GC-rich Motifs Research Articles

Estrogen Regulates KiSS1 Gene Expression through Estrogen Receptor α and SP Protein Complexes

Kisspeptins are natural ligands of G protein-coupled receptor-54. Activation of KiSS1/G protein-coupled receptor-54 signaling pathways results in potent activation of the hypothalamus-pituitary-gonadal axis and initiates puberty. Recent data have shown that in female mice, KiSS1 is positively regulated by estradiol (E(2)) in the anteroventral periventricular nucleus, an important reproductive neuroendocrine brain region, but negatively regulated in the arcuate nucleus. However, little is known about the molecular mechanisms governing E(2)-modulated KiSS1 expression. Here, we demonstrate that the expression level of the KiSS1 gene was up-regulated with the administration of E(2) in estrogen receptor alpha (ERalpha)-positive hypothalamic GT1-7 cells. Using transient transfection of human KiSS1 gene promoter coupled to a luciferase reporter, E(2) increases promoter activity in the presence of ERalpha. Deletion analysis of KiSS1 promoter indicates that the E(2)-regulated increase in promoter activity depends on the Sp1 sites of the proximal promoter region. Using both EMSAs and chromatin immunoprecipitation analysis, we determined that both Sp1 and Sp3 proteins constitutively associate with the four putative Sp1 sites in vitro, whereas the association of ERalpha with the KiSS1 promoter is dependent on E(2) exposure. Sp1 and ERalpha form a complex in vivo to mediate the E(2)-induced activation of KiSS1 promoter. Interestingly, Sp1 transactivates KiSS1 promoter activity, whereas Sp3 functions as a transcriptional repressor. Together, these results demonstrate that E(2)-dependent transcriptional activation of KiSS1 gene is mediated by ERalpha through the interaction of Sp1/Sp3 proteins with the GC-rich motifs of KiSS1 promoter, providing a molecular mechanism of how steroid hormone feedback regulates KiSS1 expression.

Coactivation of estrogen receptor α (ERα)/Sp1 by vitamin D receptor interacting protein 150 (DRIP150)

Vitamin D receptor interacting protein (DRIP150) coactivates estrogen receptor α (ERα)-mediated transactivation in breast cancer cell lines transfected with a construct (pERE 3) containing three estrogen responsive elements (EREs). In this study, we show that DRIP150 also coactivates ERα/Sp1-mediated transactivation in ZR-75, MCF-7, and MDA-MB-231 breast cancer cells transfected with a construct (pSp1 3) containing three consensus GC-rich motifs. Studies on coactivation of wild-type and variant ERα/Sp1 by DRIP150 indicates that the DNA-binding domain and helix 12 in the ligand binding domain of ERα are required and the coactivation response is squelched by overexpressing an NR-box peptide that contains two LXXLL motifs from GRIP2. In contrast, coactivation of ERα/Sp1 by wild-type and mutant DRIP150 expression plasmids show that coactivation of ERα/Sp1 by DRIP150 is independent of the NR-boxes. Deletion analysis of DRIP150 demonstrates that coactivation requires an α-helical NIFSEVRVYN (amino acids 795–804) motif within 23 amino acid sequence (789–811) in the central region of DRIP150 and similar results were obtained for coactivation of ERα by DRIP150. Thus, although different domains of ERα are required for hormone-dependent activation of ERα and ERα/Sp1, coactivation of these transcription factors by DRIP150 requires the α-helical amino acids 795–804. This is the first report of a coactivator that enhances ERα/Sp1-mediated transactivation in breast cancer cells.

