The Department of Biological Sciences
Title, Principal Investigator and Abstract for the 2015 A Look Ahead UMBC Faculty Poster Session
Developing a Rat Model of Lethal Human Prostate Cancer
Prostate cancer is the second leading cause of cancer-related death in men in the United States. Although organ-confined prostate cancer is curable, metastatic disease is nearly always fatal. The rat is a promising animal model for human cancer because of its large litter size, and larger body size compared to mouse, making it more amenable to study by instrumentation and drug candidate testing (Mordenti, 1986). Depending on the specific drug candidates being tested, different model organisms have distinct advantages with respect to the similarity of their cytochrome P450 (CYP) activity to human. Therefore, we endeavored to develop a rat model that would recapitulate the natural history of human prostate cancer including the lethal metastatic phenotype. In this work, we have focused on the oncogene MYC and the tumor suppressor P53.
Beyond the regulon: reconstructing the SOS response of the human gut microbiome
Metagenomic projects provide a unique window into the genetic composition of microbial communities. To date, metagenomic analyses have focused primarily on studying the composition of microbial populations and inferring the species-wise distribution of shared metabolic pathways. Here we analyze how high-quality metagenomic data can be leveraged to infer the composition of transcriptional regulatory networks through a combination of in silico and in vitro methods. Using the bacterial SOS response to DNA damage as a case example, we analyze human gut microbiome data to determine the composition of the SOS meta-regulon of Gram-positive bacteria in a natural context. Our analysis shows that the existing knowledgebase on regulatory networks and reference genomes can be effectively leveraged to mine meta-genomic data and reconstruct multi-species regulatory networks. This approach allows us to identify the core functional elements of the human gut SOS meta-regulon, governed by the transcriptional repressor LexA. As inferred from previous studies, which deemphasize the role of DNA repair in the SOS response, error-prone polymerases appear to have a predominant role in the human gut SOS response. The analysis of enrichment in LexA-binding sites among clusters of orthologous genes allows us to identify putative novel SOS protein clusters involved in cell wall biogenesis, chromosome partitioning and restriction modification. We also show how in vitro validation of high-confidence LexA-binding sites can generate insights into specific SOS network components among unculturable organisms. The methodology implemented in this work can be applied to other metagenomic datasets and transcriptional systems, potentially providing the means to compare regulatory networks across metagenomes. The use of metagenomic data to analyze transcriptional regulatory networks provides a realistic snapshot of these systems in their natural context and allows probing at their extended composition in non-culturable organisms, yielding insights into their interconnection and into the overall structure of transcriptional systems in microbiomes.
In vivo analysis of the frequency of errors during protein synthesis: some errors are more frequent because they involve mismatches that mimic Watson-Crick base pairing
Our laboratory has been interested in understanding the mechanisms responsible for errors during protein synthesis and have developed a variety of reporter systems to measure their frequency in vivo. Translation errors include misreading, where one amino acid replaces another, nonsense readthrough, where an amino acid is incorporated at a nonsense codon, and frameshifting, where the translating ribosome shifts to a different reading frame while continuing to translate. Recently, we have created reporters to measure all possible errors by single tRNAs to determine the error landscape. We have found repeatedly that tRNAs make errors much more frequently at a small subset of codons. An exhaustive analysis of errors by four tRNAs has shown a common theme among these more frequent errors. The errors invariably involve a mismatched base pair of one of three types: non-canonical U-U, U-C or U-G pairs (where the first base is in the tRNA and the second in the mRNA). These base pairs are the only known non-Watson/Crick base pairs that can adopt a geometry similar to Watson/Crick pairs although to do so they must adopt unconventional tautomeric forms, enol for keto or imino for amino. The rareness of these tautomers may explain the relatively low frequency of these decoding events, suggesting that the ribosome may be categorically unable to eliminate these errors. These results call for a rethinking of the process of misreading errors during translation and suggest that these errors are more similar to replication or transcription errors than formerly believed.
Functional Genomics Identifies Novel Genes Required for Lignocellulose Degradation in Saprophytic Bacteria
Despite the substantial amount of lignocellulose that is annually recycled in the environment, estimated at 100 billion tons, surprisingly few microbial species can depolymerize lignocellulosic polysaccharides and use the resulting sugars for carbon and energy. Work in the Gardner laboratory uses systems biology approaches to understand the mechanisms and regulation of how saprophytic bacteria are able to detect lignocellulose and use it as a nutrient.
