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PubMed1999

Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos.

We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.

Actins↗
PubMed2000

Amyloid fibril formation from full-length and fragments of amylin.

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.

Amyloid↗
PubMed1997

Identification of regions that regulate the expression and activity of G protein-gated inward rectifier K+ channels in Xenopus oocytes.

1. The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein-gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild-type subunits together with the m2 muscarinic receptor in Xenopus oocytes. 2. Co-expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1-Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane. 3. Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine-induced channel activity compared with the wild-type Kir3.2 homomeric channel. Cell-attached, single-channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings. 4. Individually substituting the N- and C-terminal tails of Kir3.1 for those of Kir3.2 showed that the C-terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N-terminal or core regions of this subunit. 5. The chimeric channel, 121, displayed a higher ratio of ACh-induced to basal activity than the Kir3.1-Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for G beta gamma. Our results suggest that substituting the Kir3.1 C-terminal tail for the Kir3.2 tail promotes the opening conformational change of the G beta gamma-bound channel. 6. The core and C-terminal regions of Kir3.1 independently conferred time dependence on voltage-dependent activation. The time constant (tau) was between 5 and 10 ms and varied little over the voltage range -60 to -120 mV.

Animals↗
PubMed1996

Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex.

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.

Amino Acid Sequence↗
PubMed1999

Mapping protein-protein interactions with a library of tethered cutting reagents: the binding site of sigma 70 on Escherichia coli RNA polymerase.

Surface-exposed lysine amino groups and other reactive nucleophiles of the sigma 70 protein were conjugated with the cutting reagent iron (S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (FeBABE) via 2-iminothiolane (2IT) with low efficiency. The result is a library of sigma 70 conjugates, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein. Model calculations indicate that the conjugates in this library should be capable of cutting nearby sites on the backbone of almost any protein or nucleic acid to which sigma 70 binds. Since cutting occurs only when the protein is bound, the cleaved sites indicate proximity; since only proximal sites are cleaved, interpretation of the results is straightforward. We used this library to map the periphery of the binding site on the core enzyme (alpha 2 beta beta') of Escherichia coli RNA polymerase. The beta subunit was cut primarily within its conserved regions C, D, Rif I, and G; additional sites were also cut between A and B and near conserved regions E and H. The cut sites within the beta' subunit were intensely clustered between residues 250-450, which include its conserved regions C and D, along with two additional cut sites in conserved regions A and G. No cut sites on the alpha subunit were observed. These results recapitulate and extend those obtained using FeBABE conjugates of seven strategically placed single-Cys sigma 70 mutants [Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., and Meares, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6021-6026]. This technique provides a straightforward, general approach to mapping protein interactions without mutagenesis.

Binding Sites↗
PubMed2002

Multiple roles of the RNA polymerase beta subunit flap domain in sigma 54-dependent transcription.

Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (alpha(2)beta beta'omega; E) associates with a sigma (sigma) subunit to form the holoenzyme (E sigma). A mutation removing the beta subunit flap domain renders the Escherichia coli sigma(70) RNAP holoenzyme unable to recognize promoters. sigma(54) is the major variant sigma subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of beta flap removal on sigma(54)-dependent transcription. Our analysis shows that the role of the beta flap in sigma(54)-dependent and sigma(70)-dependent transcription is different. Removal of the beta flap does not prevent the recognition of sigma(54)-dependent promoters, but causes multiple defects in sigma(54)-dependent transcription. Most importantly, the beta flap appears to orchestrate the proper formation of the E sigma(54) regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.

Amino Acid Sequence↗
PubMed1998

The major core protein of messenger ribonucleoprotein particles (p50) promotes initiation of protein biosynthesis in vitro.

The major core protein of cytoplasmic messenger ribonucleoprotein particles (p50) has been shown previously to inhibit protein synthesis in vitro and in vivo. Furthermore, p50 is highly homologous to the Y-box-binding transcription factor family of proteins, binds DNA containing the Y-box motif, and thus may have a dual function in cells as a regulator of both transcription and translation. Here we show that binding or removal of p50 from rabbit reticulocyte lysate by monospecific antibodies to p50 strongly inhibits translation of endogenous and exogenous globin mRNAs as well as prokaryotic beta-galactosidase mRNA in a rabbit reticulocyte cell-free system. Thus, depending on the conditions, p50 not only may act as a translational repressor, but may also be required for protein synthesis. Translation inhibition with anti-p50 antibodies is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it does not affect elongation/termination of polypeptide chains. The inhibition with anti-p50 antibodies is followed by a decay of polysomes and accumulation of the 48 S preinitiation complex. These results suggest that p50 participates in initiation of protein biosynthesis. Although uninvolved in the formation of the 48 S preinitiation complex, p50 is necessary either for attachment of the 60 S ribosomal subunit or for previous 5'-untranslated region scanning by the 43 S preinitiation complex.

