Linker-scanning Mutations Research Articles

The mating-type proteins of fission yeast induce meiosis by directly activating mei3 transcription.

Cell type control of meiotic gene regulation in the budding yeast Saccharomyces cerevisiae is mediated by a cascade of transcriptional repressors, a1-alpha2 and Rme1. Here, we investigate the analogous regulatory pathway in the fission yeast Schizosaccharomyces pombe by analyzing the promoter of mei3, the single gene whose expression is sufficient to trigger meiosis. The mei3 promoter does not appear to contain a negative regulatory element that represses transcription in haploid cells. Instead, correct regulation of mei3 transcription depends on a complex promoter that contains at least five positive elements upstream of the TATA sequence. These elements synergistically activate mei3 transcription, thereby constituting an on-off switch for the meiosis pathway. Element C is a large region containing multiple sequences that resemble binding sites for Mc, an HMG domain protein encoded by the mating-type locus. The function of element C is extremely sensitive to spacing changes but not to linker-scanning mutations, suggesting the possibility that Mc functions as an architectural transcription factor. Altered-specificity experiments indicate that element D interacts with Pm, a homeodomain protein encoded by the mating-type locus. This indicates that Pm functions as a direct activator of the meiosis pathway, whereas the homologous mating-type protein in S. cerevisiae (alpha2) functions as a repressor. Thus, despite the strong similarities between the mating-type loci of S. cerevisiae and S. pombe, the regulatory logic that governs the tight control of the key meiosis-inducing genes in these organisms is completely different.

Functional analysis of regulatory sequences controlling PR-1 gene expression in Arabidopsis.

The Arabidopsis PR-1 gene is one of a suite of genes induced co-ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6-dichloroisonicotinic acid (INA). We have characterized cis-acting regulatory elements in the PR-1 promoter involved in INA induction using deletion analysis, linker-scanning mutagenesis, and in vivo footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10-fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10-bp linker-scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The -640 linker-scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the -610 linker-scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF-kappa B. Furthermore, several inducible in vivo footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This in vivo signature of protein-DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.

Nuclear factor-1 motif and redundant regulatory elements comprise phenobarbital-responsive enhancer in CYP2B1/2.

Although the induction of drug-metabolizing systems by phenobarbital has been recognized for about 40 years, the mechanism by which cytochrome P450 gene expression is increased is still not well understood. A 163-bp fragment at about -2.2 Kb in CYP2B2 has been shown to mediate phenobarbital induction in primary rat hepatocytes (Trottier, et al. [1995] Gene 158:263-268) and by an in situ transient transfection assay in rat liver (Park, Y., et al. [1996]. J. Biol. Chem. 271:23725-23728). Deletion mutations of this fragment indicated that the 88-bp stretch from -2258 to -2170 was the minimal sequence that could mediate phenobarbital induction in the in situ system if single copies of the deleted fragments fused to the CYP2C1 proximal promoter were assayed. If three copies of the fragments were present, 5' and 3' deletions defined a minimal 37-bp core fragment, which, although necessary for phenobarbital responsiveness, was not sufficient unless additional sequence was present at either end, suggesting that redundant elements were present in the two flanking regions. Site-specific mutagenesis of an NF-1 site within the 88-bp fragment and linker scanning mutagenesis across the fragment indicated that the NF-1 site and a region to the 5' side of the site contributed to the magnitude of the response, but neither the NF-1 mutations nor any of the linker scanning mutations eliminated the response to phenobarbital. Mutation in a region 3' of the NF-1 site resulted in elevated basal expression without substantial effects on phenobarbital-induced expression. Binding of NF-1 to the 37-bp core fragment was established by gel-shift competition studies and by supershifts of the protein-DNA complexes by antisera to NF-1. Additional protein-DNA complexes were detected in the regions flanking the NF-1 site. These studies indicate that the CYP2B2 phenobarbital-responsive enhancer contains multiple constitutive and phenobarbital-responsive elements. Binding of nuclear proteins from control or phenobarbital-treated animals in vitro to this region was very similar. The only difference detected was a complex that was substantially reduced by phenobarbital treatment and mapped to the 3' side of the NF-1 site.

Secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome.

The 3'-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3' nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370-4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3' NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (beta-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis-active sequence and at several distinct locations within the 3' NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3' NTR, and a base-paired structure near the 3' terminus of the 3' NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3' terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3' NTR are coadapted for genome amplification function through a requirement for base pair interactions.