Isolation and Characterization of the Rat SND p102 Gene Promoter

In this work, we report the isolation and characterization of a 1,688-bp sequence corresponding to the promoter region of the rat endoplasmic reticulum (ER) cholesterol ester hydrolase gene, renamed as staphylococcal nuclease domain-containing protein of 102 kDa (SND p102) in GenBank database according to the structural properties and molecular weight of the protein. The transcription start site was located 216 bases upstream of the ATG start codon by RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE). Bioinformatic analysis of the isolated sequence revealed a lack of typical promoter TATA box and the presence of GC-rich motifs and CCAAT boxes recognized by Sp 1 and nuclear factor-Y among other putative binding sites for a number of transcription factors implicated in both basal and regulated processes. Electrophoretic mobility shift and supershift assays using nuclear extracts from human (HepG2) and rat (McA-RH7777) hepatoma cells demonstrated that nuclear factor-Y (NF-Y) transcription factor bound to the core sequences at (-257, -253), (-290, -286), and (-370, -366) upstream translation initiation site. The absence of TATA box and the location and reverse orientation of the CCAAT boxes in the promoter region strongly suggest a role for NF-Y in the regulation of transcription of SND p102 gene.

Interplay between Chromatin and Trans-acting Factors Regulating the Hoxd4 Promoter during Neural Differentiation

Correct patterning of the antero-posterior axis of the embryonic trunk is dependent on spatiotemporally restricted Hox gene expression. In this study, we identified components of the Hoxd4 P1 promoter directing expression in neurally differentiating retinoic acid-treated P19 cells. We mapped three nucleosomes that are subsequently remodeled into an open chromatin state upon retinoic acid-induced Hoxd4 transcription. These nucleosomes spanned the Hoxd4 transcriptional start site in addition to a GC-rich positive regulatory element located 3' to the initiation site. We further identified two major cis-acting regulatory elements. An autoregulatory element was shown to recruit HOXD4 and its cofactor PBX1 and to positively regulate Hoxd4 expression in differentiating P19 cells. Conversely, the Polycomb group (PcG) protein Ying-Yang 1 (YY1) binds to an internucleosomal linker and represses Hoxd4 transcription before and during transcriptional activation. Sequential chromatin immunoprecipitation studies revealed that the PcG protein MEL18 was co-recruited with YY1 only in undifferentiated P19 cells, suggesting a role for MEL18 in silencing Hoxd4 transcription in undifferentiated P19 cells. This study links for the first time local chromatin remodeling events that take place during transcriptional activation with the dynamics of transcription factor association and DNA accessibility at a Hox regulatory region.

Molecular Mechanism of Inhibitory Aryl Hydrocarbon Receptor—Estrogen Receptor/Sp1 Cross Talk in Breast Cancer Cells

The trifunctional carbamoylphosphate synthetase/aspartate transcarbamyltransferase/dihydroorotase (CAD) gene is hormone responsive in MCF-7 and ZR-75 breast cancer cells, and this response is inhibited by the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Estrogen-dependent induction of CAD mRNA and reporter gene activity in cells transfected with constructs (pCAD) containing hormone-responsive GC-rich CAD promoter inserts involves estrogen receptor alpha (ERalpha)/Sp1 interactions with these proximal GC-rich motifs. TCDD also inhibits hormone-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD constructs. The mechanism of inhibitory AhR-ERalpha/Sp1 cross talk was further investigated by chromatin immunoprecipitation (ChIP), and the results show that ERalpha/Sp1 and the AhR are constitutively bound to the CAD gene promoter and only minor changes are observed after treatment with 17beta-estradiol, TCDD, or their combination. However, examination of interactions of these transcription factors by fluorescence resonance energy transfer shows that E2 enhances ERalpha-Sp1 interactions, whereas cotreatment with TCDD significantly decreases interaction of these proteins. These results suggest that inhibitory AhR-ERalpha/Sp1 cross talk is due, in part, to enhanced association of AhR and ERalpha (also determined by fluorescence resonance energy transfer), which coordinately dissociates ER and Sp1 and decreases ERalpha/Sp1-mediated transactivation, whereas remaining associated with the CAD promoter. This represents a novel interaction between two ligand activated receptors where one receptor inhibits activation of the second receptor.

Vascular Endothelial Growth Factor Receptor-2 Expression Is Induced by 17β-Estradiol in ZR-75 Breast Cancer Cells by Estrogen Receptor α/Sp Proteins

Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.

