not require hypertrophic growth, and hypertrophic growth was not blunted by lack of glucose oxidation. Taken together, these findings suggest that metabolic inflexibility in cardiomyocytes plays a role in HFrEF development, independently of LVH. Furthermore, the metabolic profile in our animal model resembles what little is known of in HFpEF patients more closely than in diabetic cardiomyopathy, paving the way for future studies

Investigating the Role of Hippo/Warts Signaling in Sarcomatoid Renal Cell Carcinoma

Renal cell carcinomas (RCCs) are the most common kidney malignancy and one of the most common cancers overall. Sarcomatoid RCC (sRCC) is a particularly aggressive subtype of RCC which displays a high rate of metastasis and poor prognosis, contributing to a disproportionately high clinical burden. Attempts to identify driver mutations of sarcomatoid transformation have revealed frequent mutations in components of the Hippo/Warts pathway, a highly conserved regulator of genes controlling proliferation and apoptosis which is frequently dysregulated in cancer. Here, I describe the generation and characterization of a GEMM which gives rise to metastatic sRCC: deletion of Hippo pathway kinases Lats1/2 in adult kidney epithelia. Lats1/2 mutant tumors respond to immune checkpoint inhibition (ICI), as is seen in some human sRCCs. Bioinformatic analysis reveals an enrichment of serum response factor (Srf) targets amongst genes upregulated following Lats1/2 deletion. Ablating Srf in the Lats mutant background causes Lats;Srf mutant cells to undergo epithelial to mesenchymal transition but not progress to fulminant tumors in vivo. RNA-sequencing, CyTOF analysis, and implantation studies in immunocompromised mice suggest that Srf drives a pro-tumor microenvironment in Lats1/2 mutant sRCC. Additionally, transcriptional analysis identifies a subset of human RCCs that express an Srf transcriptional signature, indicating that Srf may contribute to the pathogenesis of RCC. This work has elucidated a novel role for Srf in immune modulation with potential implications for the treatment of human RCC

Dynamic Regulation of Ribosome Biogenesis and Its Role in Early Brain Development

Chapter 1, including figures and a substantial amount of text, has been re-used, with or without modifications, from the following previous published works: [1] Ni, C., and Buszczak, M. (2023a). The homeostatic regulation of ribosome biogenesis. Semin Cell Dev Biol 136, 13-26. [2] Ni, C., and Buszczak, M. (2023b). Ribosome biogenesis and function in development and disease. Development 150.Chapter 2, including figures and a substantial amount of text, has been re-used, with or without modifications, from the following previous published work: Ni, C., Schmitz, D.A., Lee, J., Pawłowski, K., Wu, J., and Buszczak, M. (2022). Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly. Cell Reports 38, 110597.Chapter 3, including figures and a substantial amount of text, has been re-used, with or without modifications, from the following previous pre-print work: Chunyang Ni , Leqian Yu, Barbara Vona, Dayea Park, Yulei Wei, Daniel A Schmitz, Yudong Wei, Yi Ding, Masahiro Sakurai, Emily Ballard, Yan Liu, Ashwani Kumar, Chao Xing, Hyung-Goo Kim, Cumhur Ekmekci, Ehsan Ghayoor Karimiani, Shima Imannezhad, Fatemeh Eghbal, Reza Shervin Badv, Eva Maria Christina Schwaibold, Mohammadreza Dehghani, Mohammad Yahya Vahidi Mehrjardi, Zahra Metanat, Hosein Eslamiyeh, Ebtissal Khouj, Saleh Mohammed Nasser Alhajj, Aziza Chedrawi, César Augusto Pinheiro Ferreira Alves, Henry Houlden, Michael Kruer, Fowzan S. Alkuraya, Can Cenik, Reza Maroofian, Jun Wu, and Michael Buszczak (2023) Dynamic ribosome biogenesis shapes early brain development (Submitted).While features of ribosome assembly are shared between species, our understanding of the diversity, complexity, dynamics, and regulation of ribosome production in multicellular organisms remains incomplete. To gain insights into ribosome biogenesis in human cells, we performed a genome wide loss of function screen combined with differential labeling of pre-existing and newly assembled ribosomes. These efforts identified two functionally uncharacterized genes, C1ORF109 and SPATA5. We provide evidence that these factors, together with CINP and SPATA5L1, control a late step of human pre-60S maturation in the cytoplasm. Loss of either C1ORF109 or SPATA5 impairs global protein synthesis. These results raise the possibility that neurodevelopmental disorders associated with recessive SPATA5 mutations are caused by defects in ribosome assembly. Based on these findings, we propose the expanded repertoire of ribosome biogenesis factors likely enables multicellular organisms to regulate ribosome production in different ways in response to different developmental and environmental stimuli. Lineage specific transcriptional programs orchestrate human brain development. Many neural developmental defects, however, are linked to mutations in general regulators of housekeeping genes, such as those encoding ribosome biogenesis and mRNA translation factors, suggesting additional layers of regulation. The molecular and cellular mechanisms by which minor disruptions in global protein synthesis capacity cause neurodevelopmental disorders and when these defects first arise remain unclear. Using cerebral organoids in combination with proteomic analysis, single-cell transcriptome analysis across multiple developmental stages, and single organoid translatome analysis, we discovered a previously unappreciated mechanism linking changes in ribosome levels and the timing of cell fate specification during early brain development. We find ribosome levels decrease during neuroepithelial differentiation, and differentiating cells are particularly vulnerable to defective ribosome biogenesis during this time. Reduced ribosome availability more profoundly impacts the translation of specific transcripts, disrupting both survival and cell fate commitment of transitioning neuroepithelia. Enhancing global protein synthesis ameliorates the growth and developmental defects associated with microcephaly linked variants. Together, these findings reveal that dynamic changes in ribosome levels regulate early development of central nervous system and provide insights into how disruptions in protein synthesis machinery can result in brain-specific malformations

Structural Investigation of the Alkaline pH-Dependent Activation of Insulin Receptor-Related Receptor

The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Insulin receptor family, a subset of receptor tyrosine kinases governing metabolic balance and growth. Among them, the insulin receptor-related receptor stands out for its activation mechanism, which responds to alkaline pH rather than ligand binding. However, the precise workings of this pH-induced IRR activation have remained elusive due to the lack of high-resolution structural data. Our breakthrough comes with the cryo-EM structures of human IRR in two key states: the inactive state at neutral pH and the active state at alkaline pH. Through mutation studies and cellular experiments, we've unveiled the mechanism: as pH rises, repulsion forces between IRR's pH-sensing motifs disrupt its auto-inhibition, initiating a scissor-like rotation between two units and forming a "T"-shaped active conformation. This activated IRR dimer relies on specific interactions to stabilize itself. Our findings offer an unprecedented insight into the pH-dependent activation of IRR, paving the way for a deeper understanding of this critical receptor's structure-function relationship