REU Program Sample Project Summaries

Glycoimmunology is an emerging field focused on understanding how immune responses are mediated by glycans (carbohydrates) and their interaction with glycan-binding proteins called lectins. Evolutionary conservation is in the order of Genome > Transcriptome > Proteome > Metabolome > Lipidome > Glycome. The glycome is the least conserved and most structurally diverse form of biological information; despite this, glycans are still considered the “dark matter” of the biological universe. Technologies to tackle the complexity of the glycome in a high-throughput manner are now emerging. Recent advances in the emerging field of glycoimmunology show that the human glycome plays critical roles in driving or modulating immune responses, and in cell-cell and cell-pathogen interactions.

How glycans influence immunological functions is under active investigation; including the role played by the host glycosylation machinery in modulating the persistence and immunopathogenesis of viruses. Our laboratory uses several glycomic technologies to investigate the role of the host glycosylation machinery in regulating molecular mechanisms central to HIV infection. Our goal is to create a new paradigm for discovering novel viral/host interactions and/or glycan-based interactions. This research has the potential to expand the boundaries of current knowledge about the link between infections, chronic inflammation, and the development of chronic diseases. Projects in which REU students could be involved include:

• Sialic acid modulation of HIV-associated inflammaging: determining how HIV infection accelerates the pace of age-associated glycomic dysregulation
  
  
• Precision glyco-editing as a potential strategy for HIV immunotherapy: targeting sialic-acid/siglec interactions to enhance innate immune cell clearance of HIV+ cells
  
  
• Glycomic modulation of the gut microbiome during HIV infection: determining how hypo-sialylation and dys-fucosylation alter microbiome composition, leading to microbial translocation, inflammation, and viral persistence

Our laboratory studies mechanisms of contractile force generation in the regulation of cell migration and cell invasion. Specifically, we have been interested in how bioenergetic resources are dynamically allocated to support membrane focal adhesion turnover, changes in lamellipodia formation and extrusion, sustained phosphorylation of cell motility kinases, in particular Focal Adhesion Kinase (FAK), and overall actin cytoskeletal remodeling. We pursue a model of spatiotemporal bioenergetics as a critical determinant of cell motility, where active repositioning of mitochondria to the cortical cytoskeleton provides an efficient and concentrated energy source to fuel membrane dynamics of cell movements and sustained cell migration. Mechanistically, we aim to elucidate the role of effectors of mitochondrial dynamics, organelle quality control pathways and modulation of oxidative bioenergetics, including changes in mitochondrial superoxide production in the control of subcellular mitochondrial trafficking and regional bioenergetics. Possible projects include:

• Characterizing factors that regulate mitochondrial dynamics, including dynamin-related protein-1 (Drp1) to determine how these factors control changes in mitochondrial shape, morphology, division (or fission), and bioenergetics. We use high-end imaging approaches supported by novel algorithms to test the model that increased mitochondrial fission promotes subcellular organelle trafficking to the cortical cytoskeleton and increased cell migration.
  
  
• Evaluating how “quality control” of mitochondrial protein folding impacts oxidative phosphorylation, superoxide production and affects regional bioenergetics. This project utilizes state-of-the-art biochemical and biophysical approaches to evaluate the role of chaperone-controlled protein (re)folding in regulating OXPHOS supercomplex assembly, energy production, and ROS generation during subcellular mitochondrial trafficking.

The process of cell fate determination is at the very core of biological complexity in higher eukaryotes. Genetically identical precursor cells (i.e. stem cells) can execute different transcriptional programs resulting in a wide range of differentiated cell types that are morphologically and functionally distinct. The Gardini lab works to characterize the transcriptional mechanisms that control fate choice and differentiation of human hematopoietic stem cells. In particular, we investigate how genomic regulatory sequences, termed enhancers, work to determine cell identity and specify blood lineages from hematopoietic stem cells. We use genome-wide high-throughput sequencing technology (NGS) to identify and characterize de novo enhancer elements in primary human cells. Projects for students include:

• Mapping enhancers: use chromatin immunoprecipitation (ChIP-sequencing) in primary human monocytes before and after differentiation into macrophages. The student will assess the change in chromatin histone marks (H3K27ac, H3K27me3) along the course of cell differentiation and identify critical enhancers that change their status.
  
  
• Mining existing datasets of human monocytes to identify inflammatory genes and their cognate enhancers. The student will use bioinformatic approaches on a dataset previously generated in the lab, to identify and then validate critical genes that are activated in response to inflammation and computationally identify their closest regulatory enhancer.