Unigene derived microsatellite markers for the cereal genomes

Unigene derived microsatellite (UGMS) markers have the advantage of assaying variation in the expressed component of the genome with unique identity and positions. We characterized the microsatellite motifs present in the unigenes of five cereal species namely, rice, wheat, maize, Sorghum and barley and compared with those in Arabidopsis. The overall UGMS frequency in the five cereal species was 1/7.6 kb. The maximum UGMS frequency was in rice (1/3.6 kb) and the lowest in wheat (1/10.6 kb). GC-rich trinucleotide repeat motifs coding for alanine followed by arginine and the dinucleotide repeat motif GA were found to be abundant UGMS classes across all the five cereal species. Primers could be designed for 95% (wheat and barley) to 97% (rice) of the identified microsatellites. The proportion and frequency of occurrence of long hypervariable class I (> or = 20 nucleotides) and potentially variable class II (12-20 nucleotides) UGMS across five cereal species were characterized. The class I UGMS markers were physically mapped in silico on to the finished rice genome and bin-mapped in wheat. Comparative mapping based on class I UGMS markers in rice and wheat revealed syntenic relationships between the two genomes. High degree of conservation and cross-transferability of the class I UGMS markers were evident among the five cereal species, which was validated experimentally. The class I UGMS-conserved orthologous set (COS) markers identified in this study would be useful for understanding the evolution of genes and genomes in cereals.

Distinct Pathways Regulate Proapoptotic Nix and BNip3 in Cardiac Stress

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC alpha, but not PKC epsilon or dominant negative PKC alpha, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKC alpha-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G(q) overexpression model of hypertrophy. Luciferase activity increased in G alpha(q)-NixP hearts 3.2 +/- 0.4-fold and in TAC hearts 2.8 +/- 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by G alpha(q)-mediated hypertrophic stimuli. PKC alpha, a G(q) effector, transduced Nix transcriptional induction via Sp1.

Characterization of the promoter for the α3 integrin gene in various tumor cell lines: Roles of the Ets‐ and Sp‐family of transcription factors

The alpha3beta1 integrin is an adhesion receptor for extracellular matrix proteins, including laminin isoforms, and plays crucial roles in the organization of epithelial and endothelial tissues. The aberrant expression of this adhesion molecule on tumor cells is associated with their invasive and metastatic potentials. In the present study, we analyzed the elements essential for alpha3 integrin gene expression in various tumor cell lines with different tissue origins by luciferase assay. An approximately 0.3 kb fragment of the 5'-flanking region of the mouse alpha3 integrin gene (-260/+84, relative to the major transcription start site) showed strong promoter activity in all six examined tumor cell lines. However, we found that these cell lines could be divided into two groups according to the level of dependency on the putative Ets-transcription factor binding motif located at -133. This motif was previously shown to be crucial for alpha3 integrin expression in MKN1 gastric carcinoma cells. The gene expression in one group of cell lines was upregulated mainly by the Ets motif, whereas that in the other group was less dependent on the Ets motif. We then postulated that additional regulatory elements were responsible for the expression of alpha3 integrin, and found that a GC-rich motif at -69 was another important element. An electrophoretic mobility shift assay using specific antibodies and a Western blot analysis of nuclear proteins revealed that the Sp3-transcription factor bound to this GC-rich motif. These results suggest that the Sp3 and Ets transcription factors cooperatively regulate alpha3 integrin gene expression and that the contribution of each element depends on the type of tumor cells.

The properties of CpG islands in the putative promoter regions of human immunoglobulin (Ig) genes

CpG island is a GC-rich motif occurred in gene promoter region, which can play important roles in gene silencing and imprinting. Here, we present a set of discriminant functions that can recognize the structural and compositional features of CpG islands in the putative promoter regions (PPRs) of human and mouse immunoglobulin (Ig) genes. We showed that the PPRs of both human and mouse Ig genes irrespective of gene chromosomal localization are apparently CpG island poor, with a low percentage of the CpG islands overlapped with the transcription start site (TSS). The human Ig genes that have CpG islands in the PPRs show a very narrow range of CpG densities. 47% of the Ig genes fall in the range of 3.5–4 CpGs/100 bp. In contrast, the non-Ig genes examined have a wide range of the density of CpG island, with 10.5% having the density of 8.1–15 CpGs/100 bp. Meantime, five patterns of the CpG distributions within the CpG islands have been classified: Pat A, B, C, D, and E. 21.6% and 10.8% of the Ig genes fall into the Pat B and Pat D groups, respectively, which were significantly higher than the non-Ig genes examined (8.2% and 3.8%). Moreover, the length of CpG islands is shorter in human Ig genes than in non-Ig genes but is much longer than in mouse orthologues. These findings provide a clear picture of non-neutral and nonrandom occurrence of the CpG islands in the PPRs of human and mouse Ig genes, which facilitate rational recommendations regarding their nomenclature.

