1: Cancer Lett. 2005 Sep 28;227(2):153-62. Inhibitory effects of costunolide on the telomerase activity in human breast carcinoma cells. Choi SH, Im E, Kang HK, Lee JH, Kwak HS, Bae YT, Park HJ, Kim ND. Department of Pharmacy, College of Pharmacy, and Research Institute for Drug Development, Pusan National University, Busan 609-735, South Korea. Costunolide, a natural sesquiterpene compound, has been known having cytotoxic and chemopreventive effects on various human cancer cells. In the present study, we examined the effects of costunolide on telomerase activity and on the components of telomerase in MCF-7 (wild-type p53) and MDA-MB-231 (mutant p53) cells. We found that costunolide inhibited the growth and telomerase activity of MCF-7 and MDA-MB-231 cells in a concentration- and time-dependent manner. The expression of hTERT mRNA was also inhibited but hTR mRNA was not. In addition, the bindings of transcription factors in hTERT promoters were significantly decreased in both cells by the treatment of costunolide. These results suggest that costunolide inhibited the growth of both MCF-7 and MDA-MB-231 cells and this effect was mediated at least in part by a significant reduction in telomerase activity. PMID: 16112418 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Biochim Biophys Acta. 2005 Aug 15;1730(2):126-36. Characterization of the 5'-flanking region of the human transcription factor Sp3 gene. Tapias A, Monasterio P, Ciudad CJ, Noe V. Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Avenue Diagonal 643, Barcelona E-08028, Spain. A fragment of 1079 bp from the 5'-flanking region of the human Sp3 gene was isolated and characterized. The Sp3 promoter is a GC-rich region that contains putative binding sites for Elk-1, c-Myb, NF-1, Ap1, Sp1, NF-Y, Ap2 and USF. Several transcriptional start sites located between 70 and 132 bp upstream of the translational start site were identified. The proximal promoter was contained in the first 281 bp 5' of the translational start, whereas the region including up to -225 relative to the translational start was referred as the minimal promoter. Transient transfections and luciferase assays revealed activation of the Sp3 proximal promoter upon overexpression of either Sp1 or Sp3, alone or in combination. Gel-shift and supershift assays demonstrated specific binding of Sp1 and Sp3 proteins to the GC box located in the proximal promoter of Sp3. Overexpression of NF-YA had a synergistic effect on Sp1 overexpression and an additive effect on Sp3 overexpression. Additionally, overexpression of NF-YA, Sp1 and Sp3 altogether had a synergistic effect on Sp3 promoter activity. Furthermore, binding of the NF-Y complex to the CCAAT box located in the proximal promoter of Sp3 was observed in gel-shift assays. PMID: 16024108 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Gene. 2005 May 23;351:171-80. Regulation of the cyclin D1 and cyclin A1 promoters by B-Myb is mediated by Sp1 binding sites. Bartusel T, Schubert S, Klempnauer KH. Institut fur Biochemie, Westfalische-Wilhelms-Universitat Munster, Germany. B-Myb is a highly conserved member of the Myb family of transcription factors which plays an important role during the cell cycle. Previous work has shown that B-Myb is phosphorylated at several sites by cyclin A/Cdk2 in the early S-phase. These phosphorylations increase the transactivation potential of B-Myb by counteracting the repressive function of an inhibitory domain located at the carboxyl-terminus of B-Myb. As yet, only a few genes have been identified as B-Myb target genes. Previous work has suggested that the cyclin D1 gene might be regulated by B-Myb. Here, we have studied the effect of B-Myb on the promoter of the cyclin D1 gene. We show that B-Myb is a potent activator of the cyclin D1 promoter and that this activation is not mediated by Myb binding sites but rather by a group of Sp1 binding sites which have previously been shown to be crucial for cyclin D1 promoter activity. Our data show that the C-terminal domain of B-Myb is required for the activation of the cyclin D1 promoter and that this part of B-Myb interacts with Sp1. Finally, we have found that the promoter of the cyclin A1 gene is also activated by B-Myb by a Sp1 binding site-dependent mechanism. The effect of B-Myb on the promoters of the cyclin A1 and D1 genes is reminiscent of the mechanism that has been proposed for the autoregulation of the B-myb promoter by B-Myb, which also involves Sp1 binding sites. Taken together, our identification of two novel B-Myb responsive promoters whose activation by B-Myb does not involve Myb binding sites extends previous evidence for the existence of a distinct mechanism of transactivation by B-Myb which is dependent on Sp1 binding sites. The observation that this mechanism is not subject to the inhibitory effect of the C-terminal domain of B-Myb but rather requires this domain supports the notion that the Sp1 site-dependent mechanism is already active in the G1-phase prior to the phosphorylation of B-Myb by cyclin A/Cdk2. PMID: 15922873 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Gene. 2004 Sep 15;339:61-9. The chicken telomerase reverse transcriptase (chTERT): molecular and cytogenetic characterization with a comparative analysis. Delany ME, Daniels LM. Department of Animal Science, 2131D Meyer Hall, One Shields Avenue, University of California, Davis, CA 95616, USA. medalany@ucdavis.edu Telomerase activity is essential for maintaining the termini of linear chromosomes. Telomerase consists of both a RNA and a specialized reverse transcriptase. Our objective for this study was to determine the molecular and cytogenetic features of the chicken telomerase reverse transcriptase (chTERT) gene and protein. The TERT mRNA from gastrula stage embryos was found to be 4497 bp in length, translating into a protein of 1346 amino acids (aa). The chTERT protein shares 45% aa identity with human TERT (hTERT). A distinctive feature of chTERT, as compared to human and other vertebrate TERTs, is the larger size of the protein due mainly to a considerably longer N-terminal flexible linker region (144 aa longer than in human). Chicken TERT was mapped to chromosome 2q21 near an interstitial telomere site. Several transcription factor binding motifs in the 5' flanking/promoter region of chTERT were similar to those found associated with hTERT (E-box, Ik1, MAZ, Sp1 sites), whereas several c-Myb sites were found associated with chTERT only and c-Ets-2 and WT1 were associated with hTERT only. Results presented here should promote structure-function studies of chTERT, as well as contribute to the comparative analysis of TERT regulation and function in vertebrates utilizing the telomere clock mechanism to different degrees. PMID: 15363846 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Immunol. 2004 Jun 15;172(12):7519-29. Transcriptional regulation of human CD5: important role of Ets transcription factors in CD5 expression in T cells. Arman M, Calvo J, Trojanowska ME, Cockerill PN, Santana M, Lopez-Cabrera M, Vives J, Lozano F. Servei d'Immunologia, Institut d'Investigacions Biomediques August Pi i Sunyer, Hospital Clinic i Provincial de Barcelona, Villaroel 170, Barcelona 08036, Spain. CD5 is a surface receptor constitutively expressed on thymocytes and mature T and B-1a cells. CD5 expression is tightly regulated during T and B cell development and activation processes. In this study we shown that the constitutive expression of CD5 on human T cells correlates with the presence of a DNase I-hypersensitive (DH) site at the 5'-flanking region of CD5. Human CD5 is a TATA-less gene for which 5'-RACE analysis shows multiple transcriptional start sites, the most frequent of which locates within an initiator sequence. Luciferase reporter assays indicate that a 282-bp region upstream of the initiation ATG displays full promoter activity in human T cells. Two conserved Ets-binding sites (at positions -239 and -185) were identified as functionally relevant to CD5 expression by site-directed mutagenesis, EMSAs, and cotransfection experiments. A possible contribution of Sp1 (-115 and -95), c-Myb (-177), and AP-1-like (-151) motifs was also detected. Further DH site analyses revealed an inducible DH site 10 kb upstream of the human CD5 gene in both T and B CD5(+) cells. Interestingly, a 140-bp sequence showing high homology with a murine inducible enhancer is found within that site. The data presented indicate that the 5'-flanking region of human CD5 is transcriptionally active in T cells, and that Ets transcription factors in conjunction with other regulatory elements are responsible for constitutive and tissue-specific CD5 expression. PMID: 15187131 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Vitam Horm. 2004;67:35-49. Promoter of TRAIL-R2 gene. Yoshida T, Sakai T. Department of Molecular-Targeting Cancer Prevention Graduate School of Medical Science, Kyoto Prefectural University of Medicine Kyoto 602-8566, Japan. TRAIL-R2 promoter does not have a typical TATA-box but two functional Sp1-binding sites. TRAIL-R2 promoter belongs to the class of TATA-less and GC-box-containing promoters. The minimal promoter element is contained in the region spanning -198 to -116 upstream of translational initiation codon ATG. Computer analysis shows putative transcription factor binding sites such as c-Ets, AML-1a, c-Myb, Sp1, and GATA-1 in TRAIL-R2 promoter. Hypermethylation of TRAIL-R2 is not frequent compared with that of TRAIL-R3 and TRIAL-R4. There are no potential transcription factor binding sites in highly homologous regions between TRAIL-R2 promoter and TRAIL-R1 promoter, or between TRAIL-R2 promoter and mouse homologue mouse killer (MK) promoter. TRAIL-R2 is known to be a downstream gene of p53, a tumor-suppressor gene, and a p53-binding site in TRAIL-R2 intron 1 is responsible for p53-dependent transcription. Thapsigargin, endoplasmic reticulum Ca(2+)-ATPase inhibitor calcium releaser, upregulates TRAIL-R2 expression via the promoter region. Many regulators of TRAIL-R2 have been reported. However, it has not been demonstrated whether they regulate TRAIL-R2 via the promoter region. Here, we show a list of these regulators. Finally, we demonstrate the possibility of cancer therapy using regulation of TRAIL-R2 promoter. Publication Types: Review Review, Tutorial PMID: 15110170 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Biochem Biophys Res Commun. 