We are broadly interested in mechanisms of cell homeostatic pathways, how they communicate with one another in response to environmental cues, and their impacts on tissue homeostasis. In particular, we are fascinated by the ability of cells to use a lysosomal pathway known as autophagy to degrade and recycle self-components. Our laboratory has identified and characterized the mammalian essential autophagy protein, UV irradiation resistance associated gene (UVRAG), which functions in and beyond autophagy to regulate endocytosis, cell survival, DNA repair, and organelle integrity. Employing a combination of genetic, biochemical, molecular, cellular, and computational approaches in model organisms ranging from Drosophila to Zebrafish to mice, we have defined both canonical and non-degradative functions of UVRAG and the autophagy pathway. Student projects include:

• Melanosome regulation: UV-exposed keratinocytes secrete α-melanocyte-stimulating hormone, which signals melanocytes to produce UV-blocking melanin pigment in lysosome-like structures known as melanosomes. Impaired melanogenic traffic/function allows more UV penetration and accrues more UV-induced mutations. We are investigating the molecular mechanisms regulating melanosome formation and how it adapts to environmental UV exposure.
  
  
• UVRAG and genome integrity. We discovered a striking association between UVRAG downregulation and increased UV-induced genomic instability in human melanocytes. Understanding how UVRAG contributes to UV protection is an ongoing focus of the lab.

Our laboratory is interested in the molecular mechanisms that underlie establishment and maintenance of immune regulation in infectious disease and cancer with an emphasis on viral latency in mammalian cells. With regards to HIV latency, we use the long-terminal repeat (LTR) of HIV as a model for studies to better understand how this genetic regulatory sequence induces viral RNA production or promotes long-term transcriptional silencing. Our previous work involving in vitro phenotypic screening of naturally-occurring chemical compounds and derivatives has identified multiple chemical leads which competitively inhibit LTR-mediated transcription and/or reinforce transcriptional silencing even following their withdrawal. As these chemical entities do not appear to act through previously-established cellular pathways of LTR regulation (e.g., protein kinase C, NF-κB, or CDK9 signaling), they represent new leads by which to probe for novel facets of host-mediated viral transcription and latency. We aim to identify and elucidate the basic molecular and cellular mechanisms of these pathways in order to understand the fundamental properties of viral expression and latency in both CD4 T-cells and myeloid cells. Specific projects that students in the lab can pursue include:

• Performing RNAseq-based studies of cells (cell line and/or primary cells) in the presence of viral transcription inhibitors to identify gene expression profiles that underlie distinct cellular signaling pathways and which are altered during LTR-mediated transcription or reinforced transcriptional silencing
  
  
• Employ CRISPR and pharmacological-based techniques in cell lines and/or primary cells to modify cellular signaling pathways which are implicated in viral promoter transcription and silencing
  
  
• Investigate how genetic modifications to the LTR, genomic integration site, or host molecules regulate LTR viral transcription and/or latency

The Murphy lab studies naturally occurring variants of key regulatory genes controlling cell differentiation and fate, with the goal of understanding how these variants regulate specific cellular phenotypes. In particular, we have focused on variants of TP53, the most frequently mutated gene in human cancer, which encodes the protein p53. p53 is a transcription factor that regulates specific cellular fates, including senescence, apoptosis, and ferroptosis, thereby suppressing tumor formation in different tissues. The overriding scientific goal in the laboratory is to identify the molecular mechanism(s) by which p53 determines each cellular outcome. A secondary goal is to identify the critical genes that are transcribed by p53 to produce these cell fates. In order to achieve these goals, the Murphy laboratory employs the analysis of naturally-occurring genetic hypomorphic alleles of TP53. These specific alleles are overrepresented in different ethnic populations, and alter the amino acid sequence of this protein. The focus of the laboratory is three different genetic hypomorphs, the Y107H and P47S variants, which are overrepresented in populations of African descent, and the G334R variant, which is overrepresented in individuals of Ashkenazi origin. Selected student projects include:

• The P47S variant of TP53: This hypomorph exists in 1 million African-Americans and is selectively deficient in the ferroptosis pathway. Ferroptosis is an iron- and lipid-mediated cell death program. We have identified several p53 target genes, including SLC7A11, which play a role in ferroptosis and which are not properly regulated by the P47S variant. We will investigate the mechanisms by which P47S fails to properly bind these gene promoters, recruit the transcriptional machinery, and successfully elongate transcription.
  
  
• The Y107H variant of TP53: This hypomorph exists in over 50,000 African Americans, and it is selectively impaired for the transcriptional activation of the p53 target gene PADI4, which is a chromatin modifying protein that controls the tissue-specific decision between senescence and apoptosis by p53. We will probe the requirement for PADI4 in the regulation of p53 function, using cell culture and biochemical techniques.