Sp3 is involved in the regulation of SOCS3 gene expression

Cytokine-induced expression of SOCS (suppressor of cytokine signalling) molecules is important for the negative regulatory control of STAT (signal transduction and activators of transcription)-dependent cytokine signalling, e.g. for the signal transduction of IL-6 (interleukin-6)-type cytokines through the JAK (Janus kinase)/STAT cascade. STAT activation itself represents an important step in the transcriptional activation of SOCS3 gene expression. However, downstream of the STAT-responsive element, the SOCS3 gene contains a GC-rich element in its 5'-upstream region. The aim of the present study was to investigate the implications of this GC-rich element in the transcriptional control of SOCS3 gene expression. In the present study, we show that mutation of this GC-rich element abolishes IL-6-dependent transcriptional activation of the SOCS3 promoter and that Sp3 (specificity protein 3), a ubiquitously expressed transcription factor, but not Sp1 binds to this GC-rich motif, suggesting that Sp3 is involved in the regulation of SOCS3 expression. The results suggest that Sp3 is important for IL-6-induced transcriptional activation of the SOCS3 (gene) promoter and acts as an enhancer of basal as well as induced transcriptional activity, resulting in enhanced SOCS3 mRNA and protein expression. Mutation of Lys-483, a potential target for Sp3 acetylation, inhibited Sp3-mediated enhancement of SOCS3 mRNA expression and SOCS3 promoter activation, indicating that the acetylation of this lysine residue of Sp3 is important for the enhancing effect of Sp3 on SOCS3 expression.

The Epstein-Barr Virus Protein BMRF1 Activates Gastrin Transcription

The Epstein-Barr virus (EBV) BMRF1 gene encodes an early lytic protein that functions not only as the viral DNA polymerase processivity factor but also as a transcriptional activator. BMRF1 has been previously shown to activate transcription of an EBV early promoter, BHLF1, though a GC-rich motif which binds to SP1 and ZBP-89, although the exact mechanism for this effect is not known (D. J. Law, S. A. Tarle, and J. L. Merchant, Mamm. Genome 9:165-167, 1998). Here we demonstrate that BMRF1 activates transcription of the cellular gastrin gene in telomerase-immortalized keratinocytes. Furthermore, BMRF1 activated a reporter gene construct driven by the gastrin promoter in a variety of cell types, and this effect was mediated by two SP1/ZBP-89 binding sites in the gastrin promoter. ZBP-89 has been previously shown to negatively regulate the gastrin promoter. However, ZBP-89 can function as either a negative or positive regulator of transcription, depending upon the promoter and perhaps other, as-yet-unidentified factors. BMRF1 increased the binding of ZBP-89 to the gastrin promoter, and a ZBP-89-GAL4 fusion protein was converted into a positive transcriptional regulator by cotransfection with BMRF1. BMRF1 also enhanced the transcriptional activity of an SP1-GAL4 fusion protein. These results suggest that BMRF1 activates target promoters through its effect on both the SP1 and ZBP-89 transcription factors. Furthermore, as the EBV genome is present in up to 10% of gastric cancers, and the different forms of gastrin are growth factors for gastrointestinal epithelium, our results suggest a mechanism by which lytic EBV infection could promote the growth of gastric cells.

