scholarly journals Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

1998 ◽  
Vol 18 (10) ◽  
pp. 5621-5633 ◽  
Author(s):  
Kelly A. Johnston ◽  
Michael Polymenis ◽  
Shanping Wang ◽  
John Branda ◽  
Emmett V. Schmidt
Keyword(s):  
Protein Synthesis ◽  
Protein Interactions ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Binding Protein ◽  
Limiting Factor ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Regulate Protein

ABSTRACTRegulation of the mRNA cap binding protein (eIF4E) is critical to the control of cellular proliferation since this protein is the rate-limiting factor in translation initiation and transforms fibroblasts and since eIF4E mutants arrest budding yeast in the G1phase of the cell cycle (cdc33). We previously demonstrated regulation of eIF4E by altered transcription of its mRNA in serum-stimulated fibroblasts and in response to c-myc. To identify additional factors regulating eIF4E transcription, we used linker-scanning constructs to characterize sites in the promoter of the eIF4E gene required for its expression. Promoter activity was dependent on sites at −5, −25, −45, and −75; the site at −75 included a previously describedmycbox. Electrophoretic mobility shift assays identified DNA-protein interactions at −25 and revealed a binding site (TTACCCCCCCTT) that is unique to the eIF4E promoter. Proteins of 68 and 97 kDa bound this site in UV cross-linking and Southwestern experiments. Levels of 4E regulatory factor activities correlated with c-Myc levels, eIF4E expression levels, and protein synthesis in differentiating U937 and HL60 cells, suggesting that these activities may function to regulate protein synthesis rates during differentiation. Since the eIF4E promoter lacked typical TATA and initiator elements, further studies of this novel initiator-homologous element should provide insights into mechanisms of transcription initiation and growth regulation.

scholarly journals A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618 ◽  
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

scholarly journals An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

1996 ◽  
Vol 16 (9) ◽  
pp. 4754-4764 ◽  
Author(s):  
R M Jones ◽  
J Branda ◽  
K A Johnston ◽  
M Polymenis ◽  
M Gadd ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Promoter Region ◽  
Binding Protein ◽  
Dominant Negative ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Eukaryotic Initiation Factor 4E ◽  
E Box

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.

An Experimental Study of Gene Expressing Profile in Human Myeloma Cell Line RPMI8226 Induced by Thalidomide.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4357-4357
Author(s):  
Mei Zhang ◽  
Ting Liu ◽  
Bingjing Yang ◽  
Pengcheng He ◽  
Mengchang Wang
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Protein Synthesis ◽  
Cell Line ◽  
Cdna Microarray ◽  
Binding Protein ◽  
Expression Profiles ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Gene Encoding