Functions of novel transcription factors interacting with the promoter of the gene encoding the mRNA cap binding protein (eIF4E) in growth regulation • 771

Cell division is dependent on cell growth. Although elements regulating cell division have received intense scrutiny, factors regulating growth in the form of new protein synthesis are less well characterized. The mRNA cap-binding protein (eIF4E) is rate limiting for translation initiation and transforms fibroblasts. To identify factors regulating eIF4E, we characterized the promoter of the eIF4E gene using linker scanning mutants. Promoter activity was dependent on sites at -5, -25, -45 and -75. Sites at -5, -25, and-45 were located in areas classi-cally involved in transcription initiation; a myc box at -75 was previously described. Since the eIF4E promoter lacked both TATAA and initiator elements, we characterized potential preinitiation complexes at -5, -25 and -45 sites. EMSA experiments revealed novel factors binding the -25 site whose minimum motif was TTACCCCCCCTT. The abundance of 68 and 98 kd proteins which bound this site in UV crosslinking and Southwestern analysis correlated with protein synthesis rates and c-myc levels in differentiating U937 and HL60 cells. Correlations between the 68 and 98 kd proteins, eIF4E expression and protein synthesis rates suggested a role for the -25-binding factors in down-regulation of protein synthesis during differentiation. These novel transcription factors are also positioned to mediate the induction of eIF4E by the c-myc oncogene.

Evidence for multiple promoters of the human IL-5 receptor alpha subunit gene: a novel 6-base pair element determines cell-specific promoter function.

In addition to a previously characterized promoter (P1), we now show the existence of a second promoter for the human IL-5Ralpha gene. Initially, a genomic region (P2) 5' upstream of human IL-5Ralpha exon 2 was cloned by an inverted PCR. The transcriptional start site was then mapped to a deoxycytidine (C) residue within P2 by analyzing cellular mRNA with both the 5' rapid amplification of cDNA end-PCR and S1 nuclease protection assays. Transfection of eosinophilic HL-60 cells with reporter gene constructs in which either P1 or P2 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in CAT expression; little or no CAT expression occurred in other myeloid and nonmyeloid cell lines. Deletion studies showed that a 66-bp region, ranging from -31 to +35, was sufficient to promote CAT expression in eosinophilic HL-60 cells. Analysis of linker-scanning mutants identified a novel 6-bp element (5' CTAATT 3') spanning -19 to -14 that was essential for P2 promoter activity. In electrophoretic mobility shift assays, a P2 region from -31 to +1 containing the unique 6-bp element, when used as a probe, formed a complex with a protein(s) that was found only in the eosinophilic cell line. This binding activity was lost upon replacement of the 6-bp element with a 6-bp linker, suggesting that this element likely serves as the binding site for an eosinophilic HL-60 cell-specific transcription factor(s). Together, these data suggest an important role for P2 promoter in the regulation of eosinophil-specific expression of the human IL-5 receptor alpha gene.

Analysis of the internal replication sequence indicates that there are three elements required for efficient replication of minute virus of mice minigenomes.

Prior analysis of minigenomes of minute virus of mice carried out by our laboratory indicated that sequences within the region of nucleotides 4489 to 4695, inboard of the 5' palindrome, are required for efficient DNA replication of the virus and are the site of specific interactions with unidentified factors present in a host cell nuclear extract (P. Tam and C. R. Astell, Virology 193:812-824, 1993; P. Tam and C. R. Astell, J. Virology 68:2840-2848, 1994). In order to examine this region in finer detail, a comprehensive library of linker-scanning mutants spanning the region was tested for the ability to support replication of minigenome constructs and for the ability to interact with host cell factors. Three short discrete sequence elements critical for replication competence were observed. Binding of host cell nuclear factors was localized to four sites, with two major complexes each appearing to have two binding sites within the region. All factor binding sites were found to be directly adjacent to or overlapping with sequence elements contributing to replication competence, and evidence suggesting a correlation between factor binding and minigenome replication is presented. A possible model is proposed for function of a viral origin within the region of the internal replication sequence which addresses the still-unresolved problem of how parvoviruses overcome the thermodynamic energy barrier involved in the rearrangement of the 5'-terminal palindrome from an extended form to a hairpin conformation.

An efficient protocol for linker scanning mutagenesis: analysis of the translational regulation of an Escherichia coli RNA polymerase subunit gene.

A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.