2004 May 14;317(4):1096-102. Activation of mouse RAG-2 promoter by Myc-associated zinc finger protein. Wu CX, Zhao WP, Kishi H, Dokan J, Jin ZX, Wei XC, Yokoyama KK, Muraguchi A. Department of Immunology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630, Sugitani, Toyama 930-0194, Japan. Recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in lymphocytes undergoing the antigen receptor gene rearrangement during the lymphocyte development. Our previous study showed that the -41 to -17 nucleotides (nt) 5' -upstream region of mouse RAG-2 were pre-requisite for the core promoter activity and that Pax-5/c-Myb/LEF-1 protein-protein complex was responsible for its activity in immature B cells. In this study, we show that the -65/-42 sequence, the non-conserved sequence between human and mouse RAG-2 promoter, is necessary for the full promoter activity for mouse RAG-2. Electrophoresis mobility shift assay revealed that Myc-associated zinc finger protein (MAZ) as well as SP1/3 binds a GA box in this region. Using chromatin immunoprecipitation, we show that MAZ binds the RAG-2 promoter region in pre-B cells. Furthermore, we show that MAZ synergistically activates the murine RAG-2 promoter with Pax-5/c-Myb/LEF-1 complex. These results first demonstrate that MAZ participates in activation of mouse RAG-2 promoter. PMID: 15094381 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Cytogenet Genome Res. 2003;102(1-4):309-17. The chicken telomerase RNA gene: conservation of sequence, regulatory elements and synteny among viral, avian and mammalian genomes. Delany ME, Daniels LM. Department of Animal Science, University of California, Davis, CA 95616, USA. medelany@ucdavis.edu Telomerase RNA (TR) is essential for telomerase activity and the maintenance of telomere length in proliferating cell populations. The objective of the present research was to define the cytogenetic and molecular genomic organization of chicken TR (chTR). The chTR exists as a single copy gene (TERC, alias TR), mapping to chromosome 9 (GGA9). The loci on the q arm of GGA9 map to three chromosomes in human with five of the nine GGA9q loci mapping to HSA3q. Sequencing of the chTERC locus (3,763 bp) from the UCD 001 genome (Red Jungle Fowl) included: 604 bp 5', 465 coding, and 2,694 bp 3' (from -604 to +3159). Sequence analysis included homology searches conducted on several levels including comparisons among different chicken genotypes, Marek's disease virus (MDV) sequences, plus human and murine. We provide evidence for distal 5' and 3' sequence homology between chTERC and the MDV genome among other known regions of homology (promoter and coding), elaborate on 5' transcription factor binding motifs among the various genomes as well as show type and number of TERT-related motifs 3' of chicken TR (e.g., Sp1, c-Myb, c-Myc, AP2, among others). Surrounding the gene are more than 25 Sp1 sites, over 20 oncogene transcription factor binding motifs and numerous hormonal and other specialized binding motifs. Knowledge of 5' and 3' chTERC regulatory elements will be useful for investigating normal control mechanisms during growth and development as well as investigating the potential for dysregulation of this important gene during oncogenesis, especially among different genotypes. Copyright 2003 S. Karger AG, Basel PMID: 14970722 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Biochem J. 2004 Mar 1;378(Pt 2):609-16. B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts. Cicchillitti L, Jimenez SA, Sala A, Saitta B. Division of Rheumatology, Department of Medicine, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA. We investigated the role of B-Myb, a cell-cycle-regulated transcription factor, in the expression of the alpha1 (I) pro-collagen gene (COL1A1) in scleroderma fibroblasts. Scleroderma or SSc (systemic sclerosis) is a fibrotic disease characterized by excessive production of extracellular matrix components, especially type I collagen. Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts. Overexpression of B-Myb in SSc fibroblasts was correlated with decreased COL1A1 mRNA expression. Transient transfections localized the down-regulatory effect of B-Myb to a region containing the proximal 174 bp of the COL1A1 promoter that does not contain B-Myb consensus binding sites. Gel-shift analysis, using nuclear extracts from normal and SSc fibroblasts transfected with B-Myb, showed no differences in DNA-protein complex formation when compared with the nuclear extracts from mock-transfected cells. However, we found that B-Myb decreases Sp1 (specificity protein 1) and CBF (CCAAT-binding factor) binding for their specific sites localized in the 174 bp COL1A1 proximal promoter. These results were also confirmed using B-Myb-immunodepleted nuclear extracts. Furthermore, immunoprecipitation assays using SSc nuclear extracts demonstrated a physical interaction of B-Myb with Sp1 and CBF transcription factors, and also an interaction between Sp1 and CBF. In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain. Thus these results suggest that B-Myb may be an important factor in the pathway(s) regulating collagen production in SSc fibroblasts. PMID: 14613485 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Biochem Biophys Res Commun. 2003 Oct 3;309(4):910-6. Effect of cysteamine on redox-sensitive thiol-containing proteins in the duodenal mucosa. Khomenko T, Deng X, Jadus MR, Szabo S. Pathology and Laboratory Medicine Service, Diagnostic and Molecular Medicine Health Care Group, VA Medical Center, Long Beach, CA 90822, USA. Recent studies from our laboratory demonstrated that Egr-1 is upregulated in the rat duodenal mucosa during cysteamine-induced duodenal ulceration and that antisense egr-1 oligonucleotide aggravates the duodenal ulcers. This study was aimed to determine the effects of cysteamine on redox-sensitive Egr-1 transcriptional activity and on other thiol-containing proteins such as redox factor-1 (Ref-1) and thioredoxin (Trx). Here we demonstrate for the first time that cysteamine increases the expression and nuclear translocation of Egr-1, Ref-1, and Trx, and activates binding of Egr-1 to DNA. Moreover, we also show that Egr-1 forms a complex with other redox-sensitive transcription factors (e.g., AP-1, AP-2, NFATc, Sp1, PAX-5, MTF-1, c-Myb, and CREB) in rat duodenal mucosa and that cysteamine enhances the formation of these complexes. The antioxidant ebselen markedly elevated the nuclear Ref-1 expression and Egr-1/DNA binding, and decreased the ulcerogenic effect of cysteamine as did catalase. Thus, redox-sensitive signaling systems seem to play an important role in cysteamine-induced duodenal ulceration. PMID: 13679060 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Genes Chromosomes Cancer. 2003 Oct;38(2):157-67. Site-directed mutagenesis of the ATM promoter: consequences for response to proliferation and ionizing radiation. Gueven N, Keating K, Fukao T, Loeffler H, Kondo N, Rodemann HP, Lavin MF. Queensland Cancer Fund Research Laboratory, The Queensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Queensland, Australia. Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. Copyright 2003 Wiley-Liss, Inc. PMID: 12939743 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Lung Cancer. 2002 Dec;38(3):229-34. The promoter region of the human BUBR1 gene and its expression analysis in lung cancer. Seike M, Gemma A, Hosoya Y, Hosomi Y, Okano T, Kurimoto F, Uematsu K, Takenaka K, Yoshimura A, Shibuya M, Ui-Tei K, Kudoh S. Fourth Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers. PMID: 12445743 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Cell Death Differ. 2002 Nov;9(11):1232-9. Cyclin D1-dependent regulation of B-myb activity in early stages of neuroblastoma differentiation. Cesi V, Tanno B, Vitali R, Mancini C, Giuffrida ML, Calabretta B, Raschella G. ENEA Research Center Casaccia, Biotechnology Unit, Section of Toxicology and Biomedical Sciences, Via Angullarese, 301, 00060 S Maria di Galeria Rome, Italy. Levels of the transcription factor B-myb must be down-regulated to allow terminal differentiation of neuroectodermal cells and yet its constitutive expression induces early markers of neural differentiation. Thus, we investigated potential mechanisms of enhanced B-myb activity in early stages of neural differentiation. We report here that B-myb expression does not decrease, cyclin A and Sp1 levels remain constant while p21 levels increase continuously upon retinoic acid-induced differentiation of the LAN-5 neuroblastoma cell line. In contrast, cyclin D1 expression is down-regulated at the onset of the differentiative process by protein destabilization. Luciferase assays of promoter activity indicate that B-myb-dependent transactivation is enhanced in LAN-5 cells treated with retinoic acid (RA) for 24 h. The enhancement is independent from cyclin A but is suppressed by a degradation-resistant mutant form of cyclin D1. The importance of cyclin D1 in controlling B-myb activity is further suggested by co-immunoprecipitation experiments, showing that the amount of cyclin D1 co-immunoprecipitated with B-myb decreased after RA treatment. Thus, B-myb may play an active role in the early stages of differentiation when its transactivation activity is enhanced as a consequence of cyclin D1 down-modulation. PMID: 12404122 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Mol Immunol. 