Browsing by Subject "Gene Expression"
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Item B Cell Signaling and Bioinformatics: Revealing Components of the MHC Class II Antigen Processing and Presentation Pathway(2005-05-11) Lee, Jamie Ann; Scheuermann, Richard H.Stimulation of mature B lymphocytes by extracellular ligands induces phenotypic changes through complex signal transduction pathways. Gene expression is altered as a result of these changes and re-programs the cell to undergo differentiation, activation, effecter function, anergy, and/or apoptosis. Gene expression microarrays are used to determine expression levels of a large number (tens of thousands) of genes simultaneously, resulting in a gene expression profile of the experimental sample. Microarray data must be appended with biological information in order to be interesting, and this field of microarray bioinformatics is rapidly expanding. These studies prompted the development of a bioinformatics tool termed CLASSIFI (Cluster Assignment for Biological Inference), which identifies statistically significant co-clustering of genes with similar Gene Ontology annotation within microarray gene clusters. CLASSIFI was used to analyze microarray results from two B cell projects from the Alliance for Cellular Signaling (AfCS): 1) the BAFF/CD40L project, which evaluates the effects of BAFF and CD40L on primary mouse B cells in long-term cultures, and 2) the B cell single ligand screen project, which evaluates the effects of 32 single ligands on primary mouse B cells in short-term cultures. CLASSIFI was able to identify significant overrepresentation of related genes within gene clusters for both of these data sets and facilitates hypothesis generation as to the biological process affected by a specific ligand. As CLASSIFI is strictly a statistical tool that aids in hypothesis generation, experimental validation of hypotheses was performed. The B cell single ligand screen microarray and CLASSIFI analysis followed by experimental validation revealed a biological process specific to B cell antigen receptor stimulation but not LPS or CD40L stimulation - antigen processing and presentation - and provides the groundwork for new discoveries in this field. As a result, several putative components were identified that are not currently known to play a role in antigen processing and presentation in B cells.Item Brain-Region-Specific Contributions of FOXP1 to Autism and Intellectual Disability Phenotypes(2017-08-11) Araujo, Daniel John; Eisch, Amelia J.; Konopka, Genevieve; Powell, Craig M.; Volk, Lenora J.; Wu, Jiang I.Mutations and deletions in the transcription factor FOXP1 are causative for severe forms of autism spectrum disorder (ASD) that are often comorbid with intellectual disability (ID). FOXP1 is enriched throughout the brain, with strong expression in the pyramidal neurons of the neocortex, the CA1/CA2 subfields of the hippocampus, and the medium spiny neurons of the striatum. Understanding the role that FOXP1 plays within these brain regions could allow for management of ASD and ID symptoms. This doctoral dissertation leverages multidisciplinary techniques and Foxp1 mutant mouse models to ascertain the role of Foxp1 in the brain and its contribution to specific ASD- and ID-relevant phenotypes. In the first chapter of this dissertation, I review the literature on the characteristics, demographics, and shared genetic underpinnings of ASD and ID and I review the work linking FOXP1 to these disorders. Afterwards, I describe the regional transcriptome regulated by Foxp1 within the brain and I correlate alterations in the gene expression profile of the striatum with deficits in communication (Chapter 2). Briefly, I utilized RNA-sequencing performed on Foxp1 heterozygous knockout animals to uncover the genes regulated by Foxp1 within the neocortex, hippocampus, and striatum. I also recorded the early postnatal ultrasonic vocalizations (USVs) of these animals and I was able to correlate changes in the properties of striatal medium spiny neurons with deficits in USV production. Next, I move onto using a Foxp1 conditional knockout (Foxp1cKO) mouse model to ascertain the contributions of Foxp1 in the neocortex and the hippocampus to ASD and ID-related behaviors (Chapter 3). In brief, I show that total loss of Foxp1 in the pyramidal neurons of the neocortex and the CA1/2 hippocampal subfields results in social communication deficits as well as hyperactivity and anxiety-like behaviors. I also show that Foxp1cKO mice display gross impairments in hippocampal-based spatial-learning tasks and I correlate these deficits with alterations in the expression of genes involved in hippocampal physiology and synaptic plasticity. In my final chapter (Chapter 4), I consider the implications that these data have on our understanding of the role that Foxp1 plays within the brain and I suggest research strategies to answer the new questions that my findings have generated. I also discuss the implications that this research has on our understanding of ASD and ID