Justin Wheat NIH NRSA F30 Fellowship entitled "Uncovering Transcriptional Regulation of a Master Hematopoietic Transcription Factor at Single Molecule Resolution" (Sponsor, Ulrich Steidl & Robert Singer, Cell Biology and Anatomy & Structural Biology)
Jeet Biswas NIH NRSA F30 Fellowship entitled "The sequence recognition, structure and function of the IMP family of mRNA binding proteins" (Sponsor, Robert Singer, Anatomy & Structural Biology)
Ross Firestone NIH NRSA F30 Fellowship entitled "Designing Novel Anti-Cancer Therapeutics: Targeting Methionine Metabolism" (Sponsor, Vern Schramm, Biochemistry)
Ali Zahalka NIH NRSA F30 Fellowship entitled "Contributions of sympathetic signals to prostate cancer progression" (Sponsor, Paul Frenette, Cell Biology)
Sean Healton NIH NRSA F30 Fellowship entitled "Epigenetic activity of normal and cancer-associated mutant H1 linker histones" (Sponsor, Arthur Skolutchi, Cell Biology)
Cary Weiss NIH NRSA F30 Fellowship entitled "MicroRNA-22 and the microRNA-22/tet2 network as regulators of the cell fate decision in hematopoietic stem cells and in the development of myelodysplastic syndrome" (Sponsor, Keisuke Ito, Cell Biology)
Ruth Howe, NIH NRSA F30 Fellowship entitled "Characterizing the Novel Protein C15ORF65" (Sponsor, Ulrich Steidl, Cell Biology)
Marika Osterbur, NIH NRSA F30 Fellowship entitled "Extra-coding features of mRNA are essential for hERG channel function" (Sponsor, Thomas McDonald, Molecular Pharmacology)
Michael Willcockson, NIH NRSA F30 Fellowship entitled "Regulators of the erythroid terminal differentiation decision and their connection to the cell cycle" (Sponsor, Art Skoultchi, Cell Biology)
Karin Skalina, NIH NRSA F30 Fellowship entitled "Optimization of non-ablative focused ultrasound therapy for tumor immunity" (Sponsor, Chandan Guha, Pathology)
Nelson Gil, NIH NRSA F31 Fellowship entitled "The molecular basis of receptor-ligand recognition on the immunological synapse" (Sponsor, Andras Fiser, Systems & Computational Biology)
Odelya Kaufman, NIH NRSA F30 Fellowship entitled "The role of RBPMS2 in establishing oocyte polarity" (Sponsor, Florence Marlow, Developmental & Molecular Biology)
Kim Ohaegbulam, NIH NRSA F31 Fellowship entitled "Tumor expressed B7x accelerates disease and is a novel target for immunotherapy" (Sponsor, Xingxing Zang, Microbiology & Immunology)
Jennifer Schloss, NIH NRSA F30 Fellowship entitled "Use of beta cell epitopes in preventing type 1 diabetes in humanized mice" (Sponsor, Teresa DiLorenzo, Microbiology & Immunology)
Onyi Uchime, NIH NRSA F31 Fellowship entitled "Novel investigation of the mechanism of BAX modulation" (Sponsor, Evripidis Gavathiotis, Biochemistry)
Current Students Who Previously Held Individual Predoctoral Fellowships
- Philip Campbell, NIH NRSA F31 Fellowship entitled "Polarized transport in nervous system development and disease in zebrafish" (Sponsor, Florence Marlow, Developmental and Molecular Biology)
- Ujunwa Cynthia Okoye, NIH NRSA F31 Fellowship entitled "The Role Of c-MAF In Stem Cells In Leukemia" (Sponsor, Uli Steidl, Cell Biology)
- Robert Stanley, NIH NRSA F30 Fellowship entitled "Investigating the role of ARC in hematopoiesis and myeloproliferative neoplasms" (Sponsor, Ulrich Steidl, Cell Biology)
- Carlos Diaz-Balzac, NIH NRSA F31 Fellowship entitled "Identification of Novel Loci Interacting with the Kallmann Syndrome Gene Kal-1." (Sponsor, Hannes Buelow, Genetics)
Abstracts of Recent Projects
Justin Wheat - ABSTRACT: Gene expression, which encompasses a series of reactions from initial gene activation to final protein folding, is an inherently noisy process for any cell population under study. As each step in the process is subject to independent regulatory pressures, small between cell differences in the levels of these regulators can produce substantial transcriptional heterogeneity, which may then propagate into substantial functional diversity. It is therefore challenging to understand how complex multi-cellular tissues faithfully develop given the number of genes that must be coordinately expressed to establish cellular identity. Master regulatory transcription factors (TF) are the proposed solution to this teleological dilemma. These molecules have been shown to control cohorts of genes required for normal cell function, and achieving the appropriate level and stoichiometry between different TF appears to be critical for fate decisions during normal tissue development. Moreover, the deregulation in either the expression or function of these factors appears to play a substantial role in malignant transformation. TF have been extensively studied in hematopoiesis, the highly arborized differentiation network that robustly and dynamically produces a spectrum of functionally distinct blood cell populations responsible for hemostasis, gas exchange, and immune function. In order to achieve this complex cellular output, hematopoietic differentiation is postulated to occur as a series of nodal fate decisions in increasingly oligopotent stem and progenitor cell compartments (HSPC), each with distinct gene expression programs governed by TF. Understanding how HSPC achieve the appropriate dose and activity of these TF is therefore vital to our understanding of steady state blood differentiation and may expose novel therapeutic windows in hematological disease. Complicating these efforts, however, is the finding that HSPC are functionally and transcriptionally heterogeneous, which have limited the field's ability to uncover definitive regulation of TF based on ensemble measurements. This project is intended to quantify the origins of that heterogeneity with single molecule, quantitative techniques to uncover the regulation and expression of a master hematopoietic TF, PU.1. Our proposal is to (1) determine how PU.1 mRNA and protein production is dynamically changed during differentiation in single primary HSPC from mice by RNA FISH/IF and to (2) independently measure how a highly conserved cis regulatory element (URE) controls the rate, magnitude, and dynamics of PU.1 transcription. Our preliminary findings have indicated that not only is our experimental approach feasible, it has already revealed intriguing findings about PU.1 mRNA synthesis that were previously unknown. Using these tools and sophisticated analytical techniques, this proposal will provide the highest resolution, quantitative study to date of the regulation and activity of a master regulatory transcription factor in primary HSPC. We anticipate that our approach will provide novel and fundamental insight into the molecular paradigms regulating hematopoiesis and leukemogenesis.