Identification of cis- and trans-Active Factors Regulating Human Islet Amyloid Polypeptide Gene Expression in Pancreatic β-Cells

Islet amyloid polypeptide is expressed almost exclusively in pancreatic beta- and delta-cells. Here we report that beta cell-specific expression of the human islet amyloid polypeptide gene is principally regulated by promoter proximal sequences. The sequences that control tissue-specific expression were mapped between nucleotides -2798 and +450 of the human islet amyloid polypeptide (IAPP) gene using transgenic mice. To localize the cis-acting elements involved in this response, we examined the effects of mutations within these sequences using transfected islet amyloid polypeptide promoter expression constructs in pancreatic beta cell lines. The sequences between -222 and +450 bp were found to be necessary for beta cell-specific expression. Linker-scanning mutations of the 5'-promoter proximal region defined several key distinct control elements, including a negative-acting element at -111/-102 base pairs (bp), positive-acting elements like the basic helix-loop-helix-like binding site at -138/-131 bp, and the three A/T-rich, homeobox-like sites at -172/-163, -154/-142, and -91/-84 bp. Mutations within any one of these elements eliminated transcriptional expression by the promoter. Gel mobility shift assays revealed that the PDX-1 homeobox factor, which is required for insulin gene transcription in beta cells, interacted specifically at the -154/-142- and -91/-84-bp sites. Since PDX-1 is highly enriched in beta and delta cells, these results suggest that this factor plays a principal role in defining islet beta cell- and delta cell-specific expression of the IAPP gene.

Sequences Necessary for Nitrate-Dependent Transcription of Arabidopsis Nitrate Reductase Genes

Nitrate increases the transcription of the two Arabidopsis thaliana nitrate reductase genes. We demonstrated previously that 238 and 330 bp of the 5' flanking regions, designated as NP1 and NP2, of the two nitrate reductase genes NR1 and NR2, respectively, are sufficient for nitrate-dependent transcription (Y. Lin, C.-F. Hwang, J.B. Brown, C.-L. Cheng [1994] Plant Physiol 106: 477-484). Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (LS) analysis. In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity. To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used to measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction. The single LS mutant in NP1 lost its response to nitrate, whereas the two LS mutants in NP2 partially lost their response to nitrate. A 12-bp sequence is conserved between the NP1 site and the two NP2 sites. This sequence motif is also conserved in the 5' flanking regions of other nitrate-inducible plant genes. Gel mobility shift experiments indicate that these three regions bind to similar proteins. The binding is constitutive with respect to nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves.

Functional characterization of the promoter for the gene encoding murine CD34

Since CD34 expression is restricted to the hematopoietic stem cells and decreases in differentiating cells, the analysis of the CD34 promoter is of interest to understand regulation of gene expression in stem cells. To characterize the cis-acting elements which control murine CD34 (mCD34) gene expression, we sought to clone, sequence, and functionally analyze the mCD34 promoter. An 80% decrease in promoter activity was obtained when sequences between −119 bp and −59 bp upstream of the transcriptional start site were deleted. We identified several DNA-protein complexes which correspond to functional segments defined by the linker-scanning mutants. These findings indicated the presence of the important regulatory element between bp −119 and −100, GGTTAAAAGTGAAGTAGGAA. Furthermore, from the result of functional promoter analysis in the DNase I hypersensitive site (HS) located within 5 kb upstream of the mCD34 gene, the presence of the enhancer region in the NcoI/ PstI fragment of 5′ upstream, −2.8 kb to −1.9 kb, has been identified. These data will provide useful information on the gene transfer using the CD34 promoter and enhancer.

Functional characterization of the 5′-flanking region of the gene encoding human 2-oxoglutarate dehydrogenase

The 5′-flanking region of the gene ( OGDH) encoding human 2-oxoglutarate dehydrogenase (OGDH) does not contain TATA or CAAT boxes, but contains an inverted GC box. To identify a functional OGDH promoter, systematic transient expression analysis of the 5′-flanking region (wild type, −3276/+212) of the OGDH was performed using serially nested deletions and linker-scanning mutations. The OGDH (wild type)-luciferase (LUC) reporter vector (pGV-E) construct and its deletion or linker-mutant constructs were transfected into BHK-21 cells and LUC activity was assessed. The OGDH- LUC construct expressed reproducibly >12-fold more LUC activity than did a control pGV-E vector. The promoter activity was up-regulated by treatment with 2-oxoglutarate and 2-oxoglutarate/glutamate. Deletions of sequences between nt −563 and −62 (M6 and M7) resulted in a 2-fold increase in LUC activity. Further deletion of the sequence between nt −61 and +212 (M8–M10) abolished LUC activity. High resolution mutagenesis within the −113 to −14 region indicated that the −53 to −44 and −33 to −24 sequences were required for positive regulation and the −93 to −84 sequence for negative regulation. We have identified a nuclear factor that binds to nt −63 to −24 including two cis-acting sites.

