Medical Sciences Commons^™

Computational Identification And Functional Validation Of Regulatory Motifs In Cartilage-Expressed Genes, Sherri R Davies, Li-Wei Chang, Debabrata Patra, Xiaoyun Xing, Karen Posey, Jacqueline Hecht, Gary D Stormo, Linda J Sandell

Faculty and Staff Publications

Chondrocyte gene regulation is important for the generation and maintenance of cartilage tissues. Several regulatory factors have been identified that play a role in chondrogenesis, including the positive transacting factors of the SOX family such as SOX9, SOX5, and SOX6, as well as negative transacting factors such as C/EBP and delta EF1. However, a complete understanding of the intricate regulatory network that governs the tissue-specific expression of cartilage genes is not yet available. We have taken a computational approach to identify cis-regulatory, transcription factor (TF) binding motifs in a set of cartilage characteristic genes to better define the transcriptional regulatory …

P53 Activation By Knockdown Technologies, Mara E. Robu, Jon D. Larson, Aidas Nasevicius, Soraya Beiraghi, Charles Brenner

Dartmouth Scholarship

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected …

The Pseudomonas Aeruginosa Secreted Protein Pa2934 Decreases Apical Membrane Expression Of The Cystic Fibrosis Transmembrane Conductance Regulator, Daniel P. Maceachran, Siying Ye, Jennifer M. Bomberger, Deborah A. Hogan, Agnieszka Swiatecka-Urban, Bruce Stanton, George A. O'Toole

Dartmouth Scholarship

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2934 [corrected], and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression …

Cpg Hypomethylation In A Large Domain Encompassing The Embryonic Β-Like Globin Genes In Primitive Erythrocytes, Mei Hsu, Rodwell R. Mabaera, Christopher H. Lowrey, David I. K. Martin, Steven Fiering

Dartmouth Scholarship

There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the β-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine β-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large (∼20-kb) …

Single-Stranded Dna-Binding Proteins Regulate The Abundance Of Lim Domain And Lim Domain-Binding Proteins, Zhixiong Xu, Xianzhang Meng, Ying Cai, Hong Liang, Lalitha Nagarajan, Stephen J Brandt

Student and Faculty Publications

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In …

Expression Of The Autoimmune Fcgr2b Nzw Allele Fails To Be Upregulated In Germinal Center B Cells And Is Associated With Increased Igg Production, S.M. Ziaur Rahman, H. Niu, D. Perry, Timothy L. Manser, L. Morel

Department of Microbiology and Immunology Faculty Papers

The inhibitory receptor FcγRIIb regulates B-cell functions. Genetic studies have associated Fcgr2b polymorphisms and lupus susceptibility in both humans and murine models, in which B cells express reduced FcγRIIb levels. Furthermore, FcγRIIb absence results in lupus on the appropriate genetic background, and lentiviral-mediated FcγRIIb overexpression prevents disease in the NZM2410 lupus mouse. The NZM2410/NZW allele Fcgr2b is, however, located in-between Sle1a and Sle1b, two potent susceptibility loci, making it difficult to evaluate Fcr2bNZW independent contribution. By using two congenic strains that each carries only Sle1a (B6.Sle1a(15–353)), or Fcr2bNZW in the absence of Sle1a or Sle1b (B6.Sle1(111–148)), we show that the …

Transgenic Cyclin E Triggers Dysplasia And Multiple Pulmonary Adenocarcinomas, Yan Ma, Steven Fiering, Candice Black, Xi Liu, Ziqiang Yuan, Vincent A. Memoli, David J. Robbins, Heather A. Bentley, Gregory J. Tsongalis, Eugene Demidenko, Sarah J. Freemantle, Ethan Dmitrovsky