Jeet Biswas - ABSTRACT: In humans, insulin like growth factor 2 (IGF2) mRNA binding proteins (IMPs) have been shown to be poor prognostic indicators in cancer. Work from our lab and others indicate that the two most distantly related members, ZBP1 and IMP2, accomplish this by playing drastically different roles within cells. ZBP1 (IMP1) participates in cellular organization, motility and metastasis and knockout mice are developmentally delayed and embryonic lethal. Interestingly, IMP2 knockout mice display prolonged lifespan and resistance to obesity through upregulation of mitochondrial metabolism. Work from our lab suggests that these cellular effects are mediated by the unique RNAs targets of these highly conserved and highly homologous proteins. This recognition of RNAs by IMP members is dictated by strict rules and highly conserved binding elements within the RNA target sequences. To understand how these proteins utilize their consensus sequences to guide the fate of the cell we propose a number of structural and functional studies. After determining the consensus element for IMP2 I will query the genome to identify targets of IMP2 and compare them to published ZBP1 targets. To determine how the difference in RNA preference is generated between the two proteins, I have used NMR spectroscopy to begin solving the structure of IMP2 bound to its consensus elements. By determining which amino acids of IMP2 interact with each of the binding elements, and comparing to the solved ZBP1 structure, I will understand how these proteins generate target specificity. Directed mutagenesis will then be used to interconvert the binding of each RNA binding protein. To gain mechanistic insight into how
IMP2 regulates cellular metabolism, I use my determine target sequences to study its role as a trans-acting factor for mitochondrial RNA localization. A number of studies have isolated mRNAs that are preferentially localized and translated near the surface of the mitochondria (many of which are putative IMP2 targets). Through a combination of super registration and high speed live cell imaging I hope to tease apart the individual contributions of ribosomal translocation and IMP2 towards mRNA localization onto the mitochondrial surface. By understanding if this process is a one step co-translational process or if it is a two step sequential RBP regulated process, we can better understand how translational regulation of mitochondrial proteins can regulate metabolic function, both in healthy and diseased states. I propose a multifaceted approach to understand how the IMP family (and possibly other KH domain containing RBPs) generate sequence specificity through subtle changes in the structure of its RNA recognition element. Our approach will accomplish this by determining targets which IMP2 recognizes and by understanding how these lead to a unique role in metabolic regulation. As IMP family members have been shown to be upregulated in numerous cancers and confer poor prognosis, it is likely that the role of these RBPs in oncogenesis is an important and previously unmet area for investigation.
Ross Firestone - ABSTRACT: 5´-Methylthioadenosine (MTA) is a product of AdoMet mediated polyamine synthesis. 5´-Methylthioadenosine phosphorylase (MTAP) is the sole enzyme to metabolize MTA in humans, producing adenine and methylthioribose-1-phosphate (MTR-1-P). These are salvaged in the methionine and adenine salvage pathways for resynthesis of AdoMet. Previous transition state analysis of MTAP has led to the design and synthesis of powerful transition state analogue inhibitors of MTAP. They show selective anti-cancer effects in human cancer cell lines in culture as well as in mouse human cancer xenografts. A sensitive, continuous assay for characterizing MTAP catalytic activity is needed and will be developed. We propose a new assay with an alternative substrate, 2-amino-5´-methylthioadenosine (2AMTA), to yield an intense fluorescent product that can be precisely and continuously quantified. This new technology will provide an accurate method of characterizing kinetic properties of MTAP. It is also applicable to bacterial enzymes using MTA as a substrate. We will further assess MTAP transition state analogues as anti-cancer agents. We will use a mouse model of human Familial Adenomatous Polyposis (FAP), APCMin/+, to determine the efficacy of MTAP transition state analogues as treatments for spontaneously forming cancers in immunocompetent mice. Analysis involves potential delay of disease onset and a test of disease reversal based on early and late treatments. Progression of disease analysis uses microPET live-animal imaging. Finally we will solve the transition state structure of human methionine adenosyl transferase (MAT) enzymes, with the goal of setting the foundation for future transition state analogue design. MAT2A, the cancer-cell specific MAT isoform, is upregulated in FaDu cells made resistant to MTAP transition state analogues. We hypothesize that transition state analogue inhibitors of MAT2A will work synergistically with MTAP transition state analogues and will amplify the anti-cancer effects of both therapeutics.
