Competition Gel Mobility Shift Assays Research Articles

A novel nonsteroidal antifibrotic oligo decoy containing the TGF‐β element found in the COL1A1 gene which regulates murine schistosomiasis liver fibrosis

Schistosomiasis mansoni disseminated worm eggs in mice and humans induce granulomatous inflammations and cumulative fibrosis causing morbidity and possibly mortality. In this study, intrahepatic and I.V. injections of a double-stranded oligodeoxynucleotide decoy containing the TGF-beta regulatory element found in the distal promoter of the COL1A1 gene into worm-infected mice suppressed TGF-beta1, COL1A1, tissue inhibitor of metalloproteinase-1, and decreased COL3A1 mRNAs to a lesser extent. Sequence comparisons within the mouse genome found homologous sequences within the COL3A1, TGF-beta1, and TIMP-1 5' flanking regions. Cold competition gel mobility shift assays using these homologous sequences with 5' and 3' flanking regions found in the natural COL1A1 gene showed competition. Competitive gel mobility assays in a separate experiment showed no competition using a 5-base mutated or scrambled sequence. Explanted liver granulomas from saline-injected mice incorporated 10.45 +/- 1.7% (3)H-proline into newly synthesized collagen, whereas decoy-treated mice showed no collagen synthesis. Compared with the saline control schistosomiasis mice phosphorothioate double-stranded oligodeoxynucleotide treatment decreased total liver collagen content (i.e. hydroxy-4-proline) by 34%. This novel molecular approach has the potential to be employed as a novel antifibrotic treatment modality.

Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

The genome of equine arteritis virus (EAV) produces a 3′ coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5′ end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) strands of the EAV leader RNA. Several cell proteins with molecular masses ranging from 74 to 31 kDa and 58 to 32 kDa were detected in UV-induced cross-linking assays with the EAV leader RNA (+) and (-) strands, respectively. In both cases, intense bands were observed at the 58–52 kDa molecular weight markers. Results from competition gel mobility shift assays using overlapping cold RNA probes spanning the leader RNA (+) strand indicated that nt 140–206 are not necessary for binding to cell proteins.

Two Inr elements are important for mediating the activity of the proximal promoter of the human gonadotropin-releasing hormone receptor gene.

Differential usage of several transcription start sites in the human GnRH receptor gene was evident in human brain and pituitary. To locate the promoter responsible for a cluster of the 3' CAP sites from -635 to -578 (relative to ATG) found in the pituitary, a proximal promoter element was identified at -677/-558 by 5' and 3' deletion mutant analysis. The promoter element drove a 13.1 +/- 0.6-fold increase in reporter gene activity in an orientation-dependent manner in the mouse gonadotrope-derived alphaT3-1 cells. Within the core promoter element, two functional AT-rich Inr motifs, interacting with the same protein factor with different affinities, were identified. By Southwestern blot analysis and competitive gel mobility shift assays, multiple nuclear factors (36-150 kDa) were found to interact specifically with the core promoter element. Interestingly, these nuclear proteins also interacted with a previously identified distal promoter of the human GnRH receptor gene. Taken together, our studies suggested that these two promoters share common protein factors to regulate transcription initiations at two different regions. Additional mechanisms are needed to modulate the efficiencies of individual promoters for developmental and/or tissue-specific regulations.

Cell proteins TIA-1 and TIAR interact with the 3' stem-loop of the West Nile virus complementary minus-strand RNA and facilitate virus replication.

It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3'-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3'(-) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3'(-) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3'(-) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3'(-) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (K(d)) for the interaction between TIAR RRM2 and the WNV 3'(-) SL RNA was 1.5 x 10(-8), while that for TIA-1 RRM2 was 1.12 x 10(-7). WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

A Novel TCTG(G/C) Direct Repeat and an A/T-Rich HMG2-Binding Site Control the Expression of the Rat CardiacTroponin T Gene