Dartmouth Scholarship

Cyclin E is a critical G(1)-S cell cycle regulator aberrantly expressed in bronchial premalignancy and lung cancer. Cyclin E expression negatively affects lung cancer prognosis. Its role in lung carcinogenesis was explored. Retroviral cyclin E transduction promoted pulmonary epithelial cell growth, and small interfering RNA targeting of cyclin E repressed this growth. Murine transgenic lines were engineered to mimic aberrant cyclin E expression in the lung. Wild-type and proteasome degradation-resistant human cyclin E transgenic lines were independently driven by the human surfactant C (SP-C) promoter. Chromosome instability (CIN), pulmonary dysplasia, sonic hedgehog (Shh) pathway activation, adenocarcinomas, and metastases occurred. Notably, …

The Myc Transactivation Domain Promotes Global Phosphorylation Of The Rna Polymerase Ii Carboxy-Terminal Domain Independently Of Direct Dna Binding, Victoria H. Cowling, Michael D. Cole

Dartmouth Scholarship

Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc^−/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters …

Single Nucleotide Polymorphisms (Snps) Distinguish Indian-Origin And Chinese-Origin Rhesus Macaques (Macaca Mulatta), Betsy Ferguson, Summer L. Street, Hollis Wright, Carlo Pearson, Yibing Jia, Shaun L. Thompson, Patrick Allibone, Christopher J. Dubay, Eliot Spindel, Robert B. Norgren

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: Rhesus macaques serve a critical role in the study of human biomedical research. While both Indian and Chinese rhesus macaques are commonly used, genetic differences between these two subspecies affect aspects of their behavior and physiology, including response to simian immunodeficiency virus (SIV) infection. Single nucleotide polymorphisms (SNPs) can play an important role in both establishing ancestry and in identifying genes involved in complex diseases. We sequenced the 3' end of rhesus macaque genes in an effort to identify gene-based SNPs that could distinguish between Indian and Chinese rhesus macaques and aid in association analysis.

RESULTS: We surveyed the …

Intercenter Reliability And Validity Of The Rhesus Macaque Genechip, Fenghai Duan, Eliot R. Spindel, Yu-Hua Li, Robert B. Norgren

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: The non-human primate (NHP) research community has been intensely interested in obtaining whole-genome expression arrays for their work. Recently, novel approaches were used to generate the DNA sequence information for a rhesus GeneChip. To test the reliability of the rhesus GeneChip across different centers, RNA was isolated from five sources: cerebral cortex, pancreas, thymus, testis, and an immortalized fibroblast cell line. Aliquots of this RNA were sent to each of three centers: Yerkes National Primate Research Center, Oregon National Primate Research Center and the University of Nebraska Medical Center. Each center labeled the samples and hybridized them with two …

Department Of Pathology, Thomas Jefferson University, Identification Of Conserved Gene Expression Features Between Murine Mammary Carcinoma Models And Human Breast Tumors., Jason I Herschkowitz, Karl Simin, Victor J Weigman, Igor Mikaelian, Jerry Usary, Zhiyuan Hu, Karen E Rasmussen, Laundette P Jones, Shahin Assefnia, Subhashini Chandrasekharan, Michael G Backlund, Yuzhi Yin, Andrey I Khramtsov, Roy Bastein, John Quackenbush, Robert I Glazer, Powel H Brown, Jeffrey E Green, Levy Kopelovich, Priscilla A Furth, Juan P Palazzo, Olufunmilayo I Olopade, Philip S Bernard, Gary A Churchill, Terry Van Dyke, Charles M Perou

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: Although numerous mouse models of breast carcinomas have been developed, we do not know the extent to which any faithfully represent clinically significant human phenotypes. To address this need, we characterized mammary tumor gene expression profiles from 13 different murine models using DNA microarrays and compared the resulting data to those from human breast tumors. RESULTS: Unsupervised hierarchical clustering analysis showed that six models (TgWAP-Myc, TgMMTV-Neu, TgMMTV-PyMT, TgWAP-Int3, TgWAP-Tag, and TgC3(1)-Tag) yielded tumors with distinctive and homogeneous expression patterns within each strain. However, in each of four other models (TgWAP-T121, TgMMTV-Wnt1, Brca1Co/Co;TgMMTV-Cre;p53+/- and DMBA-induced), tumors with a variety of …