NK cell expansion requires HuR and mediates control of solid tumors and long-term virus infection

Natural killer (NK) cells are lymphocytes capable of controlling tumors and virus infections through direct lysis and cytokine production. While both T and NK cells expand and accumulate in affected tissues, the role of NK cell expansion in tumor and viral control is not well understood. Here, we show that posttranscriptional regulation by the RNA-binding protein HuR is essential for NK cell expansion without negatively affecting effector functions. HuR-deficient NK cells displayed defects in the metaphase of the cell cycle, including decreased expression and alternative splicing of Ska2, a component of the spindle and kinetochore complex. HuR-dependent NK cell expansion contributed to long-term cytomegalovirus control and facilitated control of subcutaneous tumors but not tumor metastases in two independent tumor models. These results show that posttranscriptional regulation by HuR specifically affects NK cell expansion, which is required for the control of long-term virus infection and solid tumors, but not acute infection or tumor metastases, highlighting fundamental differences with antigen-specific T cell control

Elucidating gene regulatory mechanisms of postnatal maturation and regeneration in the mammalian liver

The mammalian liver is a major metabolic organ responsible for a variety of functions and has an exceptional capacity for regeneration. To enable functional competency in postnatal and adult stages, enable survival in response to injury and support extensive remodeling in regeneration hepatocytes undergo dramatic changes in gene regulatory networks. My thesis focuses on understanding the mechanisms of gene regulation, specifically post-transcriptional gene regulation, within hepatocytes in postnatal maturation, regeneration and disease using the murine model system. At the outset, I begin by exploring gene expression in chronic toxin-mediated liver injury and repair. I use genomewide transcriptomic methodologies to probe gene expression, alternative splicing and translation within the regenerating hepatocytes and find extensive remodeling at all stages of gene regulation. We find that RNA binding proteins especially splicing factors are regulated at translation level, leading to redeployment of a postnatal splicing programme. Furthermore, to overcome limitation of bulk methods in revealing subtleties in population behavior we employed single-cell genomics in acute liver injury. In Chapter three, we probed and analyzed single cell gene expression from ~22,000 liver cells post-2/3rd partial hepatectomy to reveal the division of labor within hepatocytes in regeneration to simultaneously support proliferation and metabolism. Furthermore, we show evidence using cumulative labelling methods that the major source of new hepatocytes in acute injury is midlobular zone within the liver lobule. From analysis in Chapter two as well as previous work in the lab, we had identified Epithelial splicing regulatory protein 2 (ESRP2) as an important splicing factor that has a postnatal onset in hepatocytes and is dynamically regulated in regeneration and disease. In chapter four, I begin by exploring the genomewide splicing regulation by ESRP2 using a loss-of-function mouse model and find extensive regulation of both developmentally regulated events as well as an adult stage specific program. We also find extensive defects in hepatic proliferation, cell size and polyploidization in ESPR2 KO mice. Furthermore, we developed an acute hepatocyte specific ESRP2 loss-of-function model revealing that ESRP2 is necessary to not only bring about but also maintain the adult hepatic splicing program. Using a hepatocyte specific inducible overexpression model for ESRP2 we showed by in young pups that ESRP2 is sufficient for early induction of postnatal maturation and revealed the importance of temporal regulation of ESRP2 expression. Lastly, to identify ESRP2’s genomewide binding landscape we developed, using CRISPR-Cas9 methodology, a 2xFLAG-tagged transgenic mouse and performed eCLIP to reveal both intronic as well as surprisingly 3’UTR binding by ESRP2. Lastly, we identify miR-122 biogenesis as a direct target of ESRP2 regulation leading to reduced mature miR-122 levels in ESRP2 loss-of-function mice with eventual loss of proper postnatal hepatic polyploidization. In chapter five, I explore the role of ESRP2 in chronic liver injury wherein it is dynamically downregulated via translational regulation. Using gain- and loss-of-function models we find that ESRP2 levels directly regulate hepatocyte proliferation rates. We identify 4 cassette exons harbored within proteins of the Hippo pathway, namely, NF2, CSKN1D, YAP1 and TEAD1 that are regulated by ESRP2. These exons are upregulated in development, and in regeneration their inclusion is reduced and this correlates directly with ESRP2 expression. We show using cell culture of primary hepatocytes that exclusion of these exons augments hepatocyte proliferative potential, and this occurs due to attenuation of Hippo signaling leading to YAP1 translocation to nucleus and transcriptional activation. In conclusion, I have identified a cell-type specific and temporally coordinated gene expression program operative in postnatal liver development and regeneration. Furthermore, I demonstrated a direct role for ESRP2 in regulating a post-transcriptional gene regulatory network to support postnatal hepatocyte maturation and regeneration.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste

Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens [version 1; referees: 3 approved]

Alternative splicing, polyadenylation, and chemical modifications of RNA generate astonishing complexity within eukaryotic transcriptomes. The last decade has brought numerous advances in sequencing technologies that allow biologists to investigate these phenomena with greater depth and accuracy while reducing time and cost. A commensurate development in biochemical techniques for the enrichment and analysis of different RNA variants has accompanied the advancement of global sequencing analysis platforms. Here, we present a detailed overview of the latest biochemical methods, along with bioinformatics pipelines that have aided in identifying different RNA variants. We also highlight the ongoing developments and challenges associated with RNA variant detection and quantification, including sample heterogeneity and isolation, as well as ‘Omics’ big data handling

Lipid droplet-associated hydrolase mobilizes stores of liver X receptor sterol ligands and protects against atherosclerosis

Abstract Foam cells in atheroma are engorged with lipid droplets (LDs) that contain esters of regulatory lipids whose metabolism remains poorly understood. LD-associated hydrolase (LDAH) has a lipase structure and high affinity for LDs of foam cells. Using knockout and transgenic mice of both sexes, here we show that LDAH inhibits atherosclerosis development and promotes stable lesion architectures. Broad and targeted lipidomic analyzes of primary macrophages and comparative lipid profiling of atheroma identified a broad impact of LDAH on esterified sterols, including natural liver X receptor (LXR) sterol ligands. Transcriptomic analyzes coupled with rescue experiments show that LDAH modulates the expression of prototypical LXR targets and leads macrophages to a less inflammatory phenotype with a profibrotic gene signature. These studies underscore the role of LDs as reservoirs and metabolic hubs of bioactive lipids, and suggest that LDAH favorably modulates macrophage activation and protects against atherosclerosis via lipolytic mobilization of regulatory sterols

Cell-Membrane Coated Nanoparticles for Tumor Delineation and Qualitative Estimation of Cancer Biomarkers at Single Wavelength Excitation in Murine and Phantom Models

Real-time guidance through fluorescence imaging improves the surgical outcomes of tumor resections, reducing the chances of leaving positive margins behind. As tumors are heterogeneous, it is imperative to interrogate multiple overexpressed cancer biomarkers with high sensitivity and specificity to improve surgical outcomes. However, for accurate tumor delineation and ratiometric detection of tumor biomarkers, current methods require multiple excitation wavelengths to image multiple biomarkers, which is impractical in a clinical setting. Here, we have developed a biomimetic platform comprising near-infrared fluorescent semiconducting polymer nanoparticles (SPNs) with red blood cell membrane (RBC) coating, capable of targeting two representative cell-surface biomarkers (folate, αυβ3 integrins) using a single excitation wavelength for tumor delineation during surgical interventions. We evaluate our single excitation ratiometric nanoparticles in in vitro tumor cells, ex vivo tumor-mimicking phantoms, and in vivo mouse xenograft tumor models. Favorable biological properties (improved biocompatibility, prolonged blood circulation, reduced liver uptake) are complemented by superior spectral features: (i) specific fluorescence enhancement in tumor regions with high tumor-to-normal tissue (T/NT) ratios in ex vivo samples and (ii) estimation of cell-surface tumor biomarkers with single wavelength excitation providing insights about cancer progression (metastases). Our single excitation, dual output approach has the potential to differentiate between the tumor and healthy regions and simultaneously provide a qualitative indicator of cancer progression, thereby guiding surgeons in the operating room with the resection process