Animals↗
PubMed2006

How does the human RUNX3 gene induce apoptosis in gastric cancer? Latest data, reflections and reactions.

RUNX3 is the oldest known gene in the RUNX family. Data have demonstrated its function to be thoroughly involved the neurogenesis of the dorsal root ganglia, T-cell differentiation and tumorigenesis of gastric epithelium. As a TGF-beta target, RUNX3 protein is believed to be involved in TGF-beta-mediated tumor suppressor pathway; however, little is known about its role in apoptosis. According to recent data reported by Yamamura et al., (J Biol Chem 2006; 281:5267-76), RUNX3 interacts with FoxO3a/FKHRL1 expressed in gastric cancer cells to activate Bim and induce apoptosis. The cooperation between RUNX3 and the PI3K/Akt signaling pathway component FoxO3a/FKHRL1 suggests the putative role of RUNX3 in the homoeostasis of gastric cells and in stomach cancer control. Here we discuss recent breakthroughs in our understanding of the mechanisms of RUNX3 in gastric malignancy and comment on possible future trends and perspectives.

Animals↗
PubMed2000

Regulation of Na,K-ATPase beta 1 subunit gene transcription by low external potassium in cardiac myocytes. Role of Sp1 AND Sp3.

Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K(+). Our previous data showed that exposure of cardiac myocytes to low K(+) increased the steady-state abundance of Na,K-ATPase beta1 subunit mRNA. In the present study we determined that incubation of primary cultures of neonatal rat cardiac myocytes with low K(+) augmented Na,K-ATPase beta1 gene expression at a transcriptional level and that this effect required extracellular Ca(2+). The stimulatory effect of low K(+) on Na,K-ATPase beta1 gene transcription was not dependent on increased contractile activity of cardiac myocytes. Na,K-ATPase beta1 5'-flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides -62 to -42 of the beta1 promoter contained a low K(+) response element. Site-directed mutagenesis of a potential GC box core motif GCG in the -58/-56 region of the beta1 promoter decreased basal and low K(+)-mediated transcription. Mutation of the core sequence of a putative GC box element located between nucleotides -101 and -99 further decreased the low K(+) effect on beta1 gene transcription. Electrophoretic mobility shift assays using oligonucleotides spanning the proximal and distal GC box elements of the beta1 promoter showed enhanced binding of two complexes in response to low K(+). The inclusion of a consensus GC box sequence as a competitor in gel shift analysis reduced factor binding to the low K(+) response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding of nuclear factors was abolished in gel shift studies using GC box mutants. Together these data indicate that enhanced binding of Sp1 and Sp3 to two GC box elements in the rat Na,K-ATPase beta1 subunit gene promoter mediates beta1 gene transcription up-regulation in neonatal rat cardiac myocytes exposed to low external K(+).

Animals↗
PubMed2001

Point mutations of the RUNx1/AML1 gene in sporadic and familial myeloid leukemias.

The RUNX1/AML1 gene is known to be the most frequent target for chromosomal translocation in leukemia. In addition, recent studies have demonstrated point mutations in the RUNX1 gene as an another mode of genetic lesion resulting in leukemia. Of particular interest, sporadic point mutations of biallelic type are found in a tight association with either the acute myelogenous leukemia (AML) MO subtype or trisomy 21. Germline mutations give rise to a familial platelet disorder that results in a predisposition to acute myelogenous leukemia (FPD/AML). Most of the RUNX1 mutants were defective in DNA binding but still active in beta binding, a characteristic that is consistent with the 3-dimensional structural findings and may explain the dominant inhibitory effects. Although genuine haploinsufficiency of RUNX1 was observed in some cases, a greater majority of mutant RUNX1 proteins may also act in a dominant-negative manner, possibly creating a higher propensity for leukemia development. The stronger dominant-negative effect was also deduced to be the major mechanism of the chimeric genes created by chromosomal translocations. The decrement of RUNXI activity may be a common underlying cause for RUNX1-related leukemias. However, because these RUNX1 abnormalities per se are insufficient for leukemogenesis, cooperating genetic alteration(s) should be intensively sought for further mechanistic insights and future clinical applications.

Animals↗
PubMed1990

The HAP2 subunit of yeast CCAAT transcriptional activator contains adjacent domains for subunit association and DNA recognition: model for the HAP2/3/4 complex.

