CCAAT Element Research Articles

Inhibition of DNA topoisomerase II alpha gene expression by the p53 tumor suppressor.

DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II alpha is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II alpha gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II alpha mRNA and protein following incubation for 24 h at 32 degrees C (p53 with wt conformation) than at 39 degrees C (p53 with mutant conformation). The effect of p53 on the human topo II alpha gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II alpha promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp -557 to +90) topo II alpha promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II alpha promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (-32 to +90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (-68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II alpha expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II alpha gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II alpha and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic stability associated with neoplasia.

Characterization of the cAMP Response Element of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Promoter

A dominant negative inhibitor of the cAMP-dependent protein kinase has been shown to inhibit the basal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the human colon carcinoma cell line, T84. A functional cAMP response element (CRE) was localized at -48 in the CFTR promoter, and we have analyzed the interactions of this regulatory region with transcription factors. An adjacent inverted CCAAT element (Y box) at position -60 was also investigated. Mutation of the CRE or the Y box decreases the activity of the promoter in transient transfections of T84 or JEG-3 cells. Electrophoretic mobility shift assays demonstrate that CRE-binding protein (CREB) binds to the CFTR CRE with high affinity and independently of the adjacent Y box and that the CFTR CRE binds CREB and activating transcription factor-1 in nuclear extracts of T84 and CaLu-3 cells. In transient transfections of JEG-3 cells, activation of the CFTR promoter is blocked by a dominant negative CREB mutant. The CFTR CRE will also drive cAMP-mediated expression when placed upstream of a heterologous basal promoter. These results demonstrate that CFTR is a bona fide CRE-dependent gene, and we suggest that CFTR expression levels in vivo may be responsive to hormones or drugs that activate the cAMP-dependent protein kinase system.

Activation of the Hepatitis B Virus S Promoter by Transcription Factor NF-Y via a CCAAT Element

The middle and small surface proteins of hepatitis B virus are translated from 5′-heterogeneous transcripts specified by the S promoter. We have generated a series of linker-substitution mutants that encompass the 130 base pairs comprising this promoter and measured the amount of transcripts and protein products synthesized from each mutant. The results confirm our previous finding that a CCAAT element is an important up-stream activating element for this promoter, as mutation of this element leads to a >20-fold decrease in promoter activity.In vitrobinding assays showed that the cellular transcription factor NF-Y (CCAAT-binding factor) binds to this element, and expression of a dominant-negative NF-Y subunit in transfected cells specifically reduced surface protein expression from the S promoter via the CCAAT element. In addition, two Sp1 sites also contribute to S promoter activity by a total of approximately 6-fold. Therefore, the S promoter is activated by both NF-Y and Sp1, but more strongly by the former factor.

Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free of nucleosomes. The restricted structural change observed upon transcriptional activation may indicate that the LCR need only make a specific contact with the proximal gene promoter to activate transcription.

Regulation of rat liver glucose-6-phosphatase gene expression in different nutritional and hormonal states: gene structure and 5'-flanking sequence.

The mRNA level of the catalytic subunit of rat liver glucose-6-phosphatase (Glu-6-Pase) was regulated by hormones commensurate with activity changes in vivo. Insulin exerts a dominant negative effect on the mRNA levels of Glu-6-Pase. Both mRNA levels and activities of the enzyme are low in the fed and refed state where insulin levels are elevated. Insulin administration to diabetic rats also decreases levels of mRNA and Glu-6-Pase activity. Insulin at a concentration of 1 nmol/l completely overcomes the stimulatory effect of glucocorticoids on Glu-6-Pase message levels in FAO hepatoma cells. The stimulatory response to glucocorticoid in FAO cells is biphasic, with maxima seen at 3 and 18 h after hormone addition (respectively 1.6- and 3.3-fold). 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) causes a fourfold increase in Glu-6-Pase mRNA at 3 h in FAO cells. The gene of rat liver Glu-6-Pase is 13 kilobases in length and comprised of 5 exons. The exon-intron structure is completely conserved when compared with the mouse and human genes. A 0.5-kb 3'-untranslated region, which is present in rat and mouse liver Glu-6-Pase cDNA, is absent in the Glu-6-Pase gene reported here, indicating the possible duplication of either the terminal fifth exon or the entire gene. The promoter region contains a consensus core CCAAT element at position -207 and a TATAAA at position -31. Several possible response elements have been identified in the 5'-flanking region (from a HindIII site at position -1641). A consensus glucocorticoid response element is located at base pair -1552, a 9/10 match of the insulin response sequence is located at position -1449, and a 7/8 match of the cAMP response element is located at position -164.