Q. Wang, J. L.-C. Lin, and J. J.-C. Lin. A Novel TCTG(G/C) Direct Repeat and an A/T-Rich HMG2-Binding Site Control the Expression of the Rat Cardiac Troponin T Gene. Journal of Molecular and Cellular Cardiology (2002) 34, 1667–1679. We previously showed that the rat cardiac troponin T proximal promoter (−497 bp from the transcriptional start site) was sufficient to confer cardiac-specific expression of a reporter gene in both cultured cardiomyocytes and transgenic mice. This promoter consists of two cis-regulatory modules with high sequence similarity. Nucleotides from −319 to −289 (termed D2) and nucleotides from −249 to −209 (termed F41) contain a TCTG(G/C) direct repeat and an A/T-rich sequence. Competition gel mobility shift assays revealed that the same protein factors were bound to both D2 and F41. Deletion and religation studies of the promoter suggested that D2 acted as an enhancer but could not totally substitute for the F41 function. Mutational analyses demonstrated that the direct repeat was required for the DNA-binding and promoter activity. Moreover, cardiac-specific 42 kDa proteins and a ubiquitous high mobility group 2 (HMG2) protein were identified to be responsible for the binding to the TCTG(G/C) direct repeat and the A/T-rich sequence, respectively. Overexpression of HMG2 had differential effects on the promoter in cardiomyocytes versus fibroblasts. Our results provided the first evidence to support that HMG2 together with tissue-specific factors could direct a stimulatory or inhibitory effect on the cardiac troponin T gene expression in heart or non-heart tissue, respectively.

RNA: protein interactions associated with satellites of panicum mosaic virus

The interactions between satellite panicum mosaic virus (SPMV) capsid protein (CP) and its 824 nucleotide (nt) single stranded RNA were investigated by gel mobility shift assay and Northwestern blot assay. SPMV CP has specificity for its RNA at high affinity, but little affinity for non-viral RNA. The SPMV CP also bound a 350 nt satellite RNA (satRNA) that, like SPMV, is dependent on panicum mosaic virus for its replication. SPMV CP has the novel property of encapsidating SPMV RNA and satRNA. Competition gel mobility shift assays performed with a non-viral RNA and unlabeled SPMV RNA and satRNA revealed that these RNA:protein interactions were in part sequence specific.

Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1.

Clinical applications of GnRH agonists (GnRHa) are based primarily on the decrease in gonadotropin release after down-regulation of the GnRH receptor (GnRHR) by continuous GnRHa administration. However, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR gene after prolonged GnRH treatment remain poorly understood. In the present study GnRHa-mediated regulation of human GnRHR gene transcription was studied by transiently transfecting the mouse gonadotrope-derived (alphaT3-1) cells with a 2297-bp human GnRHR promoter-luciferase construct (p2300-LucF). A dose- and time-dependent decrease in human GnRHR promoter activity was observed after GnRHa treatment. An average 71% decrease in promoter activity was observed after 24-h treatment with 0.1 microM GnRHa, which was blocked by cotreatment of the GnRH antagonist, antide. This effect was mimicked by phorbol 12-myristate 13-acetate (TPA) administration. In addition, the GnRHa- and TPA-mediated decrease in the human GnRHR promoter activity was reversed by a specific protein kinase C (PKC) inhibitor, GF109203X, or depletion of PKC by TPA pretreatment. These findings indicate that the activation of the PKC pathway is important in regulating the human GnRHR gene expression. By progressive 5'-deletion studies, we have identified a 248-bp DNA fragment (-1018 to -771, relative to the translation start site) at the 5'-flanking region of the human GnRHR gene that is responsible for the GnRHa-mediated down-regulation of human GnRHR promoter activity. Analysis of this sequence reveals the existence of two putative activating protein-1 (AP-1) sites with 87% homology to the consensus sequence (5'-TGA(G/C)T(C/A)A-3'), located at -1000 to -994 (5'-TTAGACA-3', in complementary orientation) and -943 to 937 (5'-TGAATAA-3'). Using competitive gel mobility shift assays, AP-1 binding was observed within this 248-bp region. Site-directed mutation of the putative AP-1-binding site located at -1000 to -994 abolished the GnRHa-induced inhibition. Further competitive GMSA and supershift experiments confirmed the identity of AP-1 binding in this region. By the use of Western blot analysis, a significant increase in c-Jun (100%; P < 0.05) and c-Fos (50%; P < 0.05) protein levels was observed after GnRHa treatment in alphaT3-1 cells. In addition, our data suggested that a change in AP-1 composition, particularly c-Fos, was important in mediating GnRHa-induced inhibition of human GnRHR gene expression. We conclude that activation of the PKC pathway by GnRH is important in controlling human GnRHR gene expression. In addition, the putative AP-1-binding site located at -1000 to -994 of the human GnRHR5'-flanking region has been functionally identified to be involved in mediating this down-regulatory effect.