pathophysiology in general and I recommend future directions for work focused on managing these disorders.Item Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells(2007-08-08) Senitko, Annette Nelson; Lynch, Kristen W.Alternative splicing is a common mechanism for regulating gene expression in eukaryotic cells. This process of differentially including or excluding variable exons provides a means for increasing proteome complexity. Alternative gene splicing occurs in a cell specific manner and may be influenced by changes in the extracellular environment. Despite the importance of this method for regulating gene expression, little is known about the factors involved in regulating its function. The T cell tyrosine phosphatase CD45 provides a valuable model for investigating the factors involved in regulating alternative splicing. The CD45 gene contains three variable exons whose splicing is regulated in response to T cell activation. Studies of this gene have revealed the presence of an exonic silencer sequence within variable exon 4 that is capable of influencing exon skipping under both resting and stimulated conditions. Biochemical assays have shown that the regulatory protein hnRNP L binds to this silencer sequence and results in basal exon repression during resting conditions and undergoes modifications which further influence exon skipping upon stimulation. Furthermore, in vitro assays indicate that upon stimulation, an additional regulatory protein, PSF, binds to the regulatory complex associated with the silencer sequence. Although these studies have provided novel information regarding the regulation of splicing, biochemical assays are unable to fully mimic the signaling pathways inside a cell, thus creating a need for a cell culture system. A Jurkat derived cell line, JSL1 cells, has been identified as being able to recapitulate the signal induced alternative splicing of the CD45 gene as seen in primary human T cells. This cell line presents a cell based system for evaluating the factors involved in splicing. However, in order to conduct in vivo experiments one must be able to modify protein expression. JSL1 cells present limitations due to difficulties in being able to alter protein expression. A strong promoter, EF1-α, has been employed to drive the expression of candidate proteins in JSL1 cells. Transient transfections and stable cell lines expressing cDNAs driven by this promoter have shown little if any overexpression of candidate proteins normally expressed at high levels within the cell; however, significant overexpression has been achieved with the transfection of at least one protein that exists at a lower concentration. Initial experiments indicate that stably expressed flag-tagged proteins, driven by the EF1-α promoter, may be easily purified from JSL1 cells during resting and stimulated conditions and analyzed. Such data suggests that this promoter may afford more flexibility in altering and analyzing protein expression in JSL1 cells, thereby facilitating the investigation of signaling pathways involved in regulating alternative splicing. Furthermore, strategies for regulating protein expression, through the use of a Tet-suppressor system, are in initial stages of being developed and hold the potential for providing an additional tool for evaluating the factors involved in regulating alternative splicing.Item Cohesin Promotes the Myelination Transcriptional Program in Oligodendrocytes(December 2021) Cheng, Ningyan; O'Donnell, Kathryn A.; Buszczak, Michael; Olson, Eric N.; Yu, HongtaoThe cohesin complex is crucial for sister-chromatid cohesion and chromatin spatial organization in the interphase nucleus. Cohesin-extruded DNA loops have regulatory functions in gene expression. Mutations of cohesin subunits and regulators cause human developmental diseases termed cohesinopathies. The vertebrate cohesin consists of SMC1, SMC3, RAD21, and either STAG1 or STAG2. STAG1-cohesin and STAG2-cohesin are redundant in sister-chromatid cohesion, but appear to exert specific functions in gene regulation. How they achieve their functions in gene expression is poorly understood. I characterized the outcomes of Stag2 loss in the mouse nervous system. Conditional knockout (CKO) of Stag2 in the nervous system causes severe growth retardation, neurological defects, and premature death, in part due to insufficient myelination of nerve fibers. Expression profiling reveals that myelination-related genes are downregulated in oligodendrocytes of Stag2 CKO mice. Chromatin conformational capture experiments (Hi-C) reveal that Stag2-deficient oligodendrocytes contain fewer DNA loops than wild-type cells do. In particular, promoter-anchored DNA loops at downregulated genes are significantly reduced by Stag2 loss. Interestingly, downregulated genes exhibit promoter-anchored "stripes", indicative of strong loop extrusion. We propose that STAG2-cohesin generated promoter-anchored loops at myelination-promoting genes are critical for the proper gene expression during oligodendrocyte differentiation and brain