Abstract Multiple myeloma (MM) is one of malignant plasmacyte neoplasm in hematopietic system. Although nearly 70% patients of myeloma response to chemotherapy, but repeated therapies will induce drug resistance soon and lead to refractory or relapsed myelomas. In recent years, thalidomide is used to treat relapsed and refractory myeloma with satisfied effects and overall therapeutic rate with thalidomide is about 60%. Furthermore, the adverse effects of thalidomide is slight without myelosuppression, hepatotoxicity and renal toxicity. So thalidomide is likely to be a prospective antitumor agent. However, the mechanism of antitumor activity of the agent is still not clear. DNA microarray technology has provided us a very useful method to detect simultaneously the expression pattern of thousands of genes for investigating the molecular antitumor mechanism of thalidomide. To investigate the genes expression profiles of multiple myeloma cell line RPMI8226 treated with thalidomide, cDNA microarray were used to detect thousands of gene expression in a chip. Two cDNA probes were prepared through reverse transcription from mRNA of RPMI8226 cells with or without thalidomide treatment. The probes were labled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 1152 different human genes. Fluorescent intensity were scanned and screened by means of differential analysis between two gene expression profiles. After 72 hrs’ co-culture of RPMI8226 cells and thalidomide in 100μmol/L concentration, the expression of 18 genes were up-regulated and 4 genes were down-regulated. The up-regulated genes(GeneBank Accession) included: 1) protein synthesis-related genes:NM_004184(WARS), NM_ 003335(UBE1L); 2) immune-related protein:NM_001465(FYB), NM_004341 (CAD), NM_002388(MCM3); 3) matabolism relatedgenes: BC008861, NM_001640(APEH), NM_020040(TUBB4Q), NM_001033(RRM1), NM_ 001976(ENO3), NM_003330 (TXNRD1); 4) cell signals and transducing proteins: NM_005167(ARHC), NM_001465(FYB); 5) other genes: NM_ 017432(PTOV1), NM_003564 (TAGLN2), NM_005053(RAD23A), NM_ 001033(RRM1), AK025983, NM_015685 (CLONE24904), NM_033158 (HYAL2). The down-regulated genes includes: 1) protein synthesis-related genes: NM_000994(RPL32); 2) immune-related proteins: NM_001551 (IGBP1); 3) other genes: NM_002983(SCYA3), NM_002421(MMP1). These genes were involved in preotein synthesis and degradation, cell signal transduction, cytoskeletal movement immune cell matabolism and regulation of anti-oncogene. WARS gene encoding tryptophanyl-tRNA synthetase was up-regulated by thalidomide, while MMP1 gene encoding matrix metalloprotein 1 was down-regulated. They may be related to the inhibition of angiogenesis caused by thalidomide. SCYA3 gene encoding macrophage inflammatory protein-1alpha was down-regulated by thalidomide, as well as IGBP1 gene which encoding immunoglobulin binding protein 1. They may play a role in the inhibition of cell proliferation caused by thalidomide. TUBB4Q gene encoding tubulinβ4, UBE1L gene encoding ubiquitin- activating enzyme E1-like protein and TXNRD1 gene encoding thioredoxin reductase 1 were up-regulated by thalidomide. They may involve in apoptosis of RPMI8226 cells induced by thalidomide. FYB gene encoding Fyn-binding protein was up regulated by thalidomide. The elevated expression of this gene may play a role in the killing of RPMI8226 cells by thalidomide.

P/CAF rescues the Bhlhe40-mediated repression of MyoD transactivation

2009 ◽  
Vol 422 (2) ◽  
pp. 343-352 ◽  
Author(s):  
Sheng P. Hsiao ◽  
Kai M. Huang ◽  
Hsin Y. Chang ◽  
Shen L. Chen
Keyword(s):  
Transcription Initiation ◽  
Myogenic Differentiation ◽  
Binding Protein ◽  
Core Promoter ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Boxes

Previously, we found that MRFs (myogenic regulatory factors) regulated the expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α) by targeting a short region, from nt −49 to +2 adjacent to the transcription initiation site, that contained two E-boxes. However, only the E2-box had significant affinity for MRFs, and the E1-box was predicted to be the target of Bhlhe40 (basic helix-loop-helix family, member e40, also known as Stra13, Bhlhb2, DEC1 and Sharp2), a transcriptional repressor implicated in the regulation of several physiological processes. In the present study, by using EMSA (electrophoresis mobility-shift assay), we confirmed that Bhlhe40 targeted the E1-box and formed a complex with the basic helix-loop-helix transcription factor MyoD (myogenic differentiation factor D) on the PGC-1α core promoter. We demonstrate that Bhlhe40 binds to the promoters of PGC-1α and myogenic genes in vivo and that Bhlhe40 represses the MyoD-mediated transactivation of these promoters. Furthermore, we found that this repression could be relieved by P/CAF (p300/CBP-associated factor) in a dose-dependent manner, but not by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. Bhlhe40 interacted with P/CAF and this interaction disrupted the interaction between P/CAF and MyoD. These results suggest that Bhlhe40 functions as a repressor of MyoD by binding to adjacent E-boxes and sequestering P/CAF from MyoD.