Zinc finger transcription factor Egr-1 activates Flt-1 gene expression in THP-1 cells on induction for macrophage differentiation.

Activation of macrophages is a hallmark of atherosclerosis. Stimulation of human monocytic leukemia THP-1 cells with phorbol 12-myristate 13-acetate (PMA) is known to induce a variety of genes whose function is relevant to activated macrophages. Flt-1, a receptor tyrosine kinase for vascular endothelial growth factor, is expressed in macrophages as well as in endothelial cells and mediates the biological response to vascular endothelial growth factor. In this study, we investigated the molecular mechanisms underlying the inducible expression of the flt-1 gene during the activation of THP-1 cells. Reverse transcription-polymerase chain reaction analysis showed that exposure of THP-1 cells to PMA increases flt-1 mRNA and protein levels. A transfected reporter gene, consisting of the human flt-1 promoter region coupled to the luciferase gene, indicated a direct effect of PMA on transcriptional activity. Transfection analysis of a series of 5'-deletion constructs and site-directed mutants localized the PMA-responsive region to a DNA stretch from -174 to -166, which represents overlapping Egr-1/Sp1 transcription factor-binding sites. Competitive gel mobility shift assays and supershift assays showed that PMA induces the binding of Egr-1 to this site. Consistent with these findings, the Egr-1 expression plasmid strongly induced flt-1 promoter activity in a sequence-specific manner. Taken together, our data demonstrate that PMA induces flt-1 gene transcription through an induction of Egr-1 in THP-1 cells, thus providing new evidence that the flt-1 gene is a direct target of Egr-1, the transcription factor primarily induced on macrophage differentiation.

A novel piscine vitellogenin gene: structural and functional analyses of estrogen-inducible promoter

The Oreochromis aureus vitellogenin, OaVtg, gene spans 9 kb and contains 34 exons. Its transcription start site is located 15 bp upstream of the translational start codon. Although the OaVtg promoter has a nonconsensus TATA, transient transfection assay showed that this promoter is capable of driving basal transcription. Two imperfect estrogen response elements: ERE p (proximal) and ERE d (distal) are located in the promoter at −532 and −1352, respectively. In competition gel mobility-shift assays, only ERE p exhibited specific binding of the recombinant estrogen receptor protein, GST-C/D OaER. Another imperfect ERE (ERE exon2) was detected within exon 2 of the OaVtg gene. This is a novel finding for a vitellogenin (Vtg) gene. ERE exon2 similarly showed specific recognition of GST-C/D OaER. Both ERE p and ERE exon2 showed comparable binding affinities as consensus ERE. In transient transfections, the OaVtg promoter, ERE p and ERE d elicited significant increase in estrogen-dependent synthesis of CAT protein. Hence, we propose that the non-consensus OaVtg EREs contribute to the estrogen-dependent regulation of the OaVtg gene in vivo.

Characterization of a Novel Liver-Specific Protein/DNA Binding Site in the Human HMG CoA Reductase Promoter

These studies define a novel binding element (site C) within the human HMG CoA reductase promoter using a combination ofin vitroDNase I footprinting and gel mobility shift assays. The factor interacting with site C appears to be restricted to the liver, indicating a possible role for this protein in regulating hepatic expression of the gene. Studies based on competitive gel mobility shift assays and transient co-transfection experiments performed using a reporter construct harbouring the promoter of HMG CoA reductase suggest that the protein binding to site C may belong to the C/EBP family of transcription factors. A factor interacting with this binding element was also identified in human liver nuclear protein extracts.

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins.

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.

Cell proteins bind to a 67 nucleotide sequence within the 3' noncoding region (NCR) of simian hemorrhagic fever virus (SHFV) negative-strand RNA.