Functional characterization of novel histone methyltransferases in budding yeast
Methylation of histone lysine residues is a key regulator of chromatin structure and accessibility. In budding yeast, the most extensively characterized methylation events are catalyzed by the conserved methyltransferases Set1, Set2 and Dot1. However, the activity of other lysine methyltransferases remain largely unexplored. We identified a novel histone methyltransferase in budding yeast, Set5, which catalyzes uncharacterized methylation events on the histone H4 tail, and determined that it plays a role in repressing gene expression near telomeres and retrotranspsosons. We are further interested in defining the substrate specificity and biological role for other candidate methyltransferases in yeast. To this end, we have preliminary data indicating that the completely uncharacterized enzyme Set4 possesses important biological functions and may be a histone methyltransferase that regulates the cell’s ability to respond to stress.
Protein Domain Landscapes for Discovering Critical Cancer Somatic Mutations
The fight against cancer has been hindered by its highly heterogeneous nature. Recent genome-wide sequencing studies have shown that individual malignancies contain many mutations that range from those commonly found in tumor genomes to rare cancer somatic mutations present only in a small fraction of lesions. For instance, the genome of a colorectal cancer in one patient can have somewhere between 50 to 100 somatic mutations, but might share only 2 or 3 mutated genes with colorectal tumor genomes from other patients. Somatic mutations that are frequently found in tumor genomes often play a significant role in tumor development and are thus classified as cancer driver mutations. However, efforts to correlate somatic mutations found in one or few individual tumor genomes with critical functional roles in tumor development have so far been unsuccessful. We have designed a new approach to analyze cancer somatic mutations from cancer patients based on aggregation of mutational data at the protein domain level. Our approach creates a framework for leveraging structural genomics and evolution into the analysis of somatic cancer mutations. Our data suggest that incorporating information about classification of proteins and protein sites, in particular at the protein domain level, results on discover of clusters of cancer somatic mutations leading to new hypothesis about cancer development and maintenance .
ACE Inhibition Attenuates Frailty in Drosophila
Frailty, an indicator of age-related decline, is known to be a predictor of mortality in humans. The renin-angiotensin system (RAS) has been implicated as a contributor to frailty. Although a standardized procedure for creating a frailty index in humans has been described, no “frail fly” model has been developed. The purpose of this study was to first characterize age-related changes in flies that are comparable with indicators of frailty in mammals in order to establish a “frail fly” model and secondly, to test the effect of RAS blockade on such indices. Determinants of frailty in Drosophila were established by assessing climbing speed, endurance, and strength in three genetic lines from the Drosophila Genetic Reference Panel (DGRP); each criterion had a designated cutoff point. The prevalence of frailty was determined by having the three criteria scores in the lowest quartile. Further, we tested effects of Lisinopril, an Angiotensin-Converting Enzyme (ACE) inhibitor, on development and progression of age-related decline of physical function. We show significant decline in climbing speed, endurance, and strength in RAL_229. Prevalence of frailty in RAL_229, 73, and 304 increased by 41.5, 14.5, and 15 percent, respectively, with age. The development of frailty in line 229 was associated with the highest mortality and shortest lifespan. In the frail fly line, treatment with Lisinopril significantly increased survivorship, reduced prevalence of frailty by 14.3 percent, and significantly improved climbing speed, endurance, and strength in an age-dependent manner. Blockade of RAS improved health and life span in the frail fly model.
Nucleolar stress during repression of ribosomal protein synthesis
Mutations in or haploinsufficiency for genes for ribosomal proteins (r-proteins) or biogenesis factors increase the propensity for congenital anomalies in organisms ranging from Drosophila and Zebrafish to humans, including hypodevelopment, cancer, hematological diseases, and growth retardation. These conditions are called ribosomopathies and include Diamond Black Fan Anemia, X-linked dyskeratosis congenita, Shwachman Diamond syndrome, and other cancers.While the regulation of normal ribosome biogenesis via the TOR and ras phosphorylation cascades in the TOR and ras pathways is understood fairly well, we have only a very limited understanding of why mutations disturbing normal of ribosome synthesis (“nucleolar stress”) lead to congenital diseases. Mechanisms that are both dependent and independent of the tumor suppressor p53 have been implicated.