Neutrophils are innate immune cells that serve as the first line of defense against a wide range of pathogens including bacteria, fungi, and protozoa, by utilizing two major mechanisms: phagocytosis and degranulation. Recently, formation of neutrophil extracellular traps (NET) has been described as another host defense mechanism of neutrophils against pathogens. NETs are extracellular web-like structures composed of chromatin fibers. Proteins and antimicrobial peptides that are normally found in neutrophil granules, cytoplasm, or nuclei are also found associated with NETs. NETs are formed via a novel cell death pathway called NETosis, in which disintegration of the nuclear envelope allows mixing of chromatin with cytoplasmic and granular proteins followed by their release outside the cell. The molecular mechanisms controlling NETosis are not well understood; however, a key step involves decondensation of chromatin and citrullination of histone H3. Citrullination converts arginine residues to citrulline and is catalyzed by peptidylarginine deiminases (PADs). Citrullination of histone H3 by PAD4 plays a key role in NETosis, and PAD4-deficient mice exhibit reduced NET formation and NET-dependent responses.

Currently, the lab is investigating the effects of targeting PAD4 pharmacologically to further understand the regulation of NETosis and how NETs contribute to neutrophil function in pathogen resistance and cancer. Projects for REU students include:

• Comparing the effects of previously described PAD4 inhibitors on NETosis in vitro, and determining potential additive or synergistic interactions
  
  
• Determining how PAD4 enzymes are regulated, and what factors induce citrullination of histone H3 arginine residues
  
  
• Evaluating PAD4 inhibition in vivo, using murine models of bacterial infection and metastasis

Nucleic acid gene-encoded antibodies (DNA and mRNA) are a rapidly advancing field for direct in vivo delivery of protective antibodies. Using vectored approaches, optimized antibody genes are delivered directly to muscle cells, producing and secreting fully functional antibodies into circulation. In vivo gene-encoded antibodies can deliver complex bispecific molecules in vivo that are typically challenging to develop through recombinant bioprocesses. Building on this, our group is utilizing non-viral DNA vectors to understand the cellular mechanisms that contribute to gene expression and early innate immune responses following in vivo delivery of gene-encoded antibodies and other proteins. This stems from initial observations that amino acid modifications can have a significant impact on in vivo expression of antibody. Specific areas of interest include characterization and understanding at the transcript and translation levels. Further understanding these mechanisms could have interesting implications for both infectious diseases antibody delivery as well as different gene therapies. Possible projects include:

• Characterizing transcription of gene-encoded antibodies, including comparison between in vitro and in vivo models. We are interested in understanding how the relative expression levels of recombinant and in vivo-delivered gene encoded antibodies differ. These studies will employ new nucleic acid barcoding approaches and next-generation sequencing to study transcript expression. By evaluating a series of nucleotide and codon sequence variants, we will work towards the goal of to identify nucleic acid and protein regions that impact expression.
  
  
• Characterization of ER/Golgi proteins involved with folding and secretion of antibody. Several chaperones and other proteins involved with immunoglobulin folding are upregulated specifically in plasma cells but are turned off in most other cells including B cells and non-lymphoid tissues. We will use in vitro and in vivo models to study expression of DNA-encoded ER proteins and the contribute towards protein production. These studies will use fluorescent tagging, intracellular imagine, developing models using CRISPR/Cas9 knockout/ins to further understand the contributions of each protein of interest towards protein expression.

The Sarma laboratory is interested in understanding the basic mechanisms of epigenetic gene regulation. In particular, we examine the molecular mechanisms and functional implications of RNA interactions in gene regulation and genome organization. Our recent focus has been on triplex nucleic acid structures known as R-loops, which consist of a DNA: RNA hybrid and a displaced single strand DNA. In addition to known regulatory roles, R-loops are closely linked to increased DNA damage and genome instability. To examine the function of R-loops, we have developed new technologies that allow us to identify genome-wide locations of R-loops. Our studies incorporate a combination of biochemical, cell biological and functional genomics approaches in embryonic stem cells, neural stem cells, and cancer cells. Our work relies heavily on the production and purification of various proteins that we use for interaction studies and in our high throughput sequencing technologies. Projects to which students can contribute include:

• Protein production and purification: protein-protein and protein-RNA interaction assays will be used to analyze and optimize R-loop detection using western blot or real time PCR techniques.
  