development. Our study implicates defective myelination as a contributing factor to cohesinopathy and establishes oligodendrocytes as a relevant cell type to explore the mechanism by which cohesin regulates transcription.Item Expression Analysis of the Regenerating Utricle Sensory Epithelia : From Macroarrays to Parsing Pathways(2005-05-03) Hawkins, Raymond David; Lovett, MichaelI have used a human transcription factor microarray developed in the laboratory of Dr. Michael Lovett to study gene expression differences in the chicken inner ear sensory epithelia. In the initial study, the sensory epithelium from the utricle was compared to that of the cochlea. The purpose of this study was to identify gene expression differences between these two organs. The sensory epithelium from each organ is made up of hair cells and supporting cells. These hair cells are necessary for the detection of sound in the cochlea and for the detection of movement and acceleration in the utricle. The chicken sensory epithelia is of great research interest as it possesses the ability to fully regenerate hair cells that are damaged, whereas mammalian epithelia, once damaged, cannot regenerate. These two organs were compared because the utricle is in a constant state of hair cell turnover, and the cochlea remains quiescent, unless damaged. In order to carry out such microarray expression studies on a small number of cells, between 30,000-50,000 cells, a micro-cDNA amplification method, developed in the lab, was implemented and is described here as well. The experiments were carried out via cross-species hybridizations, and subsequently, a number of genes were validated by quantitative PCR and in situ analysis. Additionally, a library subtraction was used to identify additional genes expressed in the utricle sensory epithelia. In a second microarray expression study, the utricle sensory epithelia was damaged by two independent methods and allowed to recover for various time points for expression profiling on the same transcription factor array. The first method of damage was by laser microbeam to ablate the hair cells such that they die almost instantly. The second method of damage was using ototoxic antibiotics. In each time course, the time points were compared to time matched control epithelia (undamaged). The analysis of this data reveals some very important signaling cascades and developmental pathways involved in hair cell regeneration. Finally, in an effort to functionally validate many of the genes identified during regeneration, gene transcripts were targeted by RNA interference to reduce the expression level and determine the effect on hair cell proliferation. Through this method, several genes were identified to reduce proliferation. Additionally, these experiments were profiled as a means for networking genes into pathways by identifying putative downstream targets in the expression data. An intersection of genes downregulated following inhibitory experiments reveals how several genes potentially lie downstream of one another and form a pathway containing some common regulatory elements.Item Histone Deacetylase 7 and Transcriptional Regulatory Networks of the Vascular Endothelium(2010-05-14) Young, Bryan Daniel; Olson, Eric N.Cells respond to stimuli in part through the modulation of gene expression. Signal transduction from the environment to the nucleus culminates in the activation of factors that modify chromatin structure to either facilitate or inhibit gene transcription. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two such classes of enzymes that regulate the epigenetic code. Their opposing actions – to activate transcription by histone acetylation and to inhibit transcription by deacetylation – are tightly regulated to coordinate the vast gene programs required for cellular growth and differentiation. The class II HDACs are restricted in their expression patterns, and each have unique developmental and physiological functions. The studies described here focus on HDAC7, a class II HDAC that is expressed in vascular endothelial cells and whose function is essential for the maintenance of vascular integrity during embryogenesis. Mice lacking HDAC7 die by e11.5 with complex cardiovascular malformations including endothelial, vascular smooth muscle, and myocardial defects. By generating HDAC7 conditional knockout mice, it was observed that all of these defects are recapitulated in mice bearing an endothelial-specific deletion of HDAC7, but no defects are observed upon deletion of HDAC7 in the other cell types that were affected in the HDAC7 nulls. This in vivo evidence demonstrated that HDAC7 acts cell autonomously to maintain normal vascular development, and lead to the identification of the genetic abnormalities and mechanism leading to cardiovascular failure in the HDAC7 knockout. Further, this work begins the investigation of HDAC7 in adult vascular physiology, the findings of which will reveal new mechanisms whereby the vasculature responds to stress signals or disease. To this end, methods have been