We are using Saccharomyces cerevisiae as a model for p53-independent cellular stress caused by disruption of ribosome biogenesis. Analysis cell cycle, cell morphology, budding patterns and ribosome stability in response to repression of 54 different ribosomal protein genes show interesting correlations with ribosome structure. We are now investigating the molecular mechanisms behind the effects of ribosome biogenesis on cellular stress
Daily and Seasonal Song Patterning in Grasshopper Sparrows (Ammodramus savannarum).
We examined the daily and seasonal pattern of singing in Grasshopper Sparrows, a species that produces two classes of song: “buzz” (primary) song and “warble” (sustained) song. We counted the number of buzz, warble, and combined songs across breeding cycles throughout the 2011 and 2012 breeding seasons and found regular transitions between the two song types during the course of the day and the breeding cycle in paired males. We operated twenty-five autonomous recording units (ARUs) in male territories from 0400 – 1000 EDT (6 hours/day) during May – August. An additional nine units were operated for 18 hours/day (0400 – 2200 EDT) to record all songs sung by territorial sparrows throughout the course of the day. Digital sound files were analyzed using the Syrinx sound analysis software. In addition to identifying the major singing peak from 0400 – 0800, we found a shorter, though substantial, singing peak around 2030 – 2100 (sunset). We also found that birds gradually transitioned from buzz to warble song throughout each breeding cycle, with warble song production peaking during the nestling stage. Song type production showed a daily pattern as well, with warble song produced primarily during the first hour of the morning and during the evening singing peak.
Investigation of PHT4;6-mediated defense signaling in Arabidopsis thaliana
Plants are frequently challenged by a variety of pathogens that cause diseases and yield loss worldwide. A thorough understanding of plant defense mechanisms is a key to the success of disease intervention and treatment. Plants have developed sophisticated signaling pathways that respond to many environmental stresses including insults from pathogens. Salicylic acid (SA) is a small phenolic compound that acts as a hormone to transduce defense signaling in plants. Previously we identified a mutant disrupted in the PHOSPHATE TRANSPORTER 4;1 (PHT4;1) gene that negatively regulates plant defense. PHT4;1 belongs to a six-member gene family, of which the most distally related member is PHT4;6. A loss of function mutation in PHT4;6 confers enhanced disease resistance to P. syringae infection and cell death phenotypes. However, how PHT4;6 affects disease resistance has not been well understood in Arabidopsis. Here we report a study to further elucidate the function of PHT4;6 in defense, using genetic and molecular approaches. Our results indicate that PHT4;6 possibly acts through an SA-independent pathway in defense regulation.
The circadian clock component LUX ARRHYTHMO regulates Arabidopsis defense through salicylic acid
Recent studies showed that two morning clock genes regulate Arabidopsis defense independently of the key defense signaling mediated by salicylic acid (SA). To further understand the defense role of the circadian clock, we tested a mutant impaired in the evening clock gene LUX ARRHYTHMO in defense responses. We found that the lux-1 mutant was compromised to both basal and R-gene mediated defense against Pseudomonas syringae and expression of the LUX gene was suppressed by P. syringae. We also found that lux-1 had transiently reduced SA accumulation after infection with a virulent P. syringae strain. Consistent with this result, the double mutant acd6-1lux-1 displayed suppression on dwarfism, cell death, and constitutive defense phenotypes, compared with acd6-1, which has been used as a convenient genetic tool in gauging the change of defense levels. We further found that two downstream targets of LUX also could modulate resistance to P. syringae via the SA pathway. Together our results showed that LUX regulates Arabidopsis defense, possibly through affecting SA signaling. These data further support crosstalk between the circadian clock and plant innate immunity and also reveal different molecular mechanisms underlying clock-defense crosstalk.
Differential roles of two homologous cyclin-dependent kinase inhibitor genes in regulating cell cycle and innate immunity in Arabidopsis
Precise cell cycle control is critical for plant development and responses to pathogen invasion. Two cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and its close homolog SIM-RELATED 1 (SMR1) were recently shown to be involved in Arabidopsis defense regulation based on the phenotypes conferred by a sim-1smr1-1 mutant. However whether these two genes play differential roles in cell cycle and defense control is unknown. In this report, we examined plants impaired in SIM and/or SMR1 genes for cell cycle and defense phenotypes. Our data show that while SIM plays a major role in promoting trichome development, a process tightly associated with endoreduplication, SMR1 has stronger effect on non-trichome leaf cell ploidy. In addition, we found that a smr1-1 but not a sim-1 mutant was more susceptible to P. syringae and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, the smr1-1 mutant but not the sim-1 mutant partially suppressed dwarfism, high accumulation of SA, and cell death phenotypes exhibited by the acd6-1 mutant, a convenient genetic tool that has been used to gauge the change of defense levels. Thus SMR1 but not SIM functions at least partly through the SA pathway in defense control. Furthermore cell ploidy analysis by flow cytometry with an SA activator or with SA mutants revealed that SA signaling is necessary and sufficient to disrupt cell cycle progression. Interestingly, a mutant with disrupted CYCD3 genes and increased endoreduplication also led to compromised disease resistance to P. syringae. Together, this study further support the importance of cell cycle control during host-pathogen interactions and also reveals differential roles of two homologous CKIs in regulating cell cycle progression and innate immunity in Arabidopsis.
