CCAAT Element Research Articles

The hsp70 gene CCAAT-binding factor mediates transcriptional activation by the adenovirus E1a protein.

Expression of the human hsp70 gene is cell cycle regulated and is inducible by both serum and the adenovirus E1a protein (K. Milarski and R. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986; M. C. Simon, K. Kitchener, H.-T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986; B. Wu and R. Morimoto, Proc. Natl. Acad. Sci. USA 82:6070-6074, 1985). This regulated expression is predominantly controlled by the CCAAT element at position -70 relative to the transcriptional initiation site (G. Williams, T. McClanahan, and R. Morimoto, Mol. Cell. Biol. 9:2574-2587, 1989; B. Wu, H. Hurst, N. Jones, and R. Morimoto, Mol. Cell. Biol. 6:2994-2999, 1986). A corresponding CCAAT-binding factor (CBF) of 999 amino acids has recently been cloned and shown to stimulate transcription selectively from the hsp70 promoter in a CCAAT element-dependent manner (L. Lum, L. Sultzman, R. Kaufman, D. Linzer, and B. Wu, Mol. Cell. Biol. 10:6709-6717, 1990). We report here that the first 192 residues of CBF, when fused to the DNA-binding domain of the heterologous activator GAL-4, are necessary and sufficient to mediate E1a-dependent transcriptional activation. E1a and CBF exhibit complex formation in vitro, suggesting that an in vivo interaction between these proteins may be relevant to the well-characterized E1a-induced transcriptional activation of the hsp70 promoter.

Cloning of the rat insulin-like growth factor binding protein-1 gene and analysis of its 5′ promoter region

To understand specific mechanisms involved in the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), an important modulator of IGF bioactivity, we cloned the rat IGFBP-1 gene and sequenced a 1.5 kb Sph1-Sph1 fragment containing 1110 bases upstream from the translation start site. computer analysis reveals the presence of ATA, CACCC, and CCAAT elements, and putative homeodomain, AP-1, insulin and glucocorticoid response elements in the 5′ promoter. Primer extension and ribonuclease protection studies reveal a single cap site in RNA from rat hepatoma cells and both control and diabetic rat liver.

Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis

The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70--chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to--100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker-scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5' boundary to--188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP70 gene promoter can function during Xenopus embryogenesis.

Brefeldin A as a regulator of grp78 gene expression in mammalian cells.

We report here that brefeldin A (BFA), which specifically blocks protein transport from an endoplasmic reticulum to the Golgi apparatus and causes resorption of Golgi membrane to the endoplasmic reticulum, specifically induced the endoplasmic reticulum-resident protein GRP78. Treatment of a human hepatoma cell line Alex-PC with BFA at a concentration of 5 micrograms/ml increased the grp78 transcript level by 12-fold. Analyses of the transcriptional rate of grp78 and the transfection with grp78 promoter suggested that this cell line utilized a posttranscriptional mechanism to increase the expression of grp78 in response to BFA. The induction process was partially dependent on de novo protein synthesis. Interestingly, in a hamster lung fibroblast cell line, K12, the induction of grp78 by BFA could be mediated by a transcriptional control mechanism. We further demonstrated that in K12 cells the region of the grp78 promoter responsive to BFA was within a 40-base pair region between -169 and -130, containing the conserved grp core and a 10-base pair region between -99 to -90 that contained a proximal CCAAT element. A model of how BFA regulates grp78 expression at both the transcriptional and posttranscriptional level is presented.

Protein binding sites within the human thymidine kinase promoter

DNase I footprint analysis, using total HeLa cell nuclear extract and purified transcription factor Sp1, was carried out to determine the various protein binding sites within the human thymidine kinase promoter. The promoter has two separate CCAAT elements and multiple Sp1-binding sites, as well as at least one undefined protein binding site. Detailed analysis of protein binding to the two CCAAT elements showed that changing the spacing between the two CCAAT elements altered both protein binding to the distal CCAAT element as well as promoter activity. Both CCAAT elements can act as functional transcription elements, but are not oriented for optimal promoter strength in the human tk promoter. Our studies show that a promoter fragment that has been previously shown to be the minimal region to maintain a serum responsive promoter regulation apparently contains only a single Sp1-binding domain and the more distal of the CCAAT elements.

Tissue restricted expression and chromosomal localization of the YB-1 gene encoding a 42 kD nuclear CCAAT binding protein.