The 3'NCR of the SHFV negative-strand RNA [SHFV 3'(-)NCR RNA] is thought to be the initiation site of full-length and possibly also subgenomic positive-strand RNA and so is likely to contain cis-acting signals for viral RNA replication. Cellular and viral proteins may specifically interact with this region to form replication complexes. When in vitro transcribed SHFV 3'(-)NCR RNA was used as a probe in gel mobility shift assays, two RNA-protein complexes were detected with MA104 S100 cytoplasmic extracts. The specificity of thes RNA-protein interactions was demonstrated by competition gel mobility shift assays. Four MA104 protein (103, 86, 55, and 36 kDa) were detected by UV-induced cross-linking assays and three proteins (103, 55, and 36 kDa) were detected by northwestern blotting assays. The binding sites for these proteins were mapped to the region between nucleotides 117 to 184 on the SHFV 3'(-)NCR RNA. Four cellular proteins with identical molecular masses to those of the proteins that bind to the SHFV 3'(-)NCR RNA were detected by the 3'(-)NCR of another arterivirus, LDV-C, suggesting that divergent arteriviruses utilize the same set of conserved cell protein domains.

Translation elongation factor-1 alpha interacts with the 3' stem-loop region of West Nile virus genomic RNA.

The conserved 3'-terminal stem-loop (3' SL) of the West Nile virus (WNV) genomic RNA was previously used to probe for cellular proteins that may be involved in flavivirus replication and three cellular proteins were detected that specifically interact with the WNV 3' SL RNA (J. L. Blackwell and M. A. Brinton, J. Virol. 69:5650-5658, 1995). In this study, one of these cellular proteins was purified to apparent homogeneity by ammonium sulfate precipitation and liquid chromatography. Amino acid sequence Western blotting, and supershift analyses identified the cellular protein as translation elongation factor-1 alpha (EF-1 alpha). Competition gel mobility shift assays demonstrated that the interaction between EF-1 alpha and WNV 3' SL RNA was specific. Dephosphorylation of EF-1 alpha by calf intestinal alkaline phosphatase inhibited its binding to WNV 3' SL RNA. The apparent equilibrium dissociation constant for the interaction between EF-1 alpha and WNV 3' SL RNA was calculated to be 1.1 x 10(-9) M. Calculation of the stoichiometry of the interaction indicated that one molecule of EF-1 alpha binds to each molecule of WNV 3' SL RNA. Using RNase footprinting and nitrocellulose filter binding assays, we detected a high-activity binding site on the main stem of the WNV 3' SL RNA. Interaction with EF-1 alpha at the high-activity binding site was sequence specific, since nucleotide substitution in this region reduced the binding activity of the WNV 3' SL RNA for EF-1 alpha by approximately 60%. Two low-activity binding sites were also detected, and each accounted for approximately 15 to 20% of the binding activity. Intracellular association between the host protein and the viral RNA was suggested by coimmunoprecipitation of WNV genomic RNA and EF-1 alpha, using an anti-EF-1 alpha antibody.

Molecular Cloning and Characterization of the Promoter for the Chinese Hamster DNA Topoisomerase IIα Gene

To investigate the mechanisms governing the expression of DNA topoisomerase II alpha, the Chinese hamster topoisomerase II alpha gene has been cloned and the promoter region analyzed. There are several transcriptional start sites clustered in a region of 30 base pairs, with the major one being 102 nucleotides upstream from the ATG translation initiation site. Sequencing data reveal one GC box and a total of five inverted CCAAT elements (ICEs) within a region of 530 base pairs upstream from the major transcription start site. Sequence comparison between the human and Chinese hamster topoisomerase II alpha gene promoter regions shows a high degree of homology centered at the ICEs and GC box. In vitro DNase I footprinting results indicate protection by binding proteins at and around each ICE on both DNA strands. However, no obvious protection was observed for the GC box. Competition gel mobility shift assays with oligonucleotides containing either the wild-type or mutated ICE sequences suggest that identical or similar proteins specifically bind at each ICE, although with different affinities for individual ICE sequences. Chloramphenicol acetyltransferase assays employing nested 5'-deletions of the 5'-flanking sequence of the gene demonstrate that the sequence between -186 and +102, which contains three proximal ICEs, is sufficient for near wild-type level of promoter activity. When these three ICEs were gradually replaced with sequences which do not interact with the binding proteins, reducing promoter activity of the resulted constructs was observed. In conjunction with results from footprinting and gel mobility shift studies, the transient gene expression finding suggests that the ICEs are functionally important for the transcriptional regulation of the topoisomerase II alpha gene.