Human T-cell leukemia virus type 1 Tax transactivates the human proliferating cell nuclear antigen promoter.

The human T-cell leukemia virus type 1 (HTLV-1) transforming protein, Tax, is a potent transactivator of both viral and cellular gene expression. The ability of Tax to transform cells is believed to depend on its transactivation of cellular-growth-regulatory genes. Expression of proliferating cell nuclear antigen (PCNA) is intimately linked to cell growth and DNA replication and repair. By testing a series of PCNA promoter deletion constructs, we have demonstrated that the PCNA promoter can be transactivated by Tax. The smallest construct that was activated did not include the ATF/CRE binding site at nucleotide -50, and mutations in the ATF/CRE element in the context of a larger promoter were still activated by Tax. In addition, a Tax mutant that is defective for activation of the CRE pathway retained the ability to activate the -397 promoter construct. When a series of linker scanner mutations that span the region from nucleotide -45 to -7 were assayed, mutations in and around a repeat sequence were found to abolish Tax transactivation. Multimerized copies of either half of the repeat were Tax responsive. A single protein complex was shown to bind specifically to the Tax-responsive region, and the binding of this complex was enhanced in the presence of Tax. These results demonstrate that the PCNA promoter contains a Tax-responsive element located between nucleotides -45 and -7 whose sequence is different from those of other, previously identified Tax-responsive elements. The ability of Tax to activate the PCNA promoter may play an important role in cellular transformation by HTLV-1.

Analysis of the promoter elements necessary for IL-4 and anti-CD40 antibody induction of murine Fc epsilon RII (CD23): comparison with the germline epsilon promoter.

IL-4 and CD40 ligand stimulate transcription of CD23 (Fc epsilonRII) in B cells and are necessary for the expression of germline epsilon mRNA and production of IgE. Because in vivo studies have shown that the Fc epsilonRII is involved in the regulation of IgE, a study was initiated to compare how IL-4 and engagement of CD40 up-regulate the Fc epsilonRII and epsilon genes. Herein, we describe the preparation of a series of linker-scanning mutants that cover the IL-4 response region in the murine Fc epsilonRII promoter, and their function when transfected into M12.4.5 and M12.4.1 B lymphoma cell lines. Several discrete elements were found to be necessary for IL-4 induction of the Fc epsilonRII gene, some of which have homology with the binding sites of known transcription factors, including NF-IL-4 and NF-kappaB. In contrast, the response element for anti-CD40 (plus IL-4) mapped to a single discrete sequence, a NF-kappaB-like site. Aligning the Fc epsilonRII and germline epsilon promoters in the region that is highly conserved between the human and mouse homologues of both genes reveals a high degree of identity, particularly within discrete clusters. Comparing the function of linker-scanning mutants of the Fc epsilonRII promoter with a similar report for germline epsilon shows that both genes require at least two homologous and similarly located DNA elements in their promoters for a full IL-4 induction. Moreover, the similar response of Fc epsilonRII and epsilon promoter-driven chloramphenicol acetyl transferase plasmids to several cytokines and other agents suggests that the two proximal promoter regions are activated by a similar cassette of factors.

Ethylene control of E4 transcription during tomato fruit ripening involves two cooperative cis elements.

E4 gene transcription is controlled by ethylene during tomato fruit ripening. To define the ethylene-responsive promoter elements, we have tested the activity of mutations of the E4 promoter, and of chimeric genes in transient assay. Using a set of linker scan mutations of the region from -160 to -91, we determined that sequences located between -150 and -121 bp from the transcription start site are required for normal levels of ethylene-regulated transcription. However, E4 sequences from -193 to -40 were not able to confer ethylene-responsiveness to the minimal (-46) 35S promoter. The E4/E8 binding protein (E4/E8 BP) interacts with sequences in the 5'-flanking regions of both E4 and the coordinately regulated E8 gene, and its role in regulation of E4 transcription was investigated. The E4 binding site spans the E4 TATA box, and so mutations of this site were limited to those that did not disrupt the E4 TATA box. Mutations of this site which reduced affinity for the E4/E8 BP also resulted in reduced activity in transient assay, supporting a role for this element in normal regulation of the gene. Fusion of the 35S enhancer to E4 sequences from -85 to +65 did not result in an ethylene-responsive promoter, indicating that the E4/E8 BP-binding site is not sufficient for ethylene response. We conclude that at least two cis elements are required for ethylene-responsive transcription of the E4 gene during fruit ripening, one between -150 and -121 and the other between -40 and +65.

Activation of NF-kappaB/Rel by CD40 engagement induces the mouse germ line immunoglobulin Cgamma1 promoter.