The transcription factor Nrf2 enhances myeloid-derived suppressor cell-mediated immune suppression and tumor progression
Rosenberg Lab (Daniel Beury)
Tumor-induced myeloid-derived suppressor cells (MDSC) contribute to immune suppression in tumor-bearing individuals and are a major obstacle to effective immunotherapy. Reactive oxygen species (ROS) are one of the mechanisms used by MDSC to suppress T cell activation. Although ROS are toxic to most cells, MDSC are not negatively impacted by their production of ROS. Nuclear factor erythroid derived 2-like 2 (Nrf2) is a transcription factor that regulates a battery of genes which attenuate oxidative stress and therefore we hypothesized that MDSC resistance to ROS may be due to their up-regulation of Nrf2. To test this hypothesis, we utilized BALB/c and C57BL/6 mice bearing 4T1 mammary carcinoma and MC38 colon carcinoma, respectively. MDSC from the peripheral blood of Nrf2-/- mice with 4T1-tumors were more oxidatively stressed and apoptotic, produced less H2O2, and were less suppressive than MDSC from wild type mice, indicating that Nrf2 sustains MDSC survival and suppressive activity. Primary tumors and levels of MDSC were similar in Nrf2-/- and wild type mice, but Nrf2-/- mice survived longer. Since Nrf2-/- MDSC were more apoptotic, but Nrf2-/- and wild type tumor-bearing mice had similar levels of MDSC, we hypothesized that MDSC differentiate more rapidly from Nrf2-/- than from wild type progenitor cells. This hypothesis was confirmed because Nrf2-/- bone marrow cells cultured with IL-6 and GM-CSF, produced more MDSC than similar cultures of wild type progenitor cells. These data demonstrate that Nrf2 facilitates tumor progression by increasing MDSC-mediated suppression and by delaying MDSC turnover, and identify Nrf2 as a potential therapeutic target for reducing tumor-induced immune suppression and enhancing cancer immunotherapy.
High Mobility Group Box Protein 1 Enhances Immune Suppression By Facilitating The Differentiation And Suppressive Activity of Myeloid-Derived Suppressor Cells
Rosenberg Lab (Katherine Parker)
Chronic inflammation frequently precedes malignant transformation and is associated with tumor progression. Chronic inflammation also promotes the expansion of immune suppressive cells that aide in the process of tumor escape. A subset of these immune suppressive cells are myeloid-derived suppressor cells (MDSC) which are elevated in most individuals with cancer where their accumulation and suppressive activity are driven by inflammation. Therefore inflammation can promote tumor progression and impede anti-tumor immunity through the induction of MDSC. The alarmin High Mobility Group Box Protein I (HMGB1) is pro-inflammatory and is a binding partner, inducer, and/or chaperone for many of the pro-inflammatory molecules that drive MDSC. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. In vitro and in vivo experiments have found that HMGB1 drives MDSC expansion from bone marrow progenitor cells as well as MDSC accumulation in tumor-bearing mice. HMGB1 activates MDSC through NF-κB and facilitates several of the immune suppressive mechanisms used by MDSC to inhibit anti-tumor immunity. Neutralization of HMGB1 impedes cross-talk between MDSC and macrophages by decreasing MDSC production of IL-10, and increases the expression of L-selectin on circulating T cells. Collectively, these results demonstrate that HMGB1, which is ubiquitously present in the tumor microenvironment, is a significant contributor to tumor-induced immune suppression.