Ruth Howe - ABSTRACT: The uncharacterized locus C15orf65, which we previously identified as part of a translocation in Hodgkins lymphoma, encodes a small, highly conserved, 15kDa protein of completely unknown function. We generated a monoclonal antibody against the C terminus of C15orf65 and demonstrated that the protein is expressed in a cell-cycle-dependent fashion, with levels peaking in the G1/S phase of the cell cycle. Overexpression of the C15orf65 protein resulted in increased cell cycling, whereas knockdown decreased cell cycling and ablated the ability of myeloid cells to form tumors upon xenotransplantation. In myeloid and kidney cell lines, C15orf65 localized to the nucleus and to the chromatin fraction in particular, raising the consideration that it may participate in a histon-binding complex. However, its mechanism of action remains unexplored. Analysis of public microarray datasets showed C15orf65 mRNA expression in many tissues, with particularly high expression in the breast, trabecular bone osteoblasts, and hematopoietic stem and progenitor cells. C15orf65 mRNA levels are significantly upregulated in the PML-RARα subtype of acute myeloid leukemia (AML) and the RARS subtype of myelodysplastic syndrome (MDS), indicating a potential role in malignant as well as normal hematopoiesis. C15orf65 is highly conserved across the animal kingdom, especially within the vertebrate lineage, and has additional homologs outside of the animal kingdom. This degree of conservation indicates selective pressure for a conserved function, which we postulate is mediated by C15orf65's principal domain, DUF4490. The closest relative of DUF4490 for which both structure and function have been determined is the histone-binding Tudor domain of Sgf29. However, the structure and function of DUF4490 are as yet completely uncharacterized and thus represent a potentially informative target for structural determination. The combination of C15orf65's conservation and localization with a cell cycling phenotype and involvement in multiple hematopoietic malignancies suggests that C15orf65 may be a previously undescribed epigenetic regulator with a particular role in governing cell cycling in hematopoietic cells. We propose to 1) Identify binding partners for C15orf65 to characterize its pathway interactions; 2) Determine crystal and NMR structures for C15orf65; and 3) Delineate the functional role of C15orf65 in hematopoiesis using a newly generated conditional knockout model for the murine homolog of C15orf65, Gm5918. Our investigation of C15orf65 will provide insight into the biophysical, biochemical, and functional properties of this novel gene, and may also lead us to a new pathway for cell cycle regulation and identification of a novel histone binding domain.
Nelson Gil - ABSTRACT: Immunoglobulin superfamily (IgSF) proteins play important roles in protecting the human body from infectious diseases and tumorigenesis; on the other hand, their malfunction can lead to automimmune diseases. Because IgSF proteins function in immunity by specific trans-cellular noncovalent interactions between antigen-presenting cells and T cells, a molecular-level understanding of IgSF:IgSF binding interfaces would be of great aid to the design of novel immunomodulatory therapeutics. Excluding antibodies, the human proteome currently contains 477 extracellular IgSF proteins, of which only a quarter have documented binding partners. Given the volume of unexplored extracellular IgSF:IgSF interactions, a purely wet-lab approach to completing the IgSF interactome—the network of all known IgSF:IgSF interactions—would be prohibitively expensive. On the other hand, current computational molecular interaction prediction approaches are unsuitable for interactome prediction as they are either computationally intractable when attempted on large molecules such as proteins due to their inability to sample the entire conformational space or produce inaccurate results due to their inability to distinguish binding from non-binding protein pairs. Our goal is to develop a computational method that can be used to identify interacting IgSF receptor-ligand pairs. To accomplish this goal, we will first combine structural similarity-based and sequence-based approaches along with hidden Markov model profile-based functional sub-classification of the IgSF to identify the binding interfaces of IgSF proteins. Next, using molecular dynamics simulations, we will sample the potential energy landscape of target receptor IgSF protein binding interfaces and design an optimal complementary ligand protein interface, which will then be evaluated to fit existing IgSF proteins. We hypothesize that each receptor interface can be characterized by a unique spatial fingerprint—an extended pharmacophore which we will call the residue-specific functional atom field (rsFAF)—which represents the energetically favorable positions of key functional atoms and can be used to identify cognate ligands. Our methods will be validated using a test set of known IgSF:IgSF complexes with available crystallographic structures.