Expression of the human cytochrome c1 gene is controlled through multiple Sp1-binding sites and an initiator region.

It is widely accepted that nuclear genes that encode proteins of the oxidative-phosphorylation system are regulated by nuclear factors believed to be specific for such genes. In the present study we show that the promoter for the human cytochrome c1 gene is an exception, in that it involves only conserved Sp1 core elements and an initiator region. Maximal promoter activity within a 1.4-kb 5' flanking region of the cytochrome c1 gene is contained in a fragment (-72 to +18) that lacks TATA and CCAAT elements. The transcriptional start site was mapped to an initiator region by RNase protection of mRNA from human HepG2 cells, and by primer extension of in vitro-generated transcripts, to a sequence that is highly similar to the dihydrofolate reductase family of initiators. Deletion of this region (+1 to +18) severely impairs transcription initiation. Sp1 core elements centered at nucleotides -21 and -39 define the activation domain of the proximal promoter. Only the -39 element is protected from DNase I in the presence of crude nuclear extracts. However, transfection, gel-mobility-shift, supershift and in vitro-transcription experiments show that the -21 element binds Sp1 protein and contributes to transcription activation. No other functional oxidative-phosphorylation-specific response elements have been identified. These data implicate Sp1 as a single activating factor for an oxidative-phosphorylation gene.

Genomic Organization and the 5′ Flanking Region of the γ Subunit of the Human Amiloride-sensitive Epithelial Sodium Channel

The amiloride-sensitive epithelial sodium channel (ENaC) complex is made up of at least three different subunits alpha, beta, and gamma, which are developmentally regulated, selectively expressed, and variously up-regulated by steroid hormones. To understand mechanisms involved in regulation of the gamma subunit, we have determined the structure of the human gammaENaC gene. By 5' rapid amplification of cDNA ends, primer extension analysis, and nuclease protection assay, we identified transcription start sites in human brain, kidney, and lung. A human genomic library was screened and overlapping cosmid clones that span approximately 50 kilobases and contain the hgammaENaC gene were identified. The 5'-untranslated region is 141 bases long, and the translation start codon is contained within the second exon. The human gene spans at least 35 kilobases. The 5' end of the gene including portions of 5' flanking genomic DNA and the first intron are G + C rich and contain several CpG dinucleotides, consistent with a CpG island. The 5' flanking region contains no CCAAT or TATA-like elements but does contain two GC boxes as well as several putative transcription factor binding sites including AP-2, Sp1, CRE, PEA-3, and NF-IL6. This is the first description of the structural organization and the 5' flanking region of a member of the epithelial sodium channel complex.

The CCAAT-binding Proteins CP1 and NF-I Cooperate with ATF-2 in the Transcription of the Fibronectin Gene

We have previously proposed a molecular interaction between the liver factors that bind to the cyclic AMP response element (CRE) and CCAAT sites of the fibronectin (FN) gene based on the following evidence: (i) the close spacing of 20 base pairs between CRE and CCAAT elements is conserved in the FN genes from rats, mice, and humans; (ii) footprinting competitions showed that CRE oligonucleotides are able to detach both liver factors; (iii) CCAAT binding and transcriptional activity of liver extracts are reduced when the distance between the CRE and CCAAT elements is increased; and (iv) CCAAT-binding is stimulated by the addition of a liver extract fraction containing the CRE-binding factor ATF-2. This report provides binding and immunochemical evidence that nuclear factor I (CTF/NF-I) and CP1 (NF-Y or CBF) are the only liver factors that bind to the -150 CCAAT element of the FN gene, forming distinct complexes. We show that these factors bind less efficiently to the CCAAT site of a FN promoter in which the -170 CRE has been disrupted by site-directed mutagenesis and that each element contributes positively to the liver transcriptional activity assessed in vitro with a G-less cassette construct and in vivo by transfection of hepatoma cells with CAT constructs. Furthermore, using a method that combines UV cross-linking and immunoprecipitation, we show that antibodies specific to ATF-2 are able to specifically precipitate protein-protein-DNA complexes containing NF-I and CP1. This simple method preserves weak macromolecular interactions, avoiding the disruptive electrophoresis conditions of gel mobility shifts assays.

Activation of codependent transcription factors is required for transcriptional induction of the vgf gene by nerve growth factor and Ras.