BCL11A-dependent recruitment of SIRT1 to a promoter template in mammalian cells results in histone deacetylation and transcriptional repression

The B cell leukemia 11A protein (BCL11A/Evi9/CTIP1) has been implicated in hematopoietic cell development and malignancies. BCL11A is a transcriptional repressor that binds directly to a GC-rich motif and is also recruited to a promoter template via interaction with the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor II. In both cases, BCL11A-mediated transcriptional repression is only minimally reversed by trichostatin A, suggesting the possible lack of involvement of class I or II histone deacetylases. Nonetheless, chromatin immunoprecipitation assays revealed that expression of BCL11A in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene. BCL11A-mediated transcriptional repression, as well as deacetylation of histone H3/H4 in BCL11A-transfected cells, was partially reversed by nicotinamide, an inhibitor of class III histone deacetylases such as SIRT1. SIRT1 was found to interact directly with BCL11A and was recruited to the promoter template in a BCL11A-dependent manner leading to transcriptional repression. These findings define a role for SIRT1 in transcriptional repression mediated by BCL11A in mammalian cells.

Role of Sp proteins in regulation of vascular endothelial growth factor expression and proliferation of pancreatic cancer cells.

Sp proteins play an important role in angiogenesis and growth of cancer cells, and specificity protein 1 (Sp1) has been linked to vascular endothelial growth factor (VEGF) expression in pancreatic cancer cells. RNA interference was used to investigate the role of Sp family proteins on regulation of VEGF expression and proliferation of Panc-1 pancreatic cancer cells. Using a series of constructs containing VEGF promoter inserts, it was initially shown that Sp1 and Sp3 were required for transactivation, and this was primarily dependent on proximal GC-rich motifs. We also showed that Sp4 was expressed in Panc-1 cells, and RNA interference assays suggested that Sp4 cooperatively interacted with Sp1 and Sp3 to activate VEGF promoter constructs in these cells. However, the relative contributions of Sp proteins to VEGF expression were variable among different pancreatic cancer cell lines. Small inhibitory RNAs for Sp3, but not Sp1 or Sp4, inhibited phosphorylation of retinoblastoma protein, blocked G0/G1-->S-phase progression, and up-regulated p27 protein/promoter activity of Panc-1 cells; similar results were observed in other pancreatic cancer cells, suggesting that Sp3-dependent growth of pancreatic cancer cells is caused by inhibition of p27 expression.

Indian Hedgehog Gene Is a Target of the Bone Morphogenetic Protein Signaling Pathway

Bone morphogenetic proteins (BMPs), a large subgroup of the transforming growth factor-beta family of growth factors, are key mediators of many fundamental processes in embryonic development. The BMP target genes mediating these effects and the mechanism of their selective regulation are poorly characterized. In this study, we used a chromatin immunoprecipitation-based gene cloning method to identify BMP target genes in a mouse embryonic carcinoma cell line. We identified the Indian hedgehog (Ihh) gene as a target of BMP signaling. Both reporter and reverses transcription-PCR assays revealed that Ihh is up-regulated in embryonic cells upon BMP treatment. The BMP response element of the Ihh promoter contains multiple GC-rich motifs known as the "Mad/Medea binding box" found in the Drosophila tinman gene and in the mouse Id1 gene. DNA binding studies revealed that Smad4 binds to these GC-rich motifs. These findings indicate that BMP stimulates Ihh expression. We also suggest expression of the Ihh gene, which contains multiple Smad binding sites, might be finely regulated by a gradient of BMP concentrations.

Unlocking hidden genomic sequence.

Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.

Characterization of microsatellites revealed by genomic sequencing ofPopulus trichocarpa