  
• Generation of stable cell lines that variably express key regulatory proteins of interest: the student will transfect mammalian cells, pick individual colonies and screen for protein expression using western blot. This project will also allow students to map R-loops using ChIP-seq, Cut-and-Run, and other cutting-edge functional genomics techniques.

Viruses are remarkably efficient in taking control of infected cells and establishing themselves in the host. While some viruses persist only for a limited period, other viruses inhabit the host continuously due to their ability to manipulate cells without triggering immune responses. Epstein-Barr Virus, or EBV, resides permanently as nuclear DNA episomes in the B cells of 90% of the world’s human population. The success of EBV infection is due to the virus’s ability to adapt its gene expression under different environmental conditions (i.e., healthy vs. immunosuppression). How EBV does this is not fully understood. Our laboratory’s goal is to test our overarching hypothesis that, over millions of years of coevolution, both host and EBV reciprocally target the other’s 3-dimensional (3D) chromatin structures in an attempt to gain control, or perhaps to achieve mutual benefit. We are now poised to test our hypothesis through two main projects. The first project investigates how EBV infection impacts the 3D structure of the host genome. Specifically, we will couple Hi-C analysis to RNA-seq studies to correlate chromatin loop changes to changes in the host’s gene expression during primary infection of B cells with EBV. The second project aims to test our hypothesis that the viral genome’s global 3D structure is an essential component of the EBV epigenome and that host cells can restrict infection by targeting the 3D viral chromatin structure. This work would add a new branch to the existing model of how epigenetics and 3D chromatin structure regulate host-viral interaction and help re-envision our understanding of the impact that viruses have on the host’s functioning genome. Specific projects in which students could participate include:

• Conducting Hi-C and RNA-seq analyses of EBV-infected and uninfected cells to correlate EBV-induced changes in 3D chromatin structure to altered gene expression
  
  
• Investigating how the altered 3D chromatin structure of EBV episomes impacts viral spread and persistence

The Tian lab studies RNA biology and post-transcriptional gene regulation using systems biology and computational biology approaches. The lab currently focuses on understanding mechanisms and consequences of alternative cleavage and polyadenylation (APA) of RNA precursors, a process by which multiple RNA isoforms are generated through alternative 3’ ends. More than half of all human protein-coding genes produce APA isoforms. Different tissues have distinct APA preferences. For example, neurons tend to express long 3’ untranslated region (UTR) isoforms, whereas blood cells typically favor short 3’UTR isoforms. The overarching questions the lab is pursuing are: 1) what are the molecular underpinnings leading to 3’UTR size variations in different cell types and conditions; and 2) how 3’UTR size differences impact mRNA metabolism and cellular differentiation. Specific projects for students include those focused on:

• APA regulation in cell proliferation, differentiation, and migration
  
  
• APA and cellular stress
  
  
• APA and protein secretion

The Villanueva lab investigates the role of the protein kinases RAF/MEK and PI3K/mTOR/S6K in melanocyte lineage specification. The MAPK and PI3K pathways are highly regulated central signaling networks that control basic processes including cell proliferation, differentiation, stress responses, metabolism and cellular transformation. In particular, we study NRAS, a poorly characterized member of the RAS GTPase family. Our goal is to identify critical NRAS effectors that are essential for melanocyte differentiation and transformation. We have mapped a critical subnetwork downstream from the convergence of PI3K and MAPK and found that a S6 kinase (S6K)-SREBP1-PPARα subnetwork is vital for cellular lipid metabolism. We are dissecting these subnetworks and their specialized functions to understand how NRAS regulates cellular metabolism and survival.

Another area of research in the lab focuses on the role of telomerase, a ribonucleoprotein complex that elongates and protects telomeres, the protective “caps” that maintain chromosome stability. Telomerase is typically not expressed in normal somatic cells, which is thought to contribute to cellular and organismal aging. Activating the expression of the telomerase catalytic subunit, TERT, makes cells “immortal”, allowing them to divide indeterminately. Telomerase expression is reactivated in over 90% of human cancers, and often correlates with mutations in the TERT promoter that inappropriately activates its expression. We have shown that depleting TERT triggers rapid cell death of melanoma cells. Nonetheless, the precise role and contribution of these non-coding mutations remain poorly understood. Our goal is to determine the mechanism whereby TERT promoter mutations alter TERT expression, and to assess the effects on cellular aging and transformation, using melanoma as a model system. Potential projects for students include:

• Investigating the key upstream and downstream effectors of NRAS
  
  
• Determining the S6K interactome in NRAS mutant cells
  
  
• Investigating the functional effects of altering canonical TERT promoter mutations on melanoma cell differentiation and survival