Budding yeast HAP2 is required in concert with HAP3 and HAP4 to form a heterotrimeric CCAAT-binding transcriptional activation complex at the UAS2 element of CYC1. Functional homologs of HAP2 and HAP3 have been conserved in HeLa cells where HAP2 activity corresponds to a chromatographic fraction designated CP1B. Here, we describe deletion and codon insertion mutagenesis of the Saccharomyces cerevisiae HAP2 subunit. DNA binding was assessed both in vitro and in vivo whereas subunit association and transcriptional activation were analyzed in vivo by using a bifunctional lexA-HAP2 fusion. The results indicate that the 265-amino-acid HAP2 protein contains an "essential core" of 65 amino acids, which is entirely sufficient for assembly and DNA binding of the HAP2/3/4 complex. The essential core can be divided further into a region of 44 amino acids, which is sufficient for subunit association, and a region of 21 amino acids, which is required specifically for DNA recognition. The remainder of the HAP2 protein is dispensable. The roles of HAP3 and HAP4 in the heterotrimeric complex are also assessed by using lexA fusions and a fusion of the GAL4 acidic activation domain to HAP2, which partially bypasses a deletion of HAP4 but not HAP3. These data indicate that subunits HAP2 and HAP3 are primarily responsible for site-specific DNA binding by the complex, whereas the HAP4 subunit provides the primary transcriptional activation domain. A model for the function of this regulated transcriptional activation complex is presented.

Amino Acids↗
PubMed2003

Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo.

The alpha1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte-specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive beta-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2-null mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

Animals↗
PubMed1992

Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase.

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.

Animals↗
PubMed2001

Essential roles of Snf5p in Snf-Swi chromatin remodeling in vivo.

Snf-Swi, the prototypical ATP-dependent nucleosome-remodeling complex, regulates transcription of a subset of yeast genes. With the exception of Snf2p, the ATPase subunit, the functions of the other components are unknown. We have investigated the role of the conserved Snf-Swi core subunit Snf5p through characterization of two conditional snf5 mutants. The mutants contain single amino acid alterations of invariant or conserved residues that abolish Snf-Swi-dependent transcription by distinct mechanisms. One mutation impairs Snf-Swi assembly and, consequently, its stable association with a target promoter. The other blocks a postrecruitment catalytic remodeling step. These findings suggest that Snf5p coordinates the assembly and nucleosome-remodeling activities of Snf-Swi.

Adenosine Triphosphate↗
PubMed2004

A conserved sequence upstream of the B29 (Ig beta, CD79b) gene interacts with YY1.

The human, murine, and rat B29 (Ig beta, CD79b) genes are highly conserved in sequence and organization and exhibit strict B cell-specific expression. In the human and rat genomes, the B29 gene is located between the skeletal muscle-specific Na-channel alpha subunit (SCN4A) gene and the pituitary-specific growth hormone (GH-N) gene. The human pituitary-specific GH-N gene is controlled by a tissue-specific locus control region (LCR) located just upstream of the B29 promoter that mediates tissue-specific enhancement, histone acetylation, and an open chromatin conformation across the B29 gene in growth hormone (GH)-expressing pituitary cells. Here we show that B29 mRNA is not detected in a GH-expressing pituitary cell line and that GH-N mRNA is not detected in B cells. This differential expression suggests that the B29 gene is insulated or otherwise protected from the regulatory influences of the closely proximal GH LCR. We searched available sequences upstream of the human, mouse, and rat B29 genes and found a highly conserved sequence that fulfills the criteria recently established for non-coding DNA elements potentially involved in gene control. This B29 conserved sequence (BCS) bound ubiquitously expressed nuclear protein complexes. DNase I protection analysis of the BCS revealed a central 'footprinted' core which was confirmed to bind the multifunctional transcription factor, YY1. However, neither the BCS nor the YY1-binding core motif exhibited silencer or enhancer activity in transient transfections or position-independent insulator activity in enhancer-blocking assays. Thus, the BCS may function as a tissue-specific LCR or position-dependent insulator specifically countering the influences of the 5' GH LCR and controlling B29 gene expression.

5' Flanking Region↗
PubMed2001

A RUNX2/PEBP2alphaA/CBFA1 mutation in cleidocranial dysplasia revealing the link between the gene and Smad.

Cleidocranial dysplasia (CCD), an autosomal dominant human bone disease, is thought to be caused by heterozygous mutations in RUNX2/PEBP2alphaA/CBFA1. To understand the mechanism underlying the pathogenesis of CCD, we studied a novel mutant of RUNX2, namely CCDalphaA376, originally identified in a CCD patient. The nonsense mutation, which resulted in a truncated RUNX2 protein, severely impaired RUNX2 transactivation activity. We showed that signal transducers of transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) receptors, Smads, interact with RUNX2 in vivo and in vitro and enhance transactivation ability. The truncated RUNX2 protein failed to interact with Smads, and was unable to induce the osteoblast-like phenotype in C2C12 myoblasts following stimulation with BMP. Exogenous expression of Smads 1 and 4 in C2C12 cells stably expressing RUNX2 showed alkaline phosphatase (ALP) activity, suggesting a possible link between Smads and RUNX2, while in C2C12 stably expressing CCDalphaA376, a detectable level of ALP activity failed to be induced. The results suggest that CCDalphaA376 inhibited RUNX2 function in a dominant negative fashion.

