Medicine and Health Sciences Commons^™

Mechanisms Of Mitochondrial Promoter Recognition In Humans And Other Mammalian Species, Angelica Zamudio-Ochoa, Yaroslav I Morozov, Azadeh Sarfallah, Michael Anikin, Dmitry Temiakov

Department of Biochemistry and Molecular Biology Faculty Papers

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for > 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of …

Ceramide Induces Human Hepcidin Gene Transcription Through Jak/Stat3 Pathway., Sizhao Lu, Sathish Kumar Natarajan, Justin L. Mott, Kusum K. Kharbanda, Duygu Dee Harrison-Findik

Journal Articles: Biochemistry & Molecular Biology

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked …

Ceramide Induces Human Hepcidin Gene Transcription Through Jak/Stat3 Pathway., Sizhao Lu, Sathish Kumar Natarajan, Justin L. Mott, Kusum K. Kharbanda, Duygu Dee Harrison-Findik

Journal Articles: Biochemistry & Molecular Biology

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked …

Disrupting Sumoylation Enhances Transcriptional Function And Ameliorates Polyglutamine Androgen Receptor-Mediated Disease., Jason P Chua, Satya L Reddy, Zhigang Yu, Elisa Giorgetti, Heather L Montie, Sarmistha Mukherjee, Jake Higgins, Richard C Mceachin, Diane M Robins, Diane E Merry, Jorge A Iñiguez-Lluhí, Andrew P Lieberman

Department of Biochemistry and Molecular Biology Faculty Papers

Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that …

Expression Of Muc17 Is Regulated By Hif1Α-Mediated Hypoxic Responses And Requires A Methylation-Free Hypoxia Responsible Element In Pancreatic Cancer., Sho Kitamoto, Seiya Yokoyama, Michiyo Higashi, Norishige Yamada, Shyuichiro Matsubara, Sonshin Takao, Surinder K. Batra, Suguru Yonezawa

Journal Articles: Biochemistry & Molecular Biology

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., …

Transcriptional Profiling Of Peripheral Blood Mononuclear Cells In Pancreatic Cancer Patients Identifies Novel Genes With Potential Diagnostic Utility., Michael J. Baine, Subhankar Chakraborty, Lynette M. Smith, Kavita Mallya, Aaron R. Sasson, Randall E. Brand, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

BACKGROUND: It is well known that many malignancies, including pancreatic cancer (PC), possess the ability to evade the immune system by indirectly downregulating the mononuclear cell machinery necessary to launch an effective immune response. This knowledge, in conjunction with the fact that the trancriptome of peripheral blood mononuclear cells has been shown to be altered in the context of many diseases, including renal cell carcinoma, lead us to study if any such alteration in gene expression exists in PC as it may have diagnostic utility.

METHODS AND FINDINGS: PBMC samples from 26 PC patients and 33 matched healthy controls were …

Regulation Of Energy Stores And Feeding By Neuronal And Peripheral Creb Activity In Drosophila., Koichi Iijima, Lijuan Zhao, Christopher Shenton, Kanae Iijima-Ando

Department of Biochemistry and Molecular Biology Faculty Papers

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), we have examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot with functions of adipose tissue and the liver in flies, on energy balance, stress resistance and feeding behavior. We found that disruption of CREB function in neurons reduced glycogen and lipid stores …

The Human Rna Polymerase Ii-Associated Factor 1 (Hpaf1): A New Regulator Of Cell-Cycle Progression., Nicolas Moniaux, Christophe Nemos, Shonali Deb, Bing Zhu, Irena Dornreiter, Michael A. Hollingsworth, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

BACKGROUND: The human PAF (hPAF) complex is part of the RNA polymerase II transcription apparatus and regulates multiple steps in gene expression. Further, the yeast homolog of hPaf1 has a role in regulating the expression of a subset of genes involved in the cell-cycle. We therefore investigated the role of hPaf1 during progression of the cell-cycle.

METHODOLOGY/FINDINGS: Herein, we report that the expression of hPaf1, a subunit of the hPAF complex, increases with cell-cycle progression and is regulated in a cell-cycle dependant manner. hPaf1 specifically regulates a subclass of genes directly implicated in cell-cycle progression during G1/S, S/G2, and G2/M. …

Human Collagen Krox Up-Regulates Type I Collagen Expression In Normal And Scleroderma Fibroblasts Through Interaction With Sp1 And Sp3 Transcription Factors., Magdalini Kypriotou, Gallic Beauchef, Christos Chadjichristos, Russell Widom, Emmanuelle Renard, Sergio A. Jimenez, Joseph Korn, François-Xavier Maquart, Thierry Oddos, Otto Von Stetten, Jean-Pierre Pujol, Philippe Galéra

Department of Medicine Faculty Papers

Despite several investigations, the transcriptional mechanisms that regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a -112/-61-bp sequence in FF, ANF, …

The Caenorhabditis Elegans Heterochronic Regulator Lin-14 Is A Novel Transcription Factor That Controls The Developmental Timing Of Transcription From The Insulin/Insulin-Like Growth Factor Gene Ins-33 By Direct Dna Binding, Marta Hristova, Darcy Birse, Yang Hong, Victor Ambros