scholarly journals Identification of a Male-Specific RNA Binding Protein That Regulates Sex-Specific Splicing of Bmdsx by Increasing RNA Binding Activity of BmPSI

2010 ◽  
Vol 30 (24) ◽  
pp. 5776-5786 ◽  
Author(s):  
Masataka G. Suzuki ◽  
Shigeo Imanishi ◽  
Naoshi Dohmae ◽  
Miwako Asanuma ◽  
Shogo Matsumoto
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Z Chromosome ◽  
Specific Factor ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Splicing Silencer ◽  
Shift Analysis ◽  
Male Specific

ABSTRACT Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI.

α-Enolase Is the c-myc Promoter-Binding Protein.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4075-4075
Author(s):  
Donald M. Miller ◽  
Hunter Campbell ◽  
Shelia D. Thomas ◽  
Kara C. Sedoris
Keyword(s):  
Electrophoretic Mobility ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Regulation Of Gene Expression ◽  
Promoter Sequence ◽  
Similar Response ◽  
Mobility Shift ◽  
Constitutive Overexpression ◽  
P2 Promoter ◽  
Electrophoretic Mobility Shift Assays

Abstract Quadruplex-forming sequences occur disproportionately in the promoters of growth regulatory genes, including c-myc. These sequences appear to play important roles in the regulation of gene expression. The c-myc quadruplex-forming sequence occurs in the c-myc P1 promoter region which is responsible for more than 80% of c-myc expression. We have used proteomic techniques used to identify quadruplex-binding proteins. Two dimensional electrophoretic gels of nuclear extracts were blotted and screened with G-rich (quadruplex forming) and C-rich (control) oligonucleotides. We were surprised to observe that α-enolase binds the G-rich, but not C-rich oligonucleotide. Using electrophoretic mobility shift assays (EMSA), we confirmed that both α-enolase and its alternative translation product, MBP-1, bind quadruplex-forming oligonucleotides. We then tested the hypothesis that both α-enolase and MBP-1 may play important physiologic roles by binding the c-myc quadruplex-forming sequence. Electrophoretic mobility shift assays were used to characterize the binding of α-enolase and MBP-1 to the c-myc quadruplex-forming sequence. Binding of AS1411, a quadruplex forming oligonucleotide, was used as a control. Both α-enolase and MBP-1 demonstrated strong binding to the c-myc P2 promoter sequence (containing the quadruplex-forming sequence). A “supershift” was seen when an anti-α-enolase antibody was included in the incubation although control antibodies did not alter the EMSA pattern. The quadruplex oligonucleotide aptamer, AS1411, competed for α-enolase and MBP-1 binding to the c-myc promoter sequence. However, a nonquadruplex-forming oligonucleotide did not compete. A mutation in the c-myc quadruplex sequence abrogated binding of both α-enolase and MBP-1. In order to test the biological effect of the α-enolase/MBP-1 interaction with the c-myc promoter, we characterized the effect of constitutive overexpression of each α-enolase product, individually. Although, overexpression of MBP-1 by transfected cells resulted in down regulation of c-myc expression and induction of apoptosis, constitutive overexpression of α-enolase did not alter c-myc expression or the rate of cellular proliferation. Cells grown in low glucose medium demonstrate increased MBP-1 levels which results in decreased c-myc expression and inhibition of cellular proliferation. A similar response was seen in hypoxic cells, indicating that enolase/MBP-1 plays an important role in cellular responses to external stress. These results suggest that MBP-1 down regulates c-myc expression by simultaneous binding to the P1 and P2 promoter sequences, while α-enolase binds only the P2 sequence. This suggests a very important regulatory role for α-enolase/MBP-1 in the relationship between cellular metabolism and growth regulation. Enolase/MBP-1 quadruplex-binding likely plays an important role in regulation of c-myc expression.