3T3 Cells↗
PubMed1998

Peptidergic activation of transcription and secretion in chromaffin cells. Cis and trans signaling determinants of pituitary adenylyl cyclase-activating polypeptide (PACAP).

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. Chromogranin A is the major soluble core component in secretory vesicles. Since chromogranin A is secreted along with catecholamines, we asked whether PACAP regulates expression of the chromogranin A gene in PC12 rat chromaffin cells, so as to resynthesize the just-secreted protein, and whether such biosynthetic regulation is coupled mechanistically to catecholamine secretion. PACAP activated the endogenous chromogranin A gene by four- to fivefold. Proportional results (seven- to eightfold activation) were obtained with a transfected 1,200-bp mouse chromogranin A promoter/luciferase reporter construct. A series of chromogranin A promoter 5' deletion mutant/luciferase reporter constructs narrowed down the PACAP response element to a proximal region containing the cAMP response element (CRE box), at (-71 bp)5'-TGACGTAA-3'(-64 bp). Site-directed point mutations of the CRE site suppressed PACAP-induced trans-activation of the promoter. Thus, the proximal CRE box is entirely necessary for the chromogranin A promoter response to PACAP. Transfer of the CRE box to a neutral, heterologous promoter also conferred activation by PACAP, suggesting that the CRE domain is also sufficient to mediate the transcriptional response to PACAP. Expression of a dominant-negative mutant (KCREB) of the CRE-binding factor CREB markedly diminished trans-activation of the chromogranin A promoter by PACAP. Cotransfection of expression plasmids encoding the protein kinase A inhibitor, or an inactive protein kinase A (PKA) catalytic beta subunit, inhibited both forskolin and PACAP activation of chromogranin A transcription, revealing that PACAP-induced trans-activation is highly dependent on PKA. By contrast, inhibition of protein kinase C (by chronic exposure to phorbol ester) had no effect on transcriptional activation by PACAP. The potent PACAP/vasoactive intestinal peptide (VIP) type I receptor antagonist PACAP6-38 impaired both chromogranin A transcription or catecholamine secretion triggered by PACAP38, while the PACAP/VIP type II receptor antagonist (p-Chloro-D-Phe6, Leu17)-VIP had little or no ability to antagonize the PACAP38 effect. The agonist VIP was approximately 100- to 1,000-fold less potent than PACAP in stimulating either secretion or transcription. Thus, PACAP-evoked chromogranin A transcription and catecholamine secretion are likely mediated by the PACAP/VIP type I receptor isoform. Although the calcium channel antagonists Zn2+ (100 microM), nifedipine (10 microM), or ruthenium red (10 microM), or the cytosolic calcium chelator BAPTA-AM (50 microM) each strongly impaired PACAP-induced secretion, transcriptional activation of chromogranin A remained unaltered. Therefore, we propose that PACAP signals to chromogranin A transcription through the CRE in cis, and through PKA and CREB in trans. By contrast, a pathway involving cytosolic calcium entry through L-type voltage-dependent channels is required for PACAP to evoke catecholamine secretion.

Animals↗
PubMed2004

The bacteriophage T4 late-transcription coactivator gp33 binds the flap domain of Escherichia coli RNA polymerase.

Transcription of bacteriophage T4 late genes requires concomitant DNA replication. T4 late promoters, which consist of a single 8-bp -10 motif, are recognized by a holoenzyme containing Escherichia coli RNA polymerase core and the T4-encoded promoter specificity subunit, gp55. Initiation of transcription at these promoters by gp55-holoenzyme is inefficient, but is greatly activated by the DNA-loaded DNA polymerase sliding clamp, gp45, and the coactivator, gp33. We report that gp33 attaches to the flap domain of the Escherichia coli RNA polymerase beta-subunit and that this interaction is essential for activation. The beta-flap also mediates recognition of -35 promoter motifs by binding to sigma(70) domain 4. The results suggest that gp33 is an analogue of sigma(70) domain 4 and that gp55 and gp33 together constitute two parts of the T4 late sigma. We propose a model for the role of the gp45 sliding clamp in activation of T4 late-gene transcription.

Amino Acid Sequence↗