2002 Jun;38(15):1151-9. Combinatorial regulation of the murine RAG-2 promoter by Sp1 and distinct lymphocyte-specific transcription factors. Miranda GA, Villalvazo M, Galic Z, Alva J, Abrines R, Yates Y, Evans CJ, Aguilera RJ. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles 405 Hilgard Ave, Los Angeles, CA 90095-1606, USA. The recombination activation genes, RAG-1 and RAG-2, encode the critical components of the recombinase complex responsible for the generation of functional antigen receptor genes. In order to gain an insight into the transcription factors and cis-acting elements that regulate the lymphocyte-specific expression of RAG-2, the promoter-region of this gene was isolated and characterized. This analysis demonstrated that a relatively small promoter fragment could confer lymphocyte-restricted expression to a reporter construct. Our work and that of others subsequently revealed that RAG-2 promoter expression is positively regulated by BSAP (PAX-5) and c-Myb transcription factors in B- and T-lineage cells, respectively. Although BSAP and c-Myb were deemed necessary for lymphocyte-specific expression, our analysis also uncovered a G-rich region at the 5'-end of the core promoter that was essential for full activity in lymphocyte cell lines. Site-directed mutagenesis revealed that a GA-box within the G-rich region was required for full promoter activity and subsequent DNA binding assays demonstrated that this element was specifically recognized by Sp1. Apart from showing that Sp1 interacts within the RAG-2 promoter, we also demonstrate that the Sp1-binding site is necessary for the high-level activation of this promoter. PMID: 12044781 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Oncogene. 2002 May 13;21(21):3377-90. Transcriptional regulation of granulocyte and monocyte development. Friedman AD. Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, MD 21231, USA. adfrdman@jhmi.edu Granulocytes and monocytes develop from a common myeloid progenitor. Early granulopoiesis requires the C/EBPalpha, PU.1, RAR, CBF, and c-Myb transcription factors, and terminal neutrophil differentiation is dependent upon C/EBPepsilon, PU.1, Sp1, CDP, and HoxA10. Monopoiesis can be induced by Maf-B, c-Jun, or Egr-1 and is dependent upon PU.1, Sp1, and ICSBP. Signals eminating from cytokine receptors modulate factor activities but do not determine cell fates. Orchestration of the myeloid developmental program is achieved via cooperative gene regulation, via synergistic and inhibitory protein-protein interactions, via promoter auto-regulation and cross-regulation, via regulation of factor levels, and via induction of cell cycle arrest: For example, c-Myb and C/EBPalpha cooperate to activate the mim-1 and NE promoters, PU.1, C/EBPalpha, and CBF, regulate the NE, MPO, and M-CSF Receptor genes. PU.1:GATA-1 interaction and C/EBP suppression of FOG transcription inhibits erythroid and megakaryocyte gene expression. c-Jun:PU.1, ICSBP:PU.1, and perhaps Maf:Jun complexes induce monocytic genes. PU.1 and C/EBPalpha activate their own promoters, C/EBPalpha rapidly induces PU.1 and C/EBPepsilon RNA expression, and RARalpha activates the C/EBPepsilon promoter. Higher levels of PU.1 are required for monopoiesis than for B-lymphopoiesis, and higher C/EBP levels may favor granulopoiesis over monopoiesis. CBF and c-Myb stimulate proliferation whereas C/EBPalpha induces a G1/S arrest; cell cycle arrest is required for terminal myelopoiesis, perhaps due to expression of p53 or hypo-phosphorylated Rb. Publication Types: Review PMID: 12032776 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: J Biochem (Tokyo). 2001 Jul;130(1):39-50. Expression and exon/intron organization of two medaka fish homologs of the mammalian guanylyl cyclase A. Yamagami S, Suzuki K, Suzuki N. Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. Two cDNA clones (OlGC2 and OlGC7) and their genomic DNA clones encoding medaka fish homologs of mammalian natriuretic peptide receptor/membrane guanylyl cyclase A (GC-A) were isolated, and their complete nucleotide sequences were determined. The open reading frame predicts a protein of 1,063 amino acids for OlGC2 cDNA (4,283 bp), and one of 1,055 amino acids for OlGC7 cDNA (3,721 bp), respectively. Northern blot analyses demonstrated 4.7 kb OlGC2 transcripts in the kidney and gill, and 4.0 kb OlGC7 transcripts in the kidney, brain, and ovary, while RNase protection analyses revealed that both genes are expressed in various adult organs. Both the OlGC2 (about 33.0 kbp) and OlGC7 (about 44.3 kbp) genes consist of 22 exons with an exon/intron organization similar to those of the human GC-A gene (about 16.6 kbp) and medaka fish GC-B homolog gene (OlGC1, about 93 kbp). Intron 4 of OlGC2 contains two repeated sequence (RS) clusters, designated as RS1 (about 1 kbp) and RS2 (about 5 kbp), consisting of nucleotide 5'-AGCCTCTGCTCCTCCTTC-3'. In addition, many identical but variably sized nucleotide sequences were found in introns in OlGC1, OlGC2, OlGC6, and OlGC7. The OlGC2 and OlGC7 genes both have no apparent TATA box in the 5' flanking region upstream of the putative transcription initiation point, but several consensus sequences for cis-regulatory elements, including C/EBP, CREB, NF-IL6, and Sp1 and AP-2, NF-IL6, c-Myb, and Sp1 are present in the 5'-flanking region of OlGC2 and OlGC7, respectively. PMID: 11432778 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: FASEB J. 2001 Jul;15(9):1507-16. The role of c-Myb and Sp1 in the up-regulation of methionine adenosyltransferase 2A gene expression in human hepatocellular carcinoma. Yang H, Huang ZZ, Wang J, Lu SC. Division of Gastroenterology and Liver Diseases, USC Liver Disease Research Center, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine USC, Los Angeles, California 90033, USA. Liver-specific and non-liver-specific methionine adenosyltransferase (MAT) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine. We showed a switch from MAT1A to MAT2A expression at the transcriptional level in human hepatocellular carcinoma (HCC) that facilitates cancer cell growth. The purpose of the present study was to better understand the molecular mechanism of increased MAT2A expression in HCC. In vitro DNase I footprinting analysis revealed two protected sites (-354 to -312 and -73 to -28) using nuclear proteins from HCC and HepG2 cells, but not normal liver. These sites are also protected in HepG2 cells on in vivo DNase I footprinting analysis. These protected sites contain consensus binding sites for c-Myb and Sp1. In HCC, the mRNA levels of c-myb and Sp1 and binding to their respective sites increased. Mutation of the c-Myb or Sp1 site reduced MAT2A promoter activity by 67% and 50%, respectively. The importance of these cis-acting elements and trans-activating factors was confirmed using heterologous promoter and expression vectors. Increased expression of c-Myb and Sp1 and binding to the MAT2A promoter contribute to transcriptional up-regulation of MAT2A in HCC.-Yang, H., Huang, Z.-Z., Wang, J., Lu, S. C. The role of c-Myb and Sp1 in the up-regulation of methionine adenosyltransferase 2A gene expression in human hepatocellular carcinoma. PMID: 11427482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Gene. 2001 May 30;270(1-2):221-9. Genomic organization and characterization of the promoter region of murine GSTM2 gene. Kumar A, Reddy EP. Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA 19140, USA. In this study, we have isolated the genomic clone of the murine GSTM2 gene and determined its sequence. Consistent with the class mu genefamily, the mGSTM2 gene consists of eight exons. The exon-intron boundaries and the distribution of coding sequences within the exons of the known GST class mu family members were found to follow a similar pattern suggesting that various members of this family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. By primer extension, the start of transcription was determined to be 40 bp upstream of the initial AUG codon. To gain an understanding of the mGSTM2 regulation, we have also cloned and analyzed its promoter region. Maximal activity was observed in a 170 bp 5'-flanking region. The activity was decreased by 3-fold in a 402 bp 5'-flanking region suggesting the presence of repressor elements. While no TATA box was identified, the presence of an SP1 site at position -38 was noted. Deletion of this SP1 site completely abrogated promoter activity. The promoter contained eight putative Myb responsive elements and its transcriptional activity was upregulated by t-Myb but not by c-Myb. PMID: 11404019 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Genomics. 