Characterization of human cathepsin L promoter and identification of binding sites for NF-Y, Sp1 and Sp3 that are essential for its activity

2001 ◽  
Vol 361 (1) ◽  
pp. 173-184 ◽  
Author(s):  
Didier JEAN ◽  
Nathalie GUILLAUME ◽  
Raymond FRADE
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Promoter Activity ◽  
Splice Variants ◽  
Cathepsin L ◽  
Melanoma Cells ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Human Cathepsin ◽  
Mobility Shift Assay

Cathepsin L is a cysteine protease whose overexpression in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic. Regulation of the transcription of the gene encoding human cathepsin L has not been yet studied and only preliminary data exist on the promoter regulation of the gene encoding rodent cathepsin L. In the present study we identified molecular elements involved in the transcriptional regulation of human cathepsin L in melanoma cells. The sequence of the 5′-flanking region of the gene encoding human cathepsin L was determined up to 3263bp upstream of the translation start site. The major transcription intiation site was located. Three mRNA splice variants, differing in their 5′ untranslated ends, were identified. Regulatory regions crucial for cathepsin L promoter activity were characterized between −1489 and −1646bp. In this region, two GC boxes (−1590/−1595 and −1545/−1550) and a CCAAT motif (−1571/−1575) were involved in specific DNA–protein interactions. An electrophoretic mobility-shift assay demonstrated that Sp1 and Sp3 transcription factors bound to these GC boxes, and only the transcription factor nuclear factor Y (NF-Y) bound to the CCAAT motif. Mutagenesis studies demonstrated that these binding sites contributed at least 85% of cathepsin L promoter activity. Thus structural and functional analysis demonstrated that binding sites for NF-Y, Sp1 and Sp3 are essential for transcription of the gene encoding human cathepsin L in melanoma cells.

scholarly journals Interaction of Native and Mutant MecI Repressors with Sequences That Regulate mecA, the Gene Encoding Penicillin Binding Protein 2a in Methicillin-Resistant Staphylococci

1998 ◽  
Vol 180 (8) ◽  
pp. 2160-2166 ◽  
Author(s):  
Vijay K. Sharma ◽  
Corinne J. Hackbarth ◽  
Tanja M. Dickinson ◽  
Gordon L. Archer
Keyword(s):  
Transcriptional Repression ◽  
Methicillin Resistance ◽  
Binding Protein ◽  
Molecular Size ◽  
Protein Oligomerization ◽  
Penicillin Binding Protein ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Biochemical Basis

ABSTRACT Methicillin resistance in staphylococci is mediated by PBP2a, a penicillin binding protein with low affinity for β-lactam antibiotics. The gene encoding PBP2a, mecA, is transcriptionally regulated in some clinical isolates bymecR1 and mecI, genes divergently transcribed from mecA that encode a signal transducer and repressor, respectively. The biochemical basis of MecI-mediatedmecA transcriptional repression was investigated by using purified MecI. In DNase I protection studies, MecI protected a 30-bp palindrome encompassing the predicted mecA −10 and themecR1 −35 promoter sequences. The larger palindrome contained 15 bp of dyad symmetry within which was a smaller 6-bp palindrome. Electrophoretic mobility shift assays established a requirement for the entire 15-bp half-site for initial repressor binding. Fragments containing the 30-bp palindrome and the entire mecA-mecR1 intergenic region were retarded in gels as multiple discrete bands varying in molecular size, characteristic of cooperative DNA binding. Glutaraldehyde cross-linking confirmed oligomerization of repressor in solution. A naturally occurring MecI mutant (MecI*; D39G) repressed mecAtranscription sixfold less well than the wild type in vivo. Although MecI* protected the same target sequences and exhibited similar gel shift patterns to MecI, 5- to 10-fold more protein was required. MecI* exhibited defective oligomerization in solution, suggesting that the MecI amino terminus is important in protein-protein interactions and that protein oligomerization is necessary for optimum repression.

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