Interaction between CD40 on B cells and CD40 ligand (CD40L) on T cells has been shown to mediate T-cell contact help for B-cell proliferation, differentiation, and immunoglobulin isotype switching. It has recently been shown that cross-linking CD40 on mouse B cells induces germ line gamma1 and epsilon transcripts and that interleukin-4 synergizes with CD40 signaling to further induce these germ line transcripts. Germ line transcripts have been shown to be required for class switch recombination. Here we show that signaling via CD40 increases expression of a transiently transfected luciferase reporter plasmid driven by the germ line Cgamma1 promoter in M12.4.1 B-lymphoma cells. By linker-scanning mutation analysis of the promoter, we have identified a CD40-responsive region (CD40RR) which is able to confer inducibility by CD40L to a minimal c-fos promoter. The CD40RR contains three binding sites for NF-kappaB/Rel proteins which are each required for maximal induction of CD40RR activity by CD40L. Binding of the NF-kappaB/Rel proteins p50, p65, c-Rel, and RelB to the CD40RR is induced by CD40 signaling in M12.4.1 cells and in splenic B cells. Cotransfection of expression plasmids for p50 and p65 or p50 and RelB, but not c-Rel, into M12.4.1 cells transactivates the CD40RR and the germ line gamma1 promoter. These data demonstrate that NF-kappaB Rel proteins activated by CD40 ligation play an important role in induction of the germ line Cgamma1 promoter.

Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested.

Regulation of the Chicken Ovalbumin Gene by Estrogen and Corticosterone Requires a Novel DNA Element That Binds a Labile Protein, Chirp-I

Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE.

The major histocompatibility complex class II Ea promoter requires TFIID binding to an initiator sequence.

The major histocompatibility complex (MHC) class II Ea promoter is dependent on the presence of conserved upstream X and Y boxes and of initiator (Inr) sequences. In vitro transcription analysis of the Inr region with linker-scanning mutants pinpoints a functionally essential element that shows homology to the terminal deoxynucleotidyltransferase (TdT) Inr; contrary to the TdT Inr and other Inrs identified so far, the key sequence, between positions +5 and +12, is located within a transcribed area. Swapping the TdT sequence into the corresponding Ea position leads to a fivefold increase in transcription rate, without altering start site selection. Inr-binding proteins LBP-1/CP2 and TIP--a TdT Inr-binding protein unrelated to YY1--recognize the Ea Inr; they interact with overlapping yet distinct sequences around the Cap site, but their binding does not coincide with Ea Inr activity. A good correlation is, rather, found with binding of immunopurified holo-TFIID to this element. TFIID interacts both with Ea TATA-like and Inr sequences, but only the latter is functionally relevant. Unlike TBP, TFIID binds in the absence of TFIIA, indicating a stabilizing role for TBP-associated factors in Ea promoter recognition. Sequence comparison with other mouse and human MHC class II promoters suggests a common mechanism of start site(s) selection for the MHC class II gene family.

Interaction of Ethanol with Inducers of Glucose-regulated Stress Proteins: ETHANOL POTENTIATES INDUCERS OF grp78 TRANSCRIPTION

GRP78, a molecular chaperone expressed in the endoplasmic reticulum, is a "glucose-regulated protein" induced by stress responses that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking. Previously we showed that chronic ethanol exposure increases the expression of GRP78. To further understand the mechanism underlying ethanol regulation of GRP78 expression, we studied the interaction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma x glioma cells. We found that, in addition to increasing basal levels of GRP78 mRNA ("induction"), ethanol produced greater than additive increases in the induction of GRP78 mRNA by the "classical" GRP inducers A23187, brefeldin A, and thapsigargin ("potentiation"). Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfection analyses with the rat grp78 promoter. Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied. In contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promoter activity. Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences required for ethanol induction differ from those mediating responses to classical GRP inducers. Furthermore, linker-scanning mutations of the grp78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in induction by ethanol or classical inducing agents. While the ethanol induction response required 16-24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h. The potentiation response also decayed rapidly after ethanol removal. In addition, the protein kinase A inhibitor Rp-cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while Sp-cAMPS, an activator of protein kinase A, decreased ethanol potentiation. Taken together, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP inducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classical GRP inducers. In contrast, GRP78 induction by ethanol involves promoter sequences and a mechanistic pathway separate from that of the ethanol potentiation response or classical GRP78 inducers. These studies show that ethanol produces a novel and complex regulation of grp78 transcription which could be of particular importance during neuronal exposure to GRP-inducing stressors as might occur with central nervous system injury.