Regulation of Xenopus laevis estrogen receptor gene expression is mediated by an estrogen response element in the protein coding region.

To investigate the 17 beta-estradiol induction of the mRNA coding for the Xenopus laevis estrogen receptor (XER), we cloned the promoter and the 5'-flanking region of the ER gene. Transcription initiation sites were identified by primer extension, and confirmed by the polymerase chain reaction. The promoter and 5'-flanking region contain an imperfect TATA box and a potential CAAT box at -51. Sequence analysis and transfections indicated that no functional estrogen response element (ERE) was present in approximately 3 kb of 5'-flanking region. An imperfect ERE, GGTCAGTTTGACG, which differs from the consensus ERE sequence by 1 nucleotide, was detected in the protein coding region of the gene, approximately 480 nucleotides downstream of the transcription initiation site. In transient transfections using a simple promoter containing two copies of this Xenopus estrogen receptor ERE (XERE), we observed an estrogen-dependent increase in CAT activity of four- to five-fold, to a level approximately 20-fold greater than the activity of the control plasmid lacking the XEREs. In competition gel mobility-shift assays, the XERE exhibited a weak, but clearly detectable, ability to compete for binding of human ER to a labeled consensus ERE. Because it exhibits sequence-specific binding to the ER in competition gel mobility-shift assays, and is able to confer estrogen-dependent transcription on a simple synthetic promoter, the novel XERE, located in the protein coding region of the XER gene appears to represent a weak, but functional, ERE.

Intrinsically Bent DNA in a Eukaryotic Transcription Factor Recognition Sequence Potentiates Transcription Activation

Many eukaryotic transcription factors induce DNA bending on binding to their recognition sequences. DNA bending could play a structural role by altering contacts between the protein and DNA. Alternatively, DNA bending could play a more direct role in transcription activation. To distinguish between these possibilities, we inserted two to eight copies of the intrinsic bending sequence, AAAAAACGTG, into a minimal promoter containing only a TATA box. The intrinsic DNA bending sequence was a potent activator of transcription in both in vivo transfection experiments and in a cell-free transcription system. A protein binds to the intrinsic bending sequence with high specificity in gel mobility shift assays and was required for its transcription in cell-free extracts. The intercalator, distamycin, which eliminates the ability of the sequence to bend, specifically reduced its transcription by about 60%. Mutations in the sequence which abolished DNA bending reduced transcription by approximately 70% in vivo. Competition gel mobility shift assays showed that the transcription factor bound equally well to mutants in which DNA bending was abolished and to the intrinsic bending sequence. These data indicate that DNA bending can play a direct role in the activation of eukaryotic transcription.

A novel cAMP-dependent regulatory region including a sequence like the cAMP-responsive element, far upstream of the human CYP21A2 gene

Deletion mutants in the 5' upstream sequence of the human CYP21A2 gene demonstrated a novel regulatory DNA element responsible for cAMP-dependent expression of the gene in the transient expression system using Y-1 cells (mouse adrenocortical tumor cell line). This regulatory element (-2574 to -2489 bp) was also found to exhibit a strong enhancer activity through heterologous promoters in response to cAMP and to contain a sequence like the cAMP-responsive element (CRE) and a CAAT-like sequence. The CRE-like sequence has a five-base motif (5'-TGACG-3') of the palindromic CRE consensus (TGACGTCA). Competitive gel mobility shift assays using nuclear extracts of bovine adrenal cortex with sequences typical of the binding sites for the binding proteins of CRE and the CAAT-like sequence revealed that these binding proteins, or related factors, bound to their cognate DNA binding sites in the upstream enhancer region of the CYP21A2 gene. These two enhancer elements and their cognate binding factors cooperate with previously identified tissue-specific enhancers (adrenal-specific protein factor and Ad4-like sequences) and their binding factors to express a high level of cAMP-responsive expression of the CYP21A2 gene.