Nerve growth factor (NGF) treatment of PC12 cells leads to the elaboration of a neuronal phenotype, including the induction of neuronally expressed genes such as vgf. To study vgf transcription, we have created chimeric vgf/beta-globin genes in which vgf promoter sequences drive the expression of the beta-globin reporter gene or of a chimeric beta-globin gene fused to 3' untranslated vgf gene sequences. We have found that the level of inducibility of the latter construct by NGF resembles that of the endogenous vgf gene. Using transient transfection of the chimeric reporter genes into PC12 cells, into PC12 subclones expressing activated or dominantly interfering mutant Ras proteins, and into PC12 variants expressing specific NGF receptor/Trk mutants, we show that transcriptional regulation of the vgf promoter by NGF is mediated through a Ras-dependent signaling pathway. By mutational analysis of the vgf promoter, we have identified three promoter elements involved in mediating transcriptional induction by NGF and Ras. In addition to the cyclic AMP-responsive element (CRE), which binds to ATF-1, ATF-2, and CRE-binding protein in PC12 nuclear extracts, a novel CCAAT element and its binding proteins were identified, which, like the CRE, is necessary but not sufficient for the Ras-dependent induction of the vgf gene by NGF. We also identify a G(S)G element unusually located between the TATA box and transcriptional start site, which binds the NGF- and Ras-induced transcription factor, NGFI-A, and amplifies the transcriptional response. Integrating data from studies of vgf promoter regulation and NGF signal transduction, we present a model for vgf gene induction in which transcriptional activation is achieved through the persistent, direct activation of multiple interacting transcription factors binding to CRE and CCAAT elements, coordinated with the delayed transcription factor action at a G(S)G element resulting from the induced expression of NGFI-A.

The Aspergillus nidulans penicillin-biosynthesis gene aat (penDE) is controlled by a CCAAT-containing DNA element.

Analysis of the promoter of the penicillin biosynthesis aat (penDE) gene of Aspergillus nidulans using band-shift assays led to the identification of a CCAAT-containing DNA element which was specifically bound by a protein (complex). The identified DNA element was localised about 250 bp upstream of the transcriptional-start sites of aat. Substitution of the CCAAT core sequence by GATCC led to a fourfold reduction of expression of an aat-lacZ gene fusion. The identified binding site thus was functional in vivo and positively influenced at expression. Partial purification of the CCAAT binding protein and cross-competition experiments provided evidence that the binding protein is identical to the identified putative penicillin-regulatory protein PENR1, binding to the CCAAT element in the bidirectional intergenic promoter region between acvA (pcbAb) and ipnA (pcbC). Hence, PENR1 seems to be involved in the regulation of all three penicillin-biosynthesis genes. Cross-competition experiments demonstrated that the promoter region of the corresponding aat (penDE) gene of Penicillium chrysogenum was capable to dilute the shift of the A. nidulans probe with PENR1, suggesting the presence of a similar regulatory mechanism in this fungus. Taken together with previous data, CCAAT-containing DNA elements thus seem to represent major cis-acting sites in the promoters of beta-lactam-biosynthesis genes.

Genomic organization and chromosomal mapping of the bovine Fas/APO-1 gene.

The cell-surface protein Fas (APO-1) is a member of the tumor necrosis factor receptor (TNFR) superfamily and transduces apoptosis following binding of Fas ligand or exposure to certain anti-Fas antibodies. We have isolated the bovine Fas (bFas) gene and determined its genomic organization and chromosomal location. Our data indicate that bFas is a single-copy gene that contains 9 exons and spans approximately 31.5 kb. The 5'-flanking region lacks conventional TATA and CCAAT elements, but contains several putative regulatory elements, including multiple copies of Sp1, AP-2, E-box, and N-box consensus sites. Linkage analysis of two (CA) dinucleotide repeat microsatellites within intron 1 and physical assignment by fluorescence in situ hybridization (FISH) placed the bFas gene on bovine chromosome 26. Collectively, these data provide a basis for understanding the regulatory mechanisms that control bFas gene expression.