developed for the deletion of HDAC7 in the adult mouse using an inducible cre recombinase system together with the HDAC7 conditional allele. Additionally, these studies present progress toward the identification of the enhancer elements driving the endothelial-specific expression pattern of HDAC7. Detailed characterization of this enhancer is likely to implicate new signaling pathways as being involved in the genetic regulation of vascular development and maintenance. Finally, this work investigates the role of microRNAmediated gene silencing in the vascular system by identifying microRNAs involved in MEF2-dependent signaling in endothelial cells.Item Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming(2019-03-06) Fernandez-Perez, Antonio; Zhang, Chun-Li; Cleaver, Ondine; Olson, Eric N.; Munshi, NikhilDirect reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.Item Regulation of Insulin and CHOP Gene Expression in Pancreatic Beta Cells(2009-01-14) Shao, Chunli; Cobb, Melanie H.Insulin is a major hormone in maintaining glucose homeostasis. It is essential to understand the mechanisms by which insulin gene expression is regulated in pancreatic beta cells. In addition to examining histone modifications on the insulin gene promoter, I focused on the effect of MafA modification on insulin expression. MafA is a transcriptional activator of the insulin gene via binding to the RIPE3b/C1 (rat insulin promoter element 3b) element. Mutagenesis showed that MafA was post-translationally modified by SUMO-1/2 (small ubiquitin-like modifier) mainly at lysine 32. Low glucose starvation or hydrogen peroxide stimulation increased sumoylation of MafA. Forced sumoylation of MafA reduced its transcriptional activity towards the insulin gene promoter and increased its suppression of the CHOP (C/EBP homologous protein) gene promoter. However, sumoylation of MafA did not alter its nuclear localization, protein stability, or apparently its DNA binding to the insulin promoter in beta cells. These studies suggest that MafA sumoylation modulates gene transcription in beta cells. In type I diabetes, beta-cell apoptosis is the major reason for immune-mediated pancreatic beta-cell death. IL-1beta (interleukin 1beta), a proinflammatory cytokine, induces ER (endoplasmic reticulum) stress and activates proapoptotic networks in beta cells, such as NF-kappaB (nuclear factor-kappaB) and JNK (c-Jun N-terminal kinase) signaling pathways. The second project focused on the mechanisms by which JNK and NF-kappaB regulate the expression of CHOP, a mediator of ER stress-induced apoptosis, upon IL-1beta stimulation. Exposure of beta cells to IL-1beta markedly increased CHOP messenger RNA and protein. Electrophoretic mobility shift assays showed that IL-1beta-activated NF-kappaB bound to the CHOP promoter. Furthermore, immunoblot data indicated that expression of c-Jun was strongly increased, and that multiple residues on c-Jun were phosphorylated after IL-1beta treatment. IL-1beta also increased c-Fos expression in beta cells. These data suggest that IL-1beta-induced activation of NF-kappaB and JNK controls CHOP gene expression in pancreatic beta cells, and that IL-1beta influences beta-cell function through a variety of signaling pathways.Item [Southwestern News](2004-03-22) O'Brien, StephenItem A Structural/Behavioral Analysis of the Regulation of Dopamine Signaling by Striatal RGS Proteins(2005-08-11) Waugh, Jeffrey Lynn; Gold, Stephen J.The regulators of G-protein signaling (RGS) proteins negatively modulate heterotrimeric G protein signaling by acting as GTPase activating proteins for Galpha subunits. In the striatum and nucleus accumbens, brain regions critical for control of movement, motivation and reward, overlapping RGS expression profiles suggested that functional specificity could not be explained by anatomical localization alone. We set out to assess striatal specificity within two distinct RGS pools, the R7 RGS subfamily and RGS10. The highly striatal-specific splice form RGS9-2 is a negative modulator of dopamine D2 receptor signaling, and has been shown to inhibit drug stimulated (cocaine or direct dopamine receptor agonists) locomotor activity. RGS9-2 is a member of the R7 subfamily, comprised of RGS6, -7, -9, and -11, which share highly similar subdomain structure. We analyzed the specificity of R7 modulation of dopamine receptor signaling using a novel behavioral assay. R7 RGS proteins were virally-overexpressed in rat or mouse accumbens via a stereotaxic injection of an engineered HSV virus. Following this surgery, drug-stimulated locomotor responses were assayed. We found that in rats and RGS9 KO mice, overexpression of R7 RGS proteins produces distinct locomotor and drug sensitization phenotypes, each of which occurs only during the period of RGS overexpression. Moreover, studies using truncation and chimeric RGS mutants demonstrated that while all tested subdomains were necessary for activity, only the C-terminus of RGS9-2 was sufficient to convey activity to RGS7. Lastly, RGS overexpression leads to distinct acute