Plant bZIP Protein DNA Binding Specificity

Plant bZIP proteins exhibit a relaxed DNA-binding specificity for DNA sequence motifs containing an ACGT core. Gel mobility shift experiments employing ten different recombinant plant bZIP proteins demonstrated that nucleotides flanking the ACGT core affected binding specificity and identified three different types of ACGT elements: G-box, CACGTG; C-box, GACGTC; and A-box, TACGTA, motifs. These ten different bZIP proteins could be categorized into three groups according to their qualitative and quantitative specificity for G-box and C-box elements. Dissociation constant values (Kd values) of these bZIP proteins for high affinity G-box and C-box elements and reciprocal competition gel mobility shift assays confirmed our classification scheme. Group 1 proteins exhibit a stronger binding affinity for G-box elements, group 2 proteins bind both G-box and C-box motifs with comparable binding affinity, whereas the group 3 proteins display a stronger binding affinity for C-box oligonucleotides. Studies using a panel of G-box and C-box oligonucleotides differing in their flanking sequences identified high affinity binding sites. All ten plant bZIP proteins examined, except TGA1a, exhibited type A G-box binding activity preferring class I G-box elements. In contrast to the situation observed for G-box elements, C-box motifs displayed a very much more stringent flanking nucleotide requirement for binding activity. Protein/DNA binding experiments using scanning mutants of a high affinity G-box element and G-box/C-box hybrid elements demonstrated that bZIP protein binding activity depends upon the affinity of protein dimer subunits for ACGT half-sites. Information provided by our systematic analysis of plant bZIP DNA binding specificity can be used to identify high affinity binding sites for the plant bZIP proteins studied here. Assuming that only high affinity bZIP binding sites are likely to function in vivo, identification of these sites will allow us to predict which genes are activated by a particular bZIP protein.

Identification and characterization of two enhancers of the human albumin gene.

A 12.5-kilobase pair (kb) segment upstream of the human albumin gene was analyzed for transcription enhancing activity using transient transfection analysis, gel mobility shift assays, DNase I footprinting, and site-specific mutagenesis. Two enhancer regions were identified, one 1.7 kb upstream of the transcription initiation site (E1.7) and the other 6 kb upstream (E6). In E1.7, a nuclear protein from HuH-7 hepatoma cells binds to an AT-rich sequence, GTTACTAATTGAC. Competition gel mobility shift assays suggested that this protein is HNF-1, which regulates the promoter of the albumin gene and several other liver-specific genes. A 60-base pair E1.7 fragment carrying the AT-rich sequence stimulates a heterologous (alpha-fetoprotein) promoter in a dose-dependent manner. In E6, a HuH-7 nuclear protein binds to a GT-rich sequence, TGTTTGGC.A 27-base pair E6 fragment carrying this sequence is able to stimulate the SV40 promoter in an orientation-independent manner. An alteration of this sequence by site-specific mutagenesis resulted in the loss of transcriptional activity as well as binding to the HuH-7 nuclear protein. Competition gel mobility shift assays showed that homologous elements exist in the albumin promoter. These results show that the promoter and enhancer of the human albumin gene are regulated by two common transcription factors through two shared cis-acting elements, one AT-rich and the other GT-rich.

BINDING OF TRANSCRIPTION FACTOR SP1 TO EXON-1 OF C-MYC IS ALTERED IN BURKITTS-LYMPHOMA

In Burkitt's lymphoma (BL) a consistent chromosomal translocation involving the c-myc proto-oncogene locus on chromosome 8 and one of the immunoglobulin loci located on chromosomes 14, 22 or 2 results in the aberrant expression of c-myc. In six out of ten published BL sequences the translocated c-myc gene contains mutations and/or deletions in an Spl site in the first exon. We show by competition gel mobility shift assays and association constant comparison that the binding of Spl to this site is altered in four of these clones. The affinity of Spl for this site is increased 5-10 fold in Burkitt's lymphoma BL22 and decreased approximately 5 fold in BL2. Mutations at this site in cell lines Raji and Daudi also show a decrease in Sp1 binding when compared with the wild type sequence.