2000 Sep 1;68(2):167-78. Isolation, characterization and targeted disruption of mouse ppia: cyclophilin A is not essential for mammalian cell viability. Colgan J, Asmal M, Luban J. Department of Microbiology, Department of Medicine, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, New York, 10032, USA. Cyclophilins (CyPs) are a family of proteins found in organisms ranging from prokaryotes to humans. These molecules exhibit peptidyl-prolyl isomerase activity in vitro, suggesting that they influence the conformation of proteins in cells. CyPs also bind with varying affinities to the immunosuppressive drug cyclosporin A (CsA), a compound used clinically to prevent allograft rejection. The founding member of the family, cyclophilin A (CyPA), is an abundant, ubiquitously expressed protein of unknown function that binds with nanomolar affinity to CsA. Here, we describe the isolation and characterization of mouse Ppia (mPpia), the gene encoding CyPA. Ppia was isolated using a PCR screen that distinguishes the expressed gene from multiple pseudogenes present in the mouse genome. mPpia consists of 5 exons and 4 introns spanning roughly 4.5 kb and maps to chromosome 11 near the centromere. Sequence analysis of a 369-bp fragment from the proximal promoter region of mPpia revealed the presence of a TATA box and sites recognized by several transcriptional regulators, including Sp1, AP-2, GATA factors, c-Myb, and NF-IL-6. This region is sufficient to drive high-level reporter gene expression in transfected cells. Both copies of Ppia were disrupted in murine embryonic stem (ES) cells via gene targeting. Ppia(-/-) ES cells grow normally and differentiate into hematopoeitic precursor cells in vitro, indicating that CyPA is not essential for mammalian cell viability. Copyright 2000 Academic Press. PMID: 10964515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Oncogene. 2000 Aug 10;19(34):3931-40. Isolation and characterization of the human A-myb promoter: regulation by NF-Y and Sp1. Facchinetti V, Lopa R, Spreafico F, Bolognese F, Mantovani R, Tavner F, Watson R, Introna M, Golay J. Department of Immunology and Cell Biology, Istituto Ricerche Farmacologiche Mario Negri, Milan, Italy. The A-myb transcription factor shows a restricted tissue distribution and is cell cycle regulated. Furthermore its deregulation has profound effects on the growth and/or differentiation of the cells in which it is normally expressed. We have therefore characterized its promoter. A 12 kb genomic clone was isolated that comprises the first exon, part of the first intron as well as upstream regulatory sequences. Multiple transcription start sites have been identified which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundaries of the minimal promoter region (-183-14), of a positive upstream (-538-183) and a negative downstream regulatory region (NRE) (+83+374) have been defined. The NRE is promoter- and orientation-independent but position specific. The A-myb minimal promoter is GC-rich, does not contain any TATA box but has a functional CCAAT box. The CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence. The CCAAT box efficiently binds the NF-Y complex and its mutation decreases basal promoter activity by 50%. Two Sp1 binding sites are present upstream from the CCAAT box which can bind Spl and contribute to A-myb promoter activity by 70 and 30%, respectively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory elements. Finally, we show that the promoter is cell cycle regulated and that the SP1 and CCAAT elements are required for S phase induction. PMID: 10951586 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Endocrinology. 2000 Feb;141(2):763-71. Characterization of the murine pituitary tumor transforming gene (PTTG) and its promoter. Wang Z, Melmed S. Cedars-Sinai Research Institute, University of California School of Medicine, Los Angeles 90048, USA. We recently isolated rat pituitary tumor transforming gene (PTTG) complementary DNA and showed its potent in vitro and in vivo transforming activity. We now characterize the mouse PTTG gene and its promoter. The entire gene is composed of five exons and four introns and spans about 7 kb. Northern analysis showed that PTTG was expressed in several tumor cell lines examined, but not in all normal tissues, implying a correlation between PTTG and tumorigenesis. Using rapid amplification of 5'-cDNA ends, the transcription start site was localized at -303 nucleotides upstream to the ATG codon in both F9 and AtT20 cells. An approximately 4.3-kb upstream region demonstrated promoter activity in AtT20 cells as well as other cell lines tested, and in vivo, the cloned promoter driving an enhanced green fluorescent protein transgene exhibited transcriptional activation in testis and embryo. Serial deletions showed that -313 bp of the 5'-flanking region was critical for promoter activity. Three elements contribute to promoter activity. Both element A (-313/-293) and element C (-180/-160), in an electrophoretic mobility shift assay using NIH-3T3 nuclear extract, formed three specific complexes, which were competed by a known Sp1 oligo; one complex was supershifted by Sp1 antibody, and the other two complexes were both supershifted by an Sp3 antibody. Two mutants disrupting element A resulted in up to 70% loss of promoter activity and abrogated formation of specific DNA-protein binding complexes, implying a more important role for element A. Element B (-249/-229) shows more than 80% homology to a consensus c-myb element, but formed two specific complexes that differed from that of c-myb in the electrophoretic mobility shift assay. Thus, the integrity and possible cooperation among these elements contribute to the basal promoter activity of the mouse PTTG oncogene homolog. PMID: 10650958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Anticancer Drug Des. 1999 Jun;14(3):179-86. Effect of ecteinascidin-743 on the interaction between DNA binding proteins and DNA. Bonfanti M, La Valle E, Fernandez Sousa Faro JM, Faircloth G, Caretti G, Mantovani R, D'Incalci M. Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy. Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, a tunicate growing in mangrove roots in Caribbean. It has been shown to bind in the minor groove of DNA forming covalent adducts by reaction of the N2 of guanine with the carbinolamine moiety. We investigated ET-743 ability to inhibit the binding of different transcription factors to their consensus sequences by using gel shift assays. We have selected three types of factors: (i) oncogene products such as MYC, c-MYB and Maf; (ii) transcriptional activators regulated during the cell cycle as E2F and SRF; and (iii) general transcription factors such as TATA binding protein (TBP), Sp1 and NF-Y. We observed no inhibition of the binding of Sp1, Maf, MYB and MYC. Inhibition of DNA binding was observed for TBP, E2F, SRF at ET-743 concentrations ranging from 50 to 300 microM. The inhibition of binding of NF-Y occurs at even lower concentrations (i.e. 10-30 microM) when the recombinant subunits of NF-Y are preincubated with the drug, indicating that the inhibition of NF-Y binding does not require previous ET-743 DNA binding. Since NF-Y is a trimer containing two subunits with high resemblance to histones H2B and H2A, we have investigated the effect of ET-743 on nucleosome reconstitution. ET-743 caused a decrease of the nucleosomal band at 100 nM, with the complete disappearance of the band at 3-10 microM. These data suggest that the mode of action of this novel anticancer drug is related to its ability to modify the interaction between some DNA binding proteins and DNA. PMID: 10500494 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: DNA Res. 1999 Feb 26;6(1):21-7. Structural organization of the human Elk1 gene and its processed pseudogene Elk2. Yamauchi T, Toko M, Suga M, Hatakeyama T, Isobe M. Department of Materials and Biosystem Engineering, Faculty of Engineering, Toyama University, Toyama-city, Japan. In the ets gene family of transcription factors, ELK1 belongs to the subfamily of Ternary Complex Factors (TCFs) which bind to the Serum Response Element (SRE) in conjunction with a dimer of Serum Response Factors (SRFs). The primary structure of the human Elk1 gene was determined by genomic cloning. The gene structure of Elk1 spans 15.2 kb and consists of seven exons and six introns. The coding sequence resides on exons 3, 4, 5, 6 and 7. Sequencing of cDNA clones isolated from human hippocampus library revealed that the second exon was often skipped by an alternative splicing event. All introns commenced with nucleotides GT at the 5' boundary and ended with nucleotides AG at the 3' boundary, in agreement with the proposed consensus sequence for intron spliced donor and acceptance sites. Sequence inspection of the 5'-flanking region revealed the absence of a 'TATA' box and the presence of putative cis-acting regulatory elements such as Sp1, GATA-1, CCAAT, and c-Myb. Moreover, the sequence analysis of Elk2 locus on 14q32.3 confirmed that Elk2 gene corresponds to a processed pseudogene of Elk1 which has been reported between alpha 1 gene (IGHA1) and pseudo gamma gene (IGHGP) of immunoglobulin heavy chain. Furthermore, the results of Southern analysis using DNAs from human-mouse hybrid cell lines carrying a part of 14q32 region revealed that there is another locus hybridizing to Elk1 cDNA on 14q32.2 --> qter region in addition to Elk2 locus between IGHA1 and IGHGP loci. PMID: 10231026 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Biochem Biophys Res Commun. 