changes in weight: loss (RGS9-2) or gain (RGS7, RGS11). To elucidate RGS10 function in the brain, we mapped RGS10 protein in rodent brain using light microscopic and electron microscopic immunohistochemical techniques. Light microscopic analyses showed that RGS10 immunoreactivity labels all subcompartments of neurons and microglia, including their nuclei. Electron microscopy confirmed the presence of dense RGS10 immunoreactivity in euchromatin and resolved dense staining on terminals at symmetric synapses onto pyramidal cell somata. Dual-labeling histochemistry showed that RGS10 is expressed in specific neuronal cell types and circuits. Taken together, these data support a role for RGS10 in diverse processes including modulation of pre- and postsynaptic G-protein signaling and a potential role in modulating gene expression.Item Study of the Mechanisms Underlying Hippocampal Neuron Synaptogenesis: The Roles of Neurotrophin Signaling and MicroRNAs(2010-11-02) Zhang, Wei; Parada, Luis F.Synapse formation requires contacts between dendrites and axons. Although this process is often viewed as axon mediated, dendritic filopodia may be actively involved in mediating synaptogenic contacts. Brain-derived neurotrophic factor (BDNF) increases the density of dendritic filopodia and the conditional deletion of tyrosine receptor kinase B (TrkB) reduces synapse density in vivo (Luikart et al., 2005). Here, we report that TrkB associates with dendritic growth cones and filopodia, mediates filopodial motility, and does so via the phosphoinositide 3 kinase (PI3K) pathway. We used genetic and pharmacological manipulations of mouse hippocampal neurons to assess signaling downstream of TrkB. Conditional knock-out of two downstream negative regulators of TrkB signaling, Pten (phosphatase with tensin homolog) and Nf1 (neurofibromatosis type 1), enhanced filopodial motility. This effect was PI3K-dependent and correlated with synapse density. Phosphatidylinositol 3,4,5- trisphosphate (PIP3) was preferentially localized in filopodia and this distribution was enhanced by BDNF application. Thus, intracellular control of filopodial dynamics converged on PI3K activation and PIP3 accumulation, a cellular paradigm conserved for chemotaxis in other cell types. Our results suggest that filopodial movement is not random, but responsive to synaptic guidance cues. In order to further elucidate the mechanisms of BDNF-TrkB-PI3K pathway downstream signaling involved in regulating dendritic filopodial motility, we used a pharmacological approach as well as a gene expression approach to show that Rac1 and RhoA may play a role in this pathway. Rac1 positively regulated dendritic filopodial motility while RhoA had a negative effect. Our data suggest that BDNF-TrkB signaling might function to regulate the balance between Rac1 and RhoA, thus controlling dendritic filopodial motility. The developing nervous system is shaped by highly orchestrated programs of gene expression. This tight regulation is regulated by various transcriptional and post-transcriptional events that control individual gene expression. The recent discovery of small, non-coding RNAs has greatly expanded our understanding of the mechanisms that regulate gene expression at the post-transcriptional level. Here, I characterized the expression pattern of one neuronal microRNA, miR-381, and used in vitro cultured hippocampal neurons to show that miR-381 regulates neurite growth, as overexpression of miR-381 promotes neuronal dendritic branching. The effect of miR-381 on neuronal dendritic branching might be through a net regulation of multiple target genes.Item Studying Physiological Functions of APP Using Mice Models(2008-09-18) Li, Hongmei; Südhof, Thomas C.Beta-amyloid precursor protein (APP) is sequentially cleaved by alpha /beta - secrease and gamma-secrease into three pieces: a soluble ectodomain (sAPPalpha or sAPPbeta ), a p3 or Abeta peptide, and an APP intracellular domain (AICD). Mounting evidence indicates the neuroprotective and neurotrophic effects of sAPP domain. In order to find out whether sAPP domain carries out the major physiological function of APP, we have generated knock-in mice that express truncated ectodomain of APP at beta-cleavage site (sAPPbeta ) with FLAG tag at C-terminus. The knock-in mice were viable and fertile, with no obvious phenotype. However, when sAPPbeta - FLAG knock-in mice were bred to APLP2 knockout background ("knockin/ knockout" mice), the expression of sAPPbeta -FLAG failed to fully rescue the postnatal lethal phenotype of APP/APLP2 double knockout pups, suggesting sAPPbeta alone cannot substitute for the function of full length APP. We quantified the expression levels of a series of synaptic proteins and AICD-interacting proteins in the brains of new born APP/APLP2 double knockout (DKO) pubs, as well as in the "knock-in/knockout" (KI/KO) pubs, and found that Fe65 protein expression level is upregulated in brains from DKO pubs but not the KI/KO pubs. Collaborating with Dr. Yi Sun's lab investigating the DNA sequences in genome that