A Novel CCAAT-binding Protein Necessary for Adhesion-dependent Cyclin A Transcription at the G1/S Boundary Is Sequestered by a Retinoblastoma-like Protein in G0

Loss of adhesion leads to cell cycle arrest at the G1/S boundary in normal, adhesion-dependent, mesenchymal cells. This arrest is accompanied by the inability to produce cyclin A. Using deletional and mutational analysis of the cyclin A promoter, we have identified a CCAAT element that mediates the adhesion-dependent transcriptional activation of cyclin A in late G1 phase of the cell cycle. Specific binding of a novel 40/115-kDa heterodimeric protein complex, which we have named CBP/cycA, to this CCAAT element was detectable in growing but not in G0-arrested or nonadherent normal rat kidney fibroblasts. During G0 CBP/cycA appears to be present but sequestered by a retinoblastoma family member. These results suggest that expression of cyclin A, which controls cell cycle progression by adhesion at the G1/S boundary, is regulated by CBP/cycA and the phosphorylation status of the retinoblastoma protein or a retinoblastoma-related protein.

Liver-enriched Transcription Factor HNF-4 and Ubiquitous Factor NF-Y Are Critical for Expression of Blood Coagulation Factor X

Blood coagulation Factor X and its activated form Factor Xa play an essential role in the midphase of the clotting cascade. To delineate the mechanisms governing the liver-specific expression of Factor X, we have previously characterized the complete 2.8 kilobase pairs of the 5'-flanking region of Factor X and demonstrated that the first 209 base pairs is sufficient to confer maximal promoter activity in HepG2 cells, a hepatoma cell line that expresses Factor X. We have also shown that mutations at ACTTTG and CCAAT elements located at -56 to -51 and -120 to -116, respectively, significantly reduce the promoter activity. In this report, we demonstrate that Factor X mRNA is primarily but not exclusively expressed in the liver. Using DNase I footprinting analysis, we determine four protein binding sites within the 209-base pair fragment, designated site 1 (-73) to -44), site 2 (-128 to -94), site 3 (-165 to -132), and site 4(-195 to -169). Using gel mobility shift assays in combination with competition and supershift experiments, we demonstrate that hepatocyte nuclear factor 4 and Sp1 bind at site 1, the site which contains the ACTTTG element. Methylation interference assays reveal that HNF-4 and Sp1 contact adjacent sites with minor overlap. HNF-4 and Sp1 appear to bind site 1 in a mutually exclusive fashion. We also demonstrate that HNF-4 can transactivate the Factor X promoter in HeLa cells; mutation at the adjacent Sp1 site further increases the transactivation. Heteromeric transcription factor NF-Y was identified as the protein that binds the CCAAT box at site 2. We conclude that HNF-4 and NF-Y play crucial roles in modulating the activity of the proximal promoter of Factor X.

The age-dependent binding of CBP/tk, a CCAAT binding protein, is deregulated in transformed and immortalized mammalian cells but absent in premature aging cells.

CBP/tk, CCAAT Binding Protein for thymidine kinase, has been shown to bind to the distal and proximal CCAAT elements in human TK gene at G1/S boundary in normal human IMR-90 cells after serum stimulation (Pang and Chen, 1993). We now show that the serum-induced binding activity of CBP/tk was inversely related to the population doubling level (PDL) of the normal IMR-90 cells. However, little or almost no CBP/tk binding activity was observed in cells derived from patients with premature aging syndromes (e.g., Werner, Hutchinson-Gilford, and Cockayne syndrome). In contrast, CBP/tk binding activity in SV-40 virus-transformed human cells and in HeLa cells was overexpressed at levels 5- to 15-fold higher than that in normal cells and appeared to be deregulated. The half-life of CBP/tk binding activity in SV-40 transformed cells was at least 10 times longer than that in normal IMR-90 cells, suggesting that posttranslational control may contribute to the deregulation. CBP/tk binding activity detected in other mammalian cells such as murine NIH3T3, an immortal cell line, did not reveal any cell cycle dependence either. Further characterization of CBP/tk binding complex indicates that the binding complex may contain NF-YA and NF-YB and that the binding activity was sensitive to oxidizing reagents. Taken together, our data showed that the age- and cell cycle-dependent nature of CBP/tk is a function of cell types and that CBP/tk binding activity may be subjected to posttranslational and redox regulation.