A Soluble Form of CD80 Enhances Anti-tumor Immunity by Neutralizing Programmed Death Ligand-1 and Costimulating through CD28
Rosenberg Lab (Lucas Horn)
Tumor cells employ various methods of immune suppression to overcome anti-tumor immunity. One such method is that of the integral membrane protein programmed death ligand-1 (PDL1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD1) on T cells. Treatment of PDL1+ tumor cells with a soluble form of the costimulatory molecule CD80 (CD80-Fc) prevented PDL1-mediated immune suppression by binding PDL1 on tumor cells and blocking it from interacting with PD1 resulting in restored T cell activation. Additionally, CD80-Fc maintained activation of T cells more effectively than either PD1 or PDL1 blocking monoclonal antibodies. Studies in which costimulation through CD28 was prevented by blocking antibodies demonstrated that in addition to inhibiting PD1 pathway suppression, CD80-Fc simultaneously facilitated T cell activation and IFNγ production by costimulating through CD28. Because CTLA-4 is also a receptor for CD80, CD80-Fc has the potential to bind and deliver inhibitory signals into T cells via CTLA-4. However, inclusion of CTLA-4 blocking antibody in co-cultures of human PBMC and PDL1+ C8161 human melanoma cells did not increase T cell production of IFNγ, suggesting that either CD80-Fc is not signaling through CTLA-4 or the signaling is negligible. To determine if CD80-Fc is efficacious in vivo, CT26 tumor-bearing mice were administered three injections of either CD80-Fc, anti-PDL1 mAb, or an IgG control antibody. CD80-Fc treated mice exhibited delayed tumor progression and increased survival, and their tumors had many more tumor infiltrating CD3+ cells as compared to anti-PDL1 or IgG treated mice. Through the dual functions of blocking PDL1 and costimulation, CD80-Fc may be more effective than PD1 or PDL1 antibody therapy for overcoming PDL1-mediated suppression and promoting anti-tumor immunity.
Myeloid-derived suppressor cells inhibit immunological synapse by reducing ICAM-1 on antigen presenting cells and LFA-1 on T cells
Myeloid derived suppressor cells (MDSC) play a crucial role in tumor progression by inhibiting the innate and adaptive immune systems. During adaptive immune responses, T lymphocytes recognize antigenic peptides presented by MHC molecules on antigen presenting cells (APCs). This recognition results in the formation of an immunological synapse (IS) at the T cell/APC interface, which is crucial for T cell activation. The interaction between an intracellular adhesion molecule-1 (ICAM-1) and leukocyte function associated antigen-1 (LFA-1) precedes mature synapse formation and facilitates signaling events that promote T cell activation. To investigate if synapse formation is defective in tumor-bearing mice, splenocytes from BALB/c tumor-free and 4T1 mammary carcinoma tumor-bearing mice were stained with fluorescently-coupled antibodies to ICAM-1, LFA-1, CD11b, Gr1, and CD3, and were analyzed by flow cytometry. Macrophages and T cells from tumor-bearing mice had reduced expression of ICAM-1 and LFA-1, respectively. Because tumor-bearing mice have increased levels of MDSC as compared to tumor-free mice, MDSC may decrease expression of ICAM-1 and LFA-1. Reduced levels of these molecules may result in a defective immunological synapse and inefficient activation of T-cells. To test this hypothesis, macrophages were co-cultured with MDSC in vitro and ICAM-1 levels of macrophages were assessed. Macrophages co-cultured with MDSC had significantly reduced ICAM-1 expression. Therefore, MDSC down-regulate ICAM-1 levels on macrophages, decreasing the ability of these APC to form functional synapses to activate T cells.
Identifying new mechanisms of JAK/STAT signaling regulation in cell motility and stem cell maintenance
The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is well-known for roles in stem cell maintenance, cell movement, and metastasis. Since the molecular components of the pathway are well-conserved, we utilize fruit flies – Drosophila melanogaster –to discover new regulatory components of signaling. We focus on three adult cell types: motile border cells of the ovary, somatic stem cells of the testis, and phagocytic blood cells in our studies. STAT signaling is activated by a diffusible cytokine molecule, and we are exploring this initial activation through genetic, imaging, and mathematical modeling strategies. We are taking advantage of genetic screens to identify new regulators of JAK/STAT signaling and cell migration. Our data suggest that a chromatin remodeling enzyme has temporally distinct roles in this process. Through genetic analysis, we also identified a novel regulator of STAT signaling, called Apontic (APT). Apontic acts as a feedback regulator in a minimal genetic circuit that defines motile cells. Our recent work supports the idea that this inhibitor is also important in stem cell maintenance.