Dartmouth Scholarship

A temporal gradient of the novel nuclear protein LIN-14 specifies the timing and sequence of stage-specific developmental events in Caenorhabditis elegans. The profound effects of lin-14 mutations on worm development suggest that LIN-14 directly or indirectly regulates stage-specific gene expression. We show that LIN-14 can associate with chromatin in vivo and has in vitro DNA binding activity. A bacterially expressed C-terminal domain of LIN-14 was used to select DNA sequences that contain a putative consensus binding site from a pool of randomized double-stranded oligonucleotides. To identify candidates for genes directly regulated by lin-14, we employed DNA microarray hybridization to compare …

The Human Paf Complex Coordinates Transcription With Events Downstream Of Rna Synthesis., Bing Zhu, Subhrangsu S. Mandal, Anh-Dung Pham, Yong Zheng, Hediye Erdjument-Bromage, Surinder K. Batra, Paul Tempst, Danny Reinberg

Journal Articles: Biochemistry & Molecular Biology

The yeast PAF (yPAF) complex interacts with RNA polymerase II and coordinates the setting of histone marks associated with active transcription. We report the isolation and functional characterization of the human PAF (hPAF) complex. hPAF shares four subunits with yPAF (hCtr9, hPaf1, hLeo1, and hCdc73), but contains a novel higher eukaryotic-specific subunit, hSki8. RNAi against hSki8 or hCtr9 reduces the cellular levels of other hPAF subunits and of mono- and trimethylated H3-Lys 4 and dimethylated H3-Lys 79. The hSki8 subunit is also a component of the human SKI (hSKI) complex. Yeast SKI complex is cytoplasmic and together with Exosome mediates …

Efficient Transcriptional Activation Of Many Simple Modular Promoters By Simian Virus 40 Large T Antigen., Philip W. Rice, Charles N. Cole

Dartmouth Scholarship

Simian virus 40 (SV40) large T antigen is a multifunctional protein which plays central roles during both lytic and transforming infections by SV40. It is a potent transcriptional activator and increases expression from the SV40 late promoter and from several cellular promoters. To understand better the transcriptional activation activity of large T antigen, we examined its ability to transactivate a set of simple modular promoters containing one of four upstream activation sequences coupled with one of three different TATA box sequences originally constructed and studied by Taylor and Kingston (Mol. Cell. Biol. 10:165-175, 1990). Large T antigen activated transcription from …

Two Factors That Bind To Highly Conserved Sequences In Mammalian Type C Retroviral Enhancers., Nancy R. Manley, Mary M. O'Connell, Wanwen Sun, Nancy A. Speck, Nancy Hopkins

Dartmouth Scholarship

The transcriptional enhancers of the Moloney and Friend murine leukemia viruses (MLV) are important determinants of viral pathogenicity. We used electrophoretic mobility shift and methylation interference assays to study nuclear factors which bind to a region of these enhancers whose sequence is identical between Moloney and Friend viruses and particularly highly conserved among 35 mammalian type C retroviruses whose enhancer sequences have been aligned (E. Golemis, N. A. Speck, and N. Hopkins, J. Virol. 64:534-542, 1990). Previous studies identified sites for the leukemia virus factor b (LVb) and core proteins in this region (N. A. Speck and D. Baltimore, Mol. …

A Phorbol Ester Response Element Within The Human T-Cell Receptor Beta-Chain Enhancer., Haydn M. Prosser, David Wotton, Anne Gegonne, Jacques Ghysdael, Shuwen Wang, Nancy A. Speck, Michael J. Owen

Dartmouth Scholarship

The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 …

Mapping The Transcriptional Transactivation Function Of Simian Virus 40 Large T Antigen., Jiyue Y. Zhu, Philip W. Rice, Michele Chamberlain, Charles N. Cole

Dartmouth Scholarship

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T …

Neurospora Crassa Clock-Controlled Genes Are Regulated At The Level Of Transcription., Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

Although an extensive number of biological processes are under the daily control of the circadian biological clock, little is known about how the clock maintains its regulatory networks within a cell. An important aspect of this temporal control is the daily control of gene expression. Previously we identified two morning-specific genes that are regulated by the clock through daily control of gene expression (J. Loros, S. Denome, and J.C. Dunlap, Science 243:385-388, 1989). We have now introduced a method for transcriptional analysis in Neurospora crassa and used this nuclear run-on procedure to show that regulation of mRNA abundance for these …

Patterns Of Polyadenylation Site Selection In Gene Constructs Containing Multiple Polyadenylation Signals., Roger Denome, Charles Cole

Dartmouth Scholarship

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 …

Fine-Structure Analysis Of The Processing And Polyadenylation Region Of The Herpes Simplex Virus Type 1 Thymidine Kinase Gene By Using Linker Scanning, Internal Deletion, And Insertion Mutations., Fang Zhang, Roger M. Denome, Charles N. Cole

Dartmouth Scholarship

Most eucaryotic mRNAs are polyadenylated. In higher eucaryotes, the sequence AATAAA is located 7 to 30 base pairs (bp) upstream from the site of processing and polyadenylation and is a critical part of the signal for processing and polyadenylation. Efficient cleavage and polyadenylation also require sequences downstream of polyadenylation sites. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT box (18 of 19 consecutive residues are G or T) previously shown to be required for efficient processing and polyadenylation of tk mRNA (C. N. Cole and T. P. Stacy, …

Two Separable Functional Domains Of Simian Virus 40 Large T Antigen: Carboxyl-Terminal Region Of Simian Virus 40 Large T Antigen Is Required For Efficient Capsid Protein Synthesis., Joanne Tornow, Maryellen Polvino-Bodnar, George Santangelo, Charles N. Cole

Dartmouth Scholarship

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T …