potentially bind to AICD binding partners has shown that various promoters of a broad set of genes can bind to Fe65 and their expressions might be influenced by Fe65/AICD.Item To Develop a Small Interfering RNA (siRNA) Design and Information Resource to Facilitate Genetic Manipulaton of Human Cells(2004-05-25) Shah, Jyoti Khetsi; Minna, John D.Part I: Small interfering RNAs (siRNAs) have revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In the past, there were a set of rules designed to develop siRNA which worked efficiently in most cases. There was further refinement performed in these rules in some modern research analyses which attempted to address the question of what most closely determines siRNA functionality. I have designed and implemented a new software tool siRNA Information Resource ('sIR') that incorporates the most recent refinements in the design algorithm in order to provide fast and efficient siRNA design. sIR is a web-based computational tool which takes these existing rules for designing synthetic siRNAs and puts them in a software architecture that allows the researcher to design siRNAs for every gene. It also provides a database containing information about already developed siRNA and thus allows the researcher to access the siRNA information database consisting of siRNA information from literature and various other sources. This will ultimately help in future siRNA related discoveries. It also includes a scoring system which helps in rational selection of efficient siRNA. sIR was successfully validated using already designed and developed target siRNA sequences. Part II: One of the major problems in using chemotherapy to treat cancer is whether patients, whose tumors do not respond to one drug, would respond to another. Thus, it would be very useful if one could rationally select the appropriate chemotherapy for each patient's tumor. We are asking is whether tumor gene "expression signatures" detected by microarray analysis could identify a set of genes correlating with sensitivity or resistance to a particular drug. A large panel of breast cancer cell lines was tested with cisplatin, paclitaxel, vinorelbine, doxorubicin and gemcitabine, in vitro using a colorimetric assay to determine the concentration of drug that gives 50% growth inhibition (IC50). Gene expression profiles were also performed using Affymetrix chips and the two data sets were merged. It was found that a panel of ~100 genes were significantly up regulated (4 fold or more) for each drug in resistant cells. As an alternative approach, Pearson correlations between each gene expression data and each drug IC50 across all cell lines analyzed were determined. A positive correlation for a pair of gene and drug indicates the gene may be associated with resistance to the drug whereas a negative correlation would associate that gene with sensitivity to the drug. Some of these genes might be associated with the drug mechanism of action. We conclude that gene expression signatures do exist for individual breast tumor cell chemosensitivity and these could be of clinical significance.Item Transcriptional Regulation of Cardiac Ventricular Development(2004-05-04) Pierce, Stephanie Angelo; Srivastava, DeepakCongenital heart disease is the leading non-infectious cause of death in children. Disruption of cardiac gene expression during development can result in congenital heart defects. Numerous transcription factors regulate specific temporo-spatial events during cardiac differentiation and morphogenesis, however the mechanisms that regulate such events are largely unknown. Using a novel modified subtractive hybridization method to identify early cardiac-specific genes, we found Bop, a histone deacetylase-dependent transcriptional repressor. Bop was expressed specifically in the myocardium of the heart and somites during development. Targeted deletion of Bop in mouse resulted in hypoplasia of the right ventricle and expansion of the extracellular matrix between the myocardium and endocardium of the embryonic heart, suggesting a persistence of immature ventricular cardiomyocytes. Expression of dHAND, the evolutionarily conserved bHLH factor necessary for proper right ventricle development, was downregulated in the heart of the Bop-null embryo. In an effort to understand the mechanism by which Bop functions in cardiac differentiation and morphogenesis, the yeast two-hybrid assay was used to identify factors that interact with m-Bop in the embryonic heart. The DNA-binding factor, skNAC, and the inhibitor of mitosis, TRB3, were identified. TRB3 enhanced repression of SV40-driven luciferase activity by m-Bop. Disruption of this interaction resulted in the inability of TRB3 to enhance m-Bop's repressive activity, suggesting a novel function for TRB3 as a corepressor of m-Bop in the developing heart. skNAC was expressed in the myocardium of the heart and somites, strikingly similar to the expression pattern of Bop. While early in vitro attempts to study m-Bop and skNAC failed, in vivo efforts to study a genetic interaction are ongoing.