Microsatellites or simple sequence repeats (SSRs) are highly polymorphic, codominant markers that have great value for the construction of genetic maps, comparative mapping, population genetic surveys, and paternity analyses. Here, we report the development and testing of a set of SSR markers derived from shotgun sequencing from Populus trichocarpa Torr. & A. Gray, a nonenriched genomic DNA library, and bacterial artificial chromosomes. Approximately 23% of the 1536 genomic clones and 48% of the 768 bacterial artificial chromosome subclones contained an SSR. Of the sequences containing an SSR, 72.4% contained a dinucleotide, 19.5% a trinucleotide, and 8.1% a tetranucleotide repeat unit; 26.6% of the sequences contained multiple SSR motifs in a complex or compound repeat structures. A survey of the genome sequence database revealed very similar proportional distribution, indicating that our limited rapid, shallow sequencing effort is representative of genome-wide patterns. In total, 492 primer pairs were designed and these yielded 77 markers that were mapped in an F2pedigree, including 26 that were sufficiently informative to be included in a Populus framework map. SSRs with GC-rich motifs mapped at a significantly higher frequency than expected, although AT-rich SSRs accounted for the majority of mapped markers due to their higher representation in the genome. SSR markers developed from P. trichocarpa showed high utility throughout the genus, with amplification rates in excess of 70% for all Populus species tested. Finally, at least 30% of the markers amplified in several willow species, suggesting that some of these SSRs will be transferable across genera.

Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor alpha and SP proteins.

Vascular endothelial growth factor (VEGF) is expressed in multiple hormone-dependent cancer cells/tumors. Treatment of ZR-75 breast cancer cells with 17beta-estradiol (E2) induced a greater than fourfold increase of VEGF mRNA levels. ZR-75 breast cancer cells were transfected with pVEGF1, a construct containing a -2018 to +50 VEGF promoter insert, and E2 induced reporter gene (luciferase) activity. Deletion and mutation analysis of the VEGF gene promoter identified a GC-rich region (-66 to -47) which was required for E2-induced transactivation of pVEGF5, a construct containing the minimal promoter (-66 to +54) that exhibited E2-responsiveness. Interactions of nuclear proteins from ZR-75 cells with the proximal GC-rich region of the VEGF gene promoter were investigated by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results demonstrate that both Sp1 and Sp3 proteins bound the GC-rich motif (-66 to -47), and estrogen receptor alpha (ERalpha) interactions were confirmed by chromatin immunoprecipitation. Moreover, E2-dependent activation of constructs containing proximal and distal GC/GT-rich regions of the VEGF promoter was inhibited in ZR-75 cells transfected with small inhibitory RNAs for Sp1 and Sp3. These results were consistent with a mechanism of hormone activation of VEGF through ERalpha/Sp1 and ERalpha/Sp3 interactions with GC-rich motifs.

DNA mismatches and GC-rich motifs target transposition by the RAG1/RAG2 transposase.

In addition to their essential role in V(D)J recombination, the RAG proteins function as a transposase capable of inserting the V(D)J recombination intermediate, the signal end DNA fragment, into target DNA. RAG-mediated transposition has been suggested to contribute to genome instability and the development of lymphoid malignancies. Previous studies suggested that the RAG transposase exhibits a target site preference for GC rich sequences and hairpin structures. Here we demonstrate that a transposition hot spot (5'-GCCGCCGGGCC-3'), smaller portions of this hot spot and other GC rich motifs are able to target RAG-mediated transposition. Tracks of GC base pairs have been shown to have an unusually high rate of base pair breathing. Intriguingly, we find that DNA mismatches can efficiently target RAG-mediated transposition and suppress the use of other target sites. Hairpins, however, are not generally preferred targets. Our results indicate that target DNA melting may be a crucial step during RAG-mediated transposition, and that target site selection by the RAG transposase may be intimately linked to mutagenic and metabolic processes that transiently present favorable DNA structures to the transposition machinery.

Biological effects induced by swift heavy ions of lithium on aqueous solution of plasmid pMTa4

Biological samples exposed to swift heavy ions sustain damage on different components. Damage to DNA, a critical component of a living system, has considerable biological implications. In this study aqueous solution of plasmid pMTa4 was exposed to varying fluence of swift 7Li ions and its different topological forms were analysed by agarose gel electrophoresis to study the induced damage. To monitor radiation labile nucleotide sequence the 7Li ions exposed plasmid was degraded by three different restriction endonucleases and also analysed by agarose gel electrophoresis. The results show that 7Li ions predominantly induced double strand breaks in the plasmid DNA in a dose-dependent manner and affected preferentially the GC-rich motifs of the DNA. The results suggest that 7Li ions induce premutagenic lesions at an enhanced frequency in segments of the DNA which are rich in CG content as compared to GC-poor segments.