1999 Mar 5;256(1):104-9. The identification and characterization of two promoters and the complete genomic sequence for the Wiskott-Aldrich syndrome gene. Hagemann TL, Kwan SP. Department of Immunology/Microbiology, Rush Medical School, Chicago, Illinois, 60612, USA. The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immunodeficiency, eczema and thrombocytopenia. The gene responsible for WAS was identified through positional cloning, and the function of the encoded protein (WASP) is still the subject of much speculation. WASP is currently thought to be involved in the regulation of actin polymerization in hematopoietic cells. To study the elements that regulate the WASP gene, we have identified the sites for transcription initiation. We found that two promoters were responsible for controlling WASP expression. Multiple transcription initiation sites were found immediately adjacent to the translation start site, however an alternate exon with a second promoter region was identified 6 kb upstream. Examination of the 5' sequence adjacent to the initiation sites in both promoters failed to reveal a TATA or CCAAT box, but numerous putative transcription factor binding sites including Sp1, Ets, c-Myb and PU.1 were apparent. Reporter constructs generated from each promoter showed functional activity in the Jurkat T-cell and HEL erythro-megakaryocytic cell lines. Although the alternate exon sequence was extremely GC rich and contained several potential binding elements, the primary promoter was stronger than the upstream promoter in the cell lines assayed. The transcription factor binding site profiles within each promoter suggested that they may play different roles in regulating WASP expression depending on the stage of differentiation and development, and the cell lineage. In this study we have also reported the complete nucleotide sequence of the coding and intervening sequences for the WASP gene. A comprehensive knowledge of the genomic structure and the further characterization of WASP gene expression will facilitate the continued investigation of mutations in WAS patients, and the eventual prospect of gene therapy. Copyright 1999 Academic Press. PMID: 10066431 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Oncogene. 1999 Feb 11;18(6):1333-9. B-MYB transactivates its own promoter through SP1-binding sites. Sala A, Saitta B, De Luca P, Cervellera MN, Casella I, Lewis RE, Watson R, Peschle C. Department of Molecular Pharmacology and Pathology, Consorzio Mario Negri Sud, S. Maria Imbaro (CH), Italy. B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family. PMID: 10022815 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Genomics. 1998 Sep 15;52(3):312-24. The human cystathionine beta-synthase (CBS) gene: complete sequence, alternative splicing, and polymorphisms. Kraus JP, Oliveriusova J, Sokolova J, Kraus E, Vlcek C, de Franchis R, Maclean KN, Bao L, Bukovsk, Patterson D, Paces V, Ansorge W, Kozich V. Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado 80262, USA. JAN.KRAUS@UCHSC.edu Cystathionine beta-synthase [CBS; l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains approximately 5 kb of the 5' flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5' UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3' UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (approximately 80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number of Alu repeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians. Copyright 1998 Academic Press. PMID: 9790750 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: J Leukoc Biol. 1998 Feb;63(2):153-68. Myeloid-specific gene expression. Clarke S, Gordon S. Sir William Dunn School of Pathology, University of Oxford, United Kingdom. The mononuclear phagocytes are recruited from bone marrow precursors to most tissues of the body, particularly during inflammation or immune stimulation. This combination of accessibility as stem cells and heterogenity of tissue locations makes the myeloid cell potentially important as a carrier of therapeutic agents. Understanding the regulation of transcription in myeloid cells is necessary for any future design of tissue-specific gene targeting vectors, particularly if there are inherent size limitations. Identified members of the C/EBP, Runt/PEBP2/CBF, and Ets families of transcription factors are critical for myeloid-specific gene expression and may have myeloid-restricted expression or myeloid-specific regulation in the hematopoietic system. AP-1, Sp1, and Myb appear to be important for myeloid-restricted expression in some cases. In addition, factors involved in the up-regulation of the level of gene expression when macrophages are activated by agents such as interferon-gamma and bacterial products have been identified. Some of the sequences to which these transcription factors bind in myeloid-restricted genes have been tested in cell lines and in transgenic mice and it is now possible to make an attempt to describe the characteristics of a myeloid-specific promoter. Publication Types: Review PMID: 9468274 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Blood. 1997 Nov 15;90(10):4135-43. Murine macrophage mannose receptor promoter is regulated by the transcription factors PU.1 and SP1. Eichbaum Q, Heney D, Raveh D, Chung M, Davidson M, Epstein J, Ezekowitz RA. Division of Infectious Diseases and Hematology/Oncology, Children's Hospital, Boston, MA 02114, USA. The mannose receptor (MR) is a transmembrane protein that functions primarily as a phagocytic receptor for a wide range of microorganisms. Its expression appears to be restricted to tissue macrophages and Langerhans cells. To gain an understanding of the regulation of the gene, we have isolated the 5' flanking sequence of the murine MR gene and have analyzed a 536-bp sequence upstream of the ATG start site for transcriptional activity. This sequence lacks a TATA box but contains an initiator (Inr) consensus element overlapping the single transcriptional start site. Transcription factor binding sites contained within this sequence include PU.1, Sp1, ETS, GATA, and MYB motifs. Serial 100-bp deletions of this promoter fragment fused to a luciferase reporter gene showed various patterns of activity when transfected into different cell types. In myeloid cells, sequence elements upstream of bp -300 appeared to have a silencing effect on promoter activity. Of the four potential PU.1 binding sites contained within the fragment, one site (at -164) bound the PU.1 factor most strongly, whereas the adjacent PU.1 site (at -177 bp) bound PU.1 to a lesser degree. Mutations of these sites decreased transcriptional activity but did not abolish it. However, promoter activity was abrogated when both the -164 bp PU.1 site and the adjacent -177 bp PU.1 site were mutated. In addition, mutation of the Sp1 site also significantly reduced promoter activity. Cotransfection studies in Drosophila Schneider cells indicated that PU.1 and Sp1 may function synergistically in transactivating the murine MR. This study indicates that MR gene expression is regulated in part by the interaction between the ubiquitously expressed factor Sp1 and the lymphoid/myeloid factor PU.1 and provides a basis for studying the regulation of this gene. PMID: 9354684 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Cells. 1997 Aug 31;7(4):537-43. Characterization of the murine cyclin D2 gene: exon/intron organization and promoter activity. Jun DY, Kim MK, Kim IG, Kim YH. Department of Microbiology, College of Natural Sciences, Kyungpook National University, Taegu, Korea. Cyclin D2 is normally expressed in G1 and promotes progression through G1 of the cell cycle. From a murine genomic library constructed with spleen DNA, two overlapping genomic clones of cyclin D2 were isolated. These clones contain most of the exon of cyclin D2 except exon 5. Characterization of these clones revealed that murine cyclin D2 mRNA spans over 18 kb and 5 exons ranging from 149 to approximately 462 bp in length, and suggested that exon 5 may be at least >5 kb downstream from exon 4. Primer extension analysis of cyclin D2 mRNA isolated from murine activated T cells detected 5 putative sites of transcription initiation. These are located at - 499, - 417, - 391, - 373, and - 349 relative to the translation start site, which is given as + 1. No consensus sequence for TATA box existed at an appropriate position within the promotor region. Instead, several putative transcriptional factor binding sites for C/EBP, PEA3, AP2, NF-Y, Sp1, c-Myc, GATA-1, AP1, v-Myb, and CREB were detected. The 5'-flanking region of the cyclin D2 gene up to nucleotide - 945 shared about 61% sequence homology between mouse and human. Functional analysis of promoter activity of the 5'-flanking region of cyclin D2 suggested that the region - 1,100 to - 805 including C/EBP, PEA3, AP2, NF-Y, c-Myc, and Sp1 may have a major positive regulatory activity for expression of cyclin D2. PMID: 9339900 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Genomics. 1997 Sep 1;44(2):195-200. Gene structure, cDNA sequence, and expression of murine Bak, a proapoptotic Bcl-2 family member. Ulrich E, Kauffmann-Zeh A, Hueber AO, Williamson J, Chittenden T, Ma A, Evan G. Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom. To facilitate the creation of Bak knockout mice and the further analysis of this Bcl-2 family member, we have isolated and sequenced the complete mouse Bak cDNA. The cDNA is 2 kb long and shares an overall nucleotide identity to the human Bak cDNA of 62%. The mouse Bak protein is 208 amino acids long with a predicted molecular weight of 23 kDa. The mouse Bak mRNA could be detected in all mouse tissues examined. In addition we mapped the murine bak gene. It consists of six exons spanning about 10 kb on chromosome 17B. The 5' region of the murine bak gene is unmethylated on the dinucleotide CpG in the area around exon 1. Furthermore, it contains potential binding sites for transcription factors such as Sp1 and c-Myb. Copyright 1997 Academic Press. PMID: 9299236 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Leuk Lymphoma. 