YB-1 cDNA clones were isolated by binding site screening of a Hela expression library using a human papillomavirus type 18 enhancer oligonucleotide. YB-1 belongs to a family of transcription factors which bind to recognition sequences containing a core CCAAT element. YB-1 bound to its single stranded recognition sequence on the sense strand but not to the anti-sense strand. A synthetic peptide antiserum derived from the predicted YB-1 amino acid sequence identified a 42 kD nuclear protein in immunoblots. A protein with the same size was detected by binding site blotting experiments using the HPV18 enhancer oligonucleotide which bound YB-1. YB-1 gene expression was restricted in tissues from a human 24 week old fetus. High levels of YB-1 mRNA were present in heart, muscle, liver, lung, adrenal gland and the brain, in contrast, low amounts of YB-1 mRNA were found in thymus, kidney, bone marrow and spleen. In pancreas, bladder, stomach and testis YB-1 mRNA could not be detected by Northern hybridization. Finally, we have identified four YB-1 related loci in the mouse genome and have mapped these loci to four different mouse chromosomes by interspecific backcross analysis.

A conserved region in intron 1 negatively regulates the expression of the PCNA gene.

The Proliferating Cell Nuclear Antigen (PCNA) gene is a growth-regulated gene, whose expression is under the control of both transcriptional and posttranscriptional mechanisms. In previous work, it was shown that the 73 bp immediately upstream of the CAP site and intron 4 are major regulatory elements. We show here that intron 1 also plays a role in determining the levels of PCNA mRNA. Specifically, we show: 1) deletion of intron 1 increases the expression of PCNA mRNA in serum-deprived cells; 2) a 35 bp sequence in intron 1, containing a reverse CCAAT element specifically binds proteins from nuclear extracts; 3) this intron 1 sequence inhibits the expression of a co-tranfected human PCNA gene in transient expression assays suggesting that it competes for positive transcription factors; 4) mutations in the CCAAT region of the 35bp intron 1 probe abrogate both its protein-binding capacity and its ability to inhibit the expression of a co-transfected wt PCNA gene; and 5) the CCAAT region of human intron 1 is highly conserved in the mouse gene. We conclude that the reverse CCAAT region of intron 1 is a negative regulatory element of PCNA gene expression, and hypothesize that its inhibitory effect is abolished when certain protein(s) bind to it and that inhibition is restored if these proteins are competed out by an homologous sequence.

The hepatitis B virus S promoter comprises A CCAAT motif and two initiation regions.

The hepatitis B virus S (major surface gene) promoter is embedded in two overlapping open reading frames and specifies several transcripts with heterogeneous 5' termini. We have identified the cis-elements necessary for S promoter function. A single upstream CCAAT element is essential for high level expression in both liver and non-liver cells. No TATA box is present, but two regions surrounding the initiation sites can function separately as initiating elements. These results show that the S promoter has a simple requirement for upstream activating sequences, but a complex initiation region. Therefore, it may provide a suitable model system for studying transcription initiation in non-TATA type promoters.

NFY or a related CCAAT binding factor can be replaced by other transcriptional activators for co-operation with HNF1 in driving the rat albumin promoter in vivo

Like many eukaryotic genes, the rat albumin promoter contains a CCAAT consensus motif at position — 80. In transfected H4II hepatoma cells the strength of this promoter depends to a large extent on the integrity of a hepatic nuclear factor 1 (HNF1) binding site located at position — 60 and to a lesser extent on the CCAAT element. However, if the affinity for HNF1 is reduced, the CCAAT-box becomes essential for high, and tissue specific, promoter activity. We wished to determine which, among the different CCAAT binding factors co-existing in eukaryotic cells, was responsible for this co-operativity with HNF1. To this end we prepared a series of mutants of the CCAAT sequence and compared their effects on albumin promoter activity in vivo and on the binding of different CCAAT binding factors in vitro. Our results strongly suggest that a ubiquitous factor NFY (also designated CBF, ACF, CP1) interacts with this CCAAT element in vivo. We propose that during development NFY could facilitate transcription of the albumin gene in hepatocytes when the concentration of HNF1 is limiting. This co-operativity in transcriptional activation is not due to strict co-operativity in DNA binding between the two proteins and is not limited to NFY or a closely related factor, as the CCAAT-box can be replaced by AP1, SP1 or E2 target sites without significantly affecting the final activity.

Analysis of the human type I insulin-like growth factor receptor promoter region

We isolated genomic fragments containing the 5′ region of the human type I insulin-like growth factor receptor gene. A unique transcription start site was identified, defining a 1038 bp 5′-untranslated region. No TATA or CCAAT elements were identified in the proximal 480 nucleotides of 5′-flanking region. The region surrounding the transcription start site was similar to a recently described “initiator” sequence. The 5′-flanking and 5′-untranslated regions were highly GC-rich, with numerous potential Sp1 binding sites. A potential AP-2 binding site was identified in the 5′-flanking region and a potential thyroid response element was identified in the 5′-untranslated region. The 5′ region of the human gene was very similar to that of the rat gene, with conservation of many of the potential regulatory elements.