Comparison of the 5′-flanking sequences of the human and bovine von Willebrand factor-encoding genes reveals alternation of highly homologous domains with species-specific Alu-type repeats

von Willebrand factor (vWF), a multimeric glycoprotein important for hemostasis, is specifically synthesized in endothelial cells and in platelet precursors (megakaryocytes). Recent studies from two laboratories, including ours, were published regarding the cell-specific transcription of reporter genes controlled by the human (hu) vWF promoter in transfected bovine (bo) endothelial cells and cells of non-endothelial origins. In order to verify that the regulatory domains previously characterized in the 5′ region of hu vWF are also present in bo vWF, we have sequenced 1.9 kb unstream from the cap site, plus five exons. The comparison of human and bovine exons two to five shows homology of 83% at the nucleotide (nt) level and 78% at the deduced amino-acid sequence level. The bovine and human exons one, which are non-coding and span 233 and 250 bp, respectively, are only 64% homologous. In the first exon, potentially involved in endothelial-cell-specific transcription, the binding site for factor Sp1 is present in bo vWF, whereas the GATA sequence is replaced by a GACA sequence. The sequence corresponding to the human basal promoter, located between nt −89 and +19, is well conserved with 82% homology. However, the human TAATTA sequence (at nt −32) considered to be a TATA box, is replaced by TCATTA, and the CCAAT element at nt −18 is replaced by CCTGT. Among domains involved in transcription, the negative regulatory domain located 5′ from the core promoter is highly conserved. The bovine sequence upstream from the first intron can be aligned with the human sequence up to nt −656 which is located in a polymorphic poly(GT) 18–26 sequence. At this site, the bovine DNA contains an insertion of 523 bp which corresponds to a bovine Alu-type art2 repeat of 331 bp flanked by bovine microsatellites. The art2 sequence is an Alu-type repeat in artiodactyls with at least 100 000 copies in the bovine genome. Upstream from this insertion, 368 bp of the bovine sequence can be aligned with the human counterpart up to a 9-bp element which flanks an human Alu repeat which is absent from the bovine DNA. Upstream of the human Alu insertion and a duplicate of the 9-bp element, the two sequences are again homologous.

Transcription of Cystic Fibrosis Transmembrane Conductance Regulator Requires a CCAAT-like Element for both Basal and cAMP-mediated Regulation

The cystic fibrosis transmembrane conductance regulator (CFTR) gene in man is controlled by a tightly regulated and weak promoter. The architecture of the CFTR promoter suggests regulatory characteristics that are consistent with the absence of a TATA-like sequence, including the ability to initiate RNA transcription at numerous positions. Detailed investigation of the most proximal region of the human CFTR gene promoter through deletion and mutational analysis reveals that expression is contingent on the conservation of the inverted CCAAT sequence. Basal expression of CFTR transcription and cAMP-mediated transcriptional regulation require the presence of an imperfect and inverted CCAAT element recognized as 5'-AATTGGAAGCAAAT-3', located between 132 and 119 nucleotides upstream of the translational start site. RNA isolated from a transfected pancreatic cell line carrying integrated wild-type and mutant CFTR-directed transgenes was used to map the 5' termini of the transgenic transcripts. Analysis of the transcript termini by ribonuclease protection analysis reflects the direct association of the conserved inverted CCAAT sequence in promoting transcript initiation. Because of the requirement for the inverted CCAAT sequence for promoting transcription of CFTR, the involvement of CCAAT-binding factors is suspected in the regulation of CFTR gene transcription. To test this, we used electrophoretic mobility shift assays to demonstrate that the majority of the binding to the inverted CCAAT element, between -135 and -116, was easily competed for by binding to cognate nucleotide sequences for CCAAT-enhancer binding protein (C/EBP). An antibody specific for the C/EBP-related protein, C/EBP delta, detected C/EBP delta as part of a nuclear protein complex bound to the inverted CCAAT sequence of the CFTR gene. Also, the detection of specific activating transcription factor/cyclic-AMP response element binding protein antigens by antibody supershift analysis of nuclear complexes suggest that species of this family of transcription factors could be involved in the formation of complexes with C/EBP delta within the CFTR gene inverted CCAAT-like element. These studies raise the possibility of interactions between individual members of the C/EBP and activating transcription factor/cyclic-AMP response element binding protein families potentially contribute to the tight transcriptional control rendered by the CFTR gene promoter.

Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene

Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide -672 to nucleotide -527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains.

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.

Major Histocompatibility Complex Class II-associated Invariant Chain Gene Expression Is Up-regulated by Cooperative Interactions of Sp1 and NF-Y

Expression of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is required for efficient and complete presentation of antigens by MHC class II molecules and a normal immune response. The Ii gene is generally co-regulated with the MHC class II molecules at the level of transcription and a shared SXY promoter element has been described. This report defines the proximal promoter region of Ii which may regulate Ii transcription distinct from MHC class II. In vivo genomic footprinting identified an occupied