1997 May;25(5-6):415-25. CD11c integrin gene promoter activity during myeloid differentiation. Corbi AL, Lopez-Rodriguez C. Instituto de Parasitologia y Biomedicina, Granada, Spain. acorbi@ipb.csic.es The integrin CD11c/CD18 functions as a cell surface receptor for numerous soluble factors and proteins (LPS, fibrinogen, iC3b), mediates leukocyte interactions with other cell types and is a signal transducing receptor. CD11c/CD18 is found primarily on myeloid cells, where its expression is regulated both during differentiation and during monocyte maturation into tissue macrophages. To determine the transcription factors and cis-acting elements driving the developmentally-regulated expression of CD11c/CD18 the proximal regulatory region of the CD11c gene has been structurally and functionally characterized using the U937 and HL-60 cell lines as myeloid differentiation models. The tissue-specific activity of the CD11c promoter is conferred by two Sp1-binding sites and an adjacent C/EBP-binding element, with a likely contribution from other transcription factors with a more limited tissue distribution (PU.1, Oct-2, Myb). The participation of Sp1 in the transcription of the CD11c gene strongly suggests that CD11c/CD18 expression is dependent on the proliferative state of the cell, thus establishing a first level of control for the regulated expression of CD11c/CD18 during myeloid differentiation. The differentiation responsiveness of the CD11c promoter has been mapped to an AP-1-binding site whose mutation greatly decreases the inducibility of the promoter during the PMA-triggered differentiation of U937 cells. Although AP-1 mediates the responsiveness to several other differentiating agents including GM-CSF, additional elements are required for induction of the CD11c promoter activity upon Sodium Butyrate-triggered differentiation. In fact, the Sodium Butyrate-responsiveness and the presence of both AP-1- and C/EBP-binding sites suggests that the proximal regulatory region of the CD11c promoter might include an extracellular matrix-response element. As a whole, the transcription of the CD11c gene appears to be controlled by the proliferative state of the cell and is tightly coupled to progression along the myeloid differentiation pathway. The differentiation inducibility of the CD11c promoter has been further demonstrated after stable transfection into U937 cells, where the -361/+43 fragment retains the capacity to drive luciferase expression upon PMA-, GM-CSF- or Sodium Butyrate-triggered myeloid differentiation. Thus, while the characterization of the transcription factors regulating CD11c expression is still in progress, the CD11c promoter has been shown to constitute a very useful tool for the identification of myeloid-differenting agents which might be of potential therapeutical interest. Publication Types: Review Review, Tutorial PMID: 9250811 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: J Cell Biochem. 1997 May;65(2):231-44. Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: evidence for the role of Sp1 and not of c-Myb or PU.1 in myelomonocytic lineage-specific expression. Kao WY, Briggs JA, Kinney MC, Jensen RA, Briggs RC. Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-5310, USA. The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelomonocytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in bone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to -16) that did not include the cluster of c-Myb sites. A 4-bp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22) including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4-bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super-shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene. PMID: 9136080 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Gene. 1996 Nov 21;180(1-2):113-20. Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1. Kwon TK, Nagel JE, Buchholz MA, Nordin AA. Clinical Immunology Section, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-2780, USA. The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression. p27Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation. We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene. The gene consists of at least three exons and spans more than 5.6 kb of DNA. Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites. The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1, CRE, Myb and NFkB located at positions -153, -178, -286, -875, and -1011, respectively. To analyze the regulatory mechanisms controlling p27Kip1 gene expression, we characterized the 5'-flanking region from nt -1609 to +178. The -326 to -615 region contained positive regulatory elements. PMID: 8973354 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Biochim Biophys Acta. 1996 Sep 11;1308(3):201-4. Characterization of the human myeloid cell nuclear differentiation antigen gene promoter. Kao WY, Dworkin LL, Briggs JA, Briggs RC. Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232-5310, USA. MNDA (myeloid cell nuclear differentiation antigen) is an interferon alpha regulated nuclear protein expressed only in cells of the human myelomonocytic lineage. To identify mechanisms responsible for this lineage-specific and interferon-regulated expression, the 5' flanking sequence of the gene has been characterized. Two interferon-stimulated response elements (ISRE) flank a multiple transcription start site region identifying MNDA as a TATA-less interferon-regulated gene. Other DNA elements present include a cluster of Myb sites, several Ets, an Ets related PU.1 site and an Sp1 site located within 600 bp of the transcription start sites. In addition, DNA methylation was revealed as one of the possible factors in establishing MNDA expression. The 5' flanking sequence has promoter activity which is elevated by interferon alpha. The findings indicate that MNDA expression is regulated by mechanisms similar to other myelomonocytic cell specific genes and genes up-regulated by interferon alpha. PMID: 8809111 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Oncogene. 1996 Sep 5;13(5):1037-42. Transactivation of the GATA-1 promoter by a myb-ets-containing mouse retrovirus is mediated by CACCC elements. Sun-Hoffman L, Aurigemma RE, Sun B, Ruscetti SK. Laboratory of Molecular Onocology, National Cancer Institute, Frederick, Maryland 21702-1201, USA. The myb-ets-containing ME26 virus causes erythroleukemia in mice by a novel mechanism involving the inappropriate activation of erythroid-specific genes in hematopoietic precursor cells. We have previously shown that the ME26 viral protein can transactivate the GATA-1 promoter in transient transactivation assays carried out in mouse fibroblasts. The mouse GATA-1 promoter, whose activity is regulated by the GATA-1 protein itself, contains a double GATA consensus sequence at its 5' end and two CACCC elements at its 3' end, both of which are crucial for promoter activity in erythroid cells, as well as a nonconsensus GATA sequence and several putative c-myb and c-ets binding sites. In order to determine which sequences in the GATA-1 promoter are crucial for activation by the ME26 viral protein, we made deletions of the promoter, cloned them into a luciferase expression vector and tested their activity in mouse fibroblasts, which do not express GATA-1. Our results indicate that sequences in the 3' end of the GATA-1 promoter, which include two CACCC elements, are essential for transactivation by ME26 virus, while other upstream sites contribute to full activation by the virus. Mutation of the CACCC sites abolishes ME26 viral transactivation. The interaction of cell extracts containing ME26 viral protein and the GATA-1 promoter fragment containing the two CACCC elements was examined by electrophoretic mobility shift analysis (EMSA) and the results showed no direct interaction between the two. However, we could detect the ubiquitous transcription factor Sp1 bound to this sequence. These data demonstrate that the CACCC element is necessary for GATA-1 promoter transactivation by ME26 virus and that the viral protein may indirectly transactivate the promoter by binding to Sp1. PMID: 8806693 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Biochim Biophys Acta. 1996 May 2;1306(2-3):133-6. Genomic structure of carp mitogen-activated protein kinase kinase 1 gene. Leu JH, Lee MS, Chen KT, Chang GD, Chou C, Huang CJ. Institute of Biological Chemistry, Academia Sinicia, Taipei, Taiwan. Carp mitogen-activated protein kinase kinase 1 (cMKK1) gene was isolated from a liver genomic library. The sequence around the exon-intron boundaries and 2 kb of the promoter region were determined. Our data indicate that this gene is composed of 11 exons and 10 introns spanning about 9 kb. Multiple potential transcription initiation sites were located by primer extension analysis. Examination of 2 kb of 5'-flanking sequence revealed potential binding sites for a variety of transcription factors such as E2F, Ets-1, GATA-1, Myb, NF-IL6, Sp1, and NF-kB. PMID: 8634328 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1935-40. Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions. Ghozi MC, Bernstein Y, Negreanu V, Levanon D, Groner Y. Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel. The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1. PMID: 8700862 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Blood. 