Identification of two protein binding sites within the varicella-zoster virus major immediate early gene promoter

Binding sites for cellular proteins in the promoter of the varicella-zoster virus (VZV) major immediate early (IE) gene were investigated. Protein binding was detected at sequence motifs possessing homology to the CCAAT element and an ATF/AP-1-like binding site, and recognition of the ATF/AP-1 site was apparently facilitated by occupation of the CCAAT site. Gene expression directed by the VZV major IE promoter was stimulated by the adenovirus 5, 289 amino acid EIA gene product. The implications of the results for VZV gene expression and replication are discussed.

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

Structure of the Gene Encoding VGF, a Nervous System-Specific mRNA That Is Rapidly and Selectively Induced by Nerve Growth Factor in PC12 Cells

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.

Concerted evolution led to high expression of a prosimian primate delta globin gene locus.

The delta globin gene in simian primates is either weakly expressed (in hominoids and New World monkeys) or silent (in Old World monkeys). In prosimian primates, however, an unequal homologous crossover between the psi eta and delta loci of lemurs produced a hybrid psi eta delta pseudogene locus, whereas in tarsier the delta locus encodes a beta-type chain found in 18% of adult tarsier hemoglobin molecules. In the present study, the nucleotide and amino acid sequences of the galago delta and beta globin genes and their encoded peptides were determined, and evidence is provided showing that the galago delta locus encodes a beta-type chain (beta 2) found in 40% of the galago fetal and postnatal hemoglobin molecules, whereas the beta locus encodes the remaining 60% of the beta-type chain (beta 1). Galago beta 1 and beta 2 chains differ from each other by only one amino acid residue. The homology between the galago delta and beta loci extends from 800 base pairs 5' of the proximal CCAAT element to near the end of exon 3 as a result of a recombination event in which beta sequence replaced delta sequence. After this initial recombination event, concerted evolution between the loci continued over their conserved coding, intron 1, and promoter regions but failed to occur between the two loci in their intron 2 and distal 5'-flanking sequences where the two loci have now diverged by 20%. Calculations based on this divergence value and on a rate of noncoding sequence evolution of 4.2 x 10(-9) to 5.5 x 10(-9) substitutions/site/year for the lorisiform lineage to galago yielded a date of 18-24 million years ago for the initial recombination event. The fact that the promoter sequences of the galago delta locus are the same as that of the galago beta locus may account for the high level of expression of the galago delta gene.

Affinity-purified CCAAT-box-binding protein (YEBP) functionally regulates expression of a human class II major histocompatibility complex gene and the herpes simplex virus thymidine kinase gene.

Efficient major histocompatibility complex class II gene expression requires conserved protein-binding promoter elements, including X and Y elements. We affinity purified an HLA-DRA Y-element (CCAAT)-binding protein (YEBP) and used it to reconstitute Y-depleted HLA-DRA in vitro transcription. This directly demonstrates a positive functional role for YEBP in HLA-DRA transcription. The ability of YEBP to regulate divergent CCAAT elements was also assessed; YEBP was found to partially activate the thymidine kinase promoter. This functional analysis of YEBP shows that this protein plays an important role in the regulation of multiple genes.

A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter.

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.

Identification of cis-Acting Regulatory Elements in the Promoter Region of the Rat Brain Creatine Kinase Gene

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.

A Cloned Human CCAAT-Box-Binding Factor Stimulates Transcription from the Human hsp70 Promoter

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

Structure, genetic mapping, and expression of the gene for pyruvate, orthophosphate dikinase from maize.

Pyruvate, orthophosphate dikinase is a key enzyme in photosynthesis in some plants that exploit the C4 photosynthetic pathway for the fixation of CO2. The gene for this enzyme has been cloned and its primary structure has been analyzed. The sequence of the cloned genes spans about 12 kilobase pairs and consists of 19 exons. The site of initiation of transcription is located 211 nucleotides upstream from the first nucleotide of the initiation codon (position -211). Typical TATA and inverted CCAAT elements are present in the anticipated regions, as well as a sequence that is homologous to the binding site of Sp-1 protein (-51 to -42). Three long, direct-repeated sequences are present contiguously in the 5'-flanking region (-286 to -172), and two of the repeated sequences include sequences homologous to the core sequence of SV40 enhancer in the 3'-end portions. Southern blot analysis, coupled with mapping by analysis of restriction fragment length polymorphism, indicates that the gene for the enzyme involved in C4 photosynthesis is encoded by a single-copy gene and is located on chromosome 6. To test the promoter activity of the isolated gene, a chimeric gene composed of the 5'-flanking region of the gene and a structural gene for bacterial beta-glucuronidase was constructed and introduced into tobacco protoplasts by electroporation or used to transform tobacco plants by Agrobacterium-mediated transfer of the gene. In both cases, expression of the beta-glucuronidase gene was observed, indicating that the 5'-flanking region of the gene can act as a promoter in tobacco cells.