1995 Nov 15;86(10):3715-24. Granulocyte-macrophage colony-stimulating factor, phorbol ester, and sodium butyrate induce the CD11c integrin gene promoter activity during myeloid cell differentiation. Rubio MA, Lopez-Rodriguez C, Nueda A, Aller P, Armesilla AL, Vega MA, Corbi AL. Hospital de la Princesa, Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas (CSIC), Madrid, Spain. To analyze the activity of the CD11c promoter during myeloid differentiation without the limitations of transient expression systems, we have stably transfected the myeloid U937 cell line with the pCD11C361-Luc plasmid, in which the expression of the firefly luciferase cDNA is driven by the CD11c promoter region -361/+43, previously shown to confer myeloid specificity to reporter genes. The stable transfectants (U937-C361) retained the ability to differentiate in response to phorbol-ester (PMA), sodium butyrate (SB), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other differentiating agents. U937-C361 differentiation correlated with increased cellular luciferase levels, showing the inducibility of the CD11c promoter during myeloid differentiation and establishing the U937-C361 cells as a suitable system for studying the myeloid differentiation-inducing capacity of cytokines, growth, factors, and other biological response modifiers. Unexpectedly, the inducibility of the CD11c gene promoter showed distinct kinetics and magnitude on the PMA-, SB-, GM-CSF-triggered differentiation. Moreover, SB synergized with either PMA or GM-CSF in enhancing both the CD11c promoter activity and the cell surface expression of p150,95 on differentiating U937 cells. Furthermore, we showed the existence of a c-Myb-binding site at -85, the importance of the -99/-61 region in the CD11c promoter inducibility during PMA- or SB-triggered differentiation, and the dependency of the GM-CSF and PMA responsiveness of the CD11c promoter on an intact AP-1-binding site located at -60. These results, together with the lack of functional effect of mutations disrupting the Sp1-and Myb-binding sites within the proximal region of the CD11c promoter, indicate that the myeloid differentiation pathways indicated by SB and phorbol esters (or GM-CSF) activate a distinct set of transcription factors and show that the myeloid differentiation-inducibility of the CD11c gene maps to the -99/-53 proximal region of the promoter. PMID: 7579338 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: J Biol Chem. 1995 Nov 10;270(45):26986-92. The mouse p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1) gene. Genomic organization and structure of the 5'-flanking regulatory region. Pages G, Stanley ER, Le Gall M, Brunet A, Pouyssegur J. Centre de Biochimie, CNRS UMR134, Nice, France. Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS) PMID: 7592946 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: J Biol Chem. 1995 Oct 6;270(40):23785-9. Repression of the c-myb gene by WT1 protein in T and B cell lines. McCann S, Sullivan J, Guerra J, Arcinas M, Boxer LM. Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs Medical Center, Stanford, California, USA. The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines. PMID: 7559553 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Genomics. 1995 Jul 20;28(2):261-72. Structure and chromosomal localization of the human gene of the phosphotyrosyl phosphatase activator (PTPA) of protein phosphatase 2A. Van Hoof C, Aly MS, Garcia A, Cayla X, Cassiman JJ, Merlevede W, Goris J. Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Belgium. The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5' flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains upstream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-kappa B, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5' of a luciferase reporter gene revealed that the 5' flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis. PMID: 8530035 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Biochem Biophys Res Commun. 1995 Mar 17;208(2):756-64. Genomic structure of human transcobalamin II: comparison to human intrinsic factor and transcobalamin I. Li N, Seetharam S, Seetharam B. Department of Medicine, Medical College of Wisconsin, Milwaukee. Human transcobalamin II (TC II) gene was isolated and partially sequenced. The gene is composed of nine exons and eight introns spanning approximately 20 kb. Multiple potential transcription start sites were revealed by primer extension analysis. The 5'-flanking region of the gene contained no TATA-like motif, but a binding motif for HIP1, which is suggested to be important in the transcription of TATA-less housekeeping genes, was identified in a region very close to the initiator methionine codon. In addition, potential binding sites for a variety of transcription factors such as SP1, AP2, CF1, NF-IL6, Ets-1, Myb and E2A were also observed. Comparison of the genomic structure of TC II to other Cbl-binding proteins, human gastric intrinsic factor (IF) and transcobalamin I (TC I) revealed similar intron-exon organizations with respect to the number, position and size of exons. These results suggest that TC II, TC I and IF genes have originated by gene duplications of an ancestral gene and TC II, unlike the other two Cbl-binding proteins, is the product of a "housekeeping" gene. PMID: 7695633 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Blood. 1995 Mar 1;85(5):1246-53. The murine interleukin-3 receptor alpha subunit gene: chromosomal localization, genomic structure, and promoter function. Miyajima I, Levitt L, Hara T, Bedell MA, Copeland NG, Jenkins NA, Miyajima A. DNAX Research Institute of Molecular and Cellular Biology, Palo Alto. The interleukin-3 receptor (IL-3R) is composed of alpha and beta subunits, members of the class I cytokine receptor family. Here we describe isolation and characterization of the chromosomal gene for the mouse IL-3R alpha subunit (mIL-3R alpha). Whereas the human IL-3R alpha gene is tightly linked with the granulocyte-macrophage colony-stimulating factor receptor alpha subunit (GM-CSFR alpha) gene in the pseudoautosomal region of the X and Y chromosomes, the mIL-3R alpha gene (II3ra) is located in the proximal region of mouse chromosome 14, separated from the mouse GM-CSFR alpha gene, which is on chromosome 19. The mIL-3R alpha gene spans about 10 kb and is divided into 12 exons. All the exon-intron boundaries possess the splicing junction consensus sequences (5'GT-AG3'), and the whole genomic structure is similar to those of the previously reported class I cytokine receptor genes. There are two major transcription initiation sites that are located at 215 and 188 nucleotides upstream of the initiator codon. The promoter region is GC-rich and contains potential binding sites for GATA, Ets, c-myb,, Sp1, Ap-2, and G-C boxes, but not a typical TATA or CAAT sequence. A fusion gene containing 0.8 kb of the 5' noncoding sequence linked to the firefly luciferase gene directed the transcription in mouse mast cells but not in fibroblasts or T cells, suggesting that this promoter functions in a cell type-specific manner. Further sequential deletion of the 5' region suggests two potential regulatory regions for transcription of the mIL-3R alpha gene. PMID: 7858255 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Exp Cell Res. 1994 Jan;210(1):94-101. Effect of tumor suppressors on cell cycle-regulatory genes: RB suppresses p34cdc2 expression and normal p53 suppresses cyclin A expression. Yamamoto M, Yoshida M, Ono K, Fujita T, Ohtani-Fujita N, Sakai T, Nikaido T. Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104. We show that expression of the p34cdc2 and cyclin A genes is induced by interleukin-2 in normal human T cells and present evidence to support the idea that these genes are deregulated in leukemic T cells. Our DNA sequencing data indicate that the promoter region of the p34cdc2 gene contains putative E2F-like binding sites which are recognized by Rb and binding sites for c-myb, Sp1, and ATF, and that the promoter region of the cyclin A gene contains binding sites for p53, Sp1, and ATF. In this study we focus on the effect of p53 and Rb on these cell cycle-regulatory genes. Cotransfection of Y79 human retinoblastoma cells with a p34cdc2 promoter-luciferase expression vector and a plasmid expressing the retinoblastoma gene (RB) indicated that RB suppresses p34cdc2 expression. Cotransfection of B104 rat neuroblastoma cells with a cyclin A promoter-luciferase expression vector and a plasmid expressing the normal or mutant p53 indicated that only the normal p53 suppresses cyclin A expression. In normal T cells, PHA stimulation reduces the amount of complexes in the p34cdc2 promoter between the E2F-like binding site and the RB gene product. These complexes were not detected in leukemic T cells. Our data support the idea that tumor suppressors modulate the expression of cell cycle-regulatory genes: RB regulates p34cdc2 expression and p53 regulates cyclin A expression. PMID: 8270002 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Jpn J Cancer Res. 1993 Nov;84(11):1136-44. Characterization of the promoter region of the human c-kit proto-oncogene. Yamamoto K, Tojo A, Aoki N, Shibuya M. Department of Genetics, University of Tokyo. The c-kit proto-oncogene encodes a tyrosine kinase receptor for stem cell factor and plays a critical role in the growth and differentiation of various types of cells including hematopoietic stem cells. To investigate the mechanisms of its transcriptional regulation, we isolated the 5' flanking region of the human c-kit gene and characterized its promoter activity in hematopoietic cells. Nucleotide sequence analysis revealed that the 1.2 kb 5' flanking region lacked a typical "TATA box," but had a relatively high G + C content and four potential Sp1-binding sites. Putative binding sites for AP-2, basic helix-loop-helix proteins, Ets-domain proteins, Myb and GATA-1 were also found. Primer extension and S1 nuclease protection analyses of hematopoietic cells indicated that the major transcription start sites are 62 bp and 58 bp upstream of the translation start site. Essentially the same start sites were detected in non-hematopoietic cells such as small cell lung carcinoma and glioblastoma: this single promoter in c-kit is different from the multiple promoter system of c-fms, a c-kit-related gene, in which at least two promoters are differently used in hematopoietic and non-hematopoietic cells. An analysis of the c-kit 5' flanking region using the bacterial chloramphenicol acetyltransferase gene (CAT assay) in human erythroleukemia HEL cells, which express the endogenous c-kit mRNA at high levels, showed that a region from -180 to -22 is important for the expression of the c-kit gene. In addition, a negative regulatory element(s) is suggested to be involved in the regulation of the c-kit gene expression in mammals. PMID: 7506248 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8514-8. Genomic organization of the melanoma-associated glycoprotein MUC18: implications for the evolution of the immunoglobulin domains. Sers C, Kirsch K, Rothbacher U, Riethmuller G, Johnson JP. Institute of Immunology, University of Munich, Germany. The cell surface glycoprotein MUC18, a member of the immunoglobulin superfamily and homologous to several cell adhesion molecules, is associated with tumor progression and the development of metastasis in human malignant melanoma. Immunohistochemical and Northern blot analysis revealed that expression of the antigen is restricted to advanced primary and metastatic melanomas and to cell lines of the neuroectodermal lineage. The genomic sequence encoding the cell surface antigen spans approximately 14 kb and consists of 16 exons. The organization of the gene, which is related to that of the neural cell adhesion molecule N-CAM, shows a structure where each immunoglobulin-related domain is encoded by more than one exon. Sequencing of the putative MUC18 promoter region revealed a G + C-rich promoter lacking conventional TATA and CAAT boxes. Several motifs for binding of transcription factor Sp1 are present in the regulatory region, and only a single transcription start site within a presumed initiator sequence was identified. Sequence elements which might confer melanocyte-specific expression were not detected. Instead, recognition sequences for the transcription factors CREB, AP-2, and c-Myb, as well as CArG-box motifs, were observed. These elements may contribute to the differential regulation of the MUC18 gene in normal and malignant tissues and suggest a role for this putative adhesion molecule in neural crest cells during embryonic development. PMID: 8378324 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Oncogene. 1993 Sep;8(9):2501-9. The v-Rel oncoprotein increases expression from Sp1 site-containing promoters in chicken embryo fibroblasts. Sif S, Capobianco AJ, Gilmore TD. Department of Biology, Boston University, Massachusetts 02215. The v-Rel oncoprotein of the avian Rev-T retrovirus is a member of a family of related transcription factors, which also includes the subunits of NF-kappa B and several other interacting cellular proteins. We show here that v-Rel specifically increased expression from a reporter plasmid containing multiple Sp1 binding sites approximately sixfold in chicken embryo fibroblasts (CEFs), even though v-Rel did not bind directly to these sites. v-Rel also increased expression from a reporter plasmid containing a human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in which the kappa B binding sites were mutated but which still contained intact Sp1 binding sites. The increase in Sp1-site transactivation does not precisely correlate with transformation by v-Rel since one non-transforming v-Rel mutant still induced expression from the Sp1 site-containing promoter. v-Rel appears to increase expression from Sp1 site-containing promoters by affecting the transactivation domain of Sp1, since v-Rel increased the activity of a Gal4-Sp1 fusion protein, which contains the Sp1 transactivation domain but lacks the Sp1 DNA-binding domain. As compared with v-Rel, c-Rel induced only a slight increase in expression from the reporter plasmid containing Sp1 sites. However, v-Ras and v-Src (but not v-Myb) induced increases in transcription from the reporter plasmid containing Sp1 sites to the same extent as v-Rel, but through pathways that appear to be independent from v-Rel. These results suggest that certain oncoproteins might increase transcription from many genes that contain Sp1 binding sites, and that this might be important for certain aspects of transformation by these proteins. PMID: 8361761 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Proc Natl Acad Sci U S A. 1992 May 15;89(10):4387-91. Cloning of the rat gene encoding choline acetyltransferase, a cholinergic neuron-specific marker. Hahn M, Hahn SL, Stone DM, Joh TH. Laboratory of Molecular Neurobiology, Burke Medical Research Institute, Cornell University Medical College, White Plains, NY 10605. The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT; EC 2.3.1.6). Since the expression of ChAT in the nervous system is restricted to cholinergic neurons, it serves as a specific marker for these neurons. In Alzheimer disease, ChAT activity is markedly reduced in the affected brain areas. Nerve growth factor can increase the ChAT activity of brain cholinergic neurons in vitro and in vivo. We have cloned the rat ChAT gene and identified one 5' noncoding exon and 14 exons that account for the entire coding sequence. The exon organization is compared with the protein domains conserved during evolution. These exons are distributed over at least 64 kilobases in the rat genome; the largest intron is at least 14 kilobases long. Within a 0.7-kilobase region immediately upstream of the confirmed sequence of the noncoding exon, TATA-like elements and numerous potential binding sites for transcription factors are found, including AP-1, Sp1, octamer-binding factor(s), CTF/NF-1, and the nuclear oncoprotein Myb. PMID: 1584771 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Oncogene. 1991 Oct;6(10):1843-50. Erratum in: Oncogene. 1992 May;7(5):1047. Characterization of the human N-ras promoter region. Thorn JT, Todd AV, Warrilow D, Watt F, Molloy PL, Iland HJ. Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia. Overexpression of ras proto-oncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer in order to determine functional regions within the human N-ras promoter. A significant proportion of promoter activity was found to lie within a 439 bp fragment comprising an untranslated exon (exon 1) with the adjacent 5' sequence and a small CpG island. A 109 bp [corrected] fragment at the 5' end of exon 1 was essential for promoter activity, while a 45 bp [corrected] deletion from within this region decreased promoter activity by two-thirds. Unlike the human H-ras and mouse K-ras promoters, the N-ras promoter did not exhibit bidirectional activity. DNAse footprinting of the 439 bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA-binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb and E4TF1). In contrast, four putative Sp1 sites did not footprint. Using purified MLTF and appropriate competitors in gel shift and DNAase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon 1. PMID: 1923508 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: Nucleic Acids Res. 1989 Dec 11;17(23):9593-611. Structure and biological activity of the transcriptional initiation sequences of the murine c-myb oncogene. Sobieszczuk PW, Gonda TJ, Dunn AR. Ludwig Institute for Cancer Research, Melbourne Branch of Tumor Biology, Parkville, Victoria, Australia. To study the control mechanism(s) that govern the transcription of c-myb, genomic clones corresponding to the 5' region of the murine c-myb gene have been isolated and characterized structurally and functionally. Primer extension and nuclease protection analysis have revealed the presence of multiple transcriptional initiation sites, that are utilized in several hemopoietic cell lines (WEHI3B(D+). FDC-P1 and RB22.2). Some of the sites are used in all cell lines but others are unique; all are located in a region of the c-myb gene that is G-C rich, contains a number of potential Sp1 binding sites and lacks classical promoter consensus sequences. Experiments in which well characterized promoters controlling expression of a reporter gene have been replaced by fragments of c-myb DNA (including the observed cap sites) were performed in an attempt to demonstrate promoter activity in various cell types. It was shown that a region of the c-myb gene (approximately 1.0 kbp upstream from the splice donor site of the first exon) contains a weak promoter that has a low level of transcriptional activity in hemopoietic as well as in fibroblastic cells. These results support the suggestion that c-myb expression is not regulated primarily at the level of initiation of transcription. PMID: 2481264 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: Nucleic Acids Res. 1989 Jul 25;17(14):5651-64. Transcription of the chicken myb proto-oncogene starts within a CpG island. Dvorak M, Urbanek P, Bartunek P, Paces V, Vlach J, Pecenka V, Arnold L, Travnicek M, Riman J. Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague. The nucleotide sequence of an 8.2-kb DNA fragment from the 5' proximal part of the chicken myb proto-oncogene spanning 1761 nucleotides upstream and 6436 nucleotides downstream from a presumed c-myb initiation codon was determined. A 3.3-kb G + C-rich region found in this sequence had also other features characterizing CpG islands, i.e. no CpG underrepresentation and lack of CpG methylation. In haematopoietic tissues c-myb mRNA synthesis starts in two major regions of the CpG island, namely 98 to 108 and 143 to 145 nucleotides upstream from the c-myb initiation codon. These two regions are in or close to the 124-bp evolutionarily conserved element located in the middle part of the CpG island. No alternative splicing of the 5' end of c-myb mRNA suggested earlier (1,2) was observed. The c-myb promoter contains neither TATA nor CAAT box-like structures at the usual positions. Instead, numerous potential Sp1 factor binding sites were found both upstream and downstream from the transcription initiation sites. Moreover, consensus v-myb protein DNA-binding sites were revealed in the promoter region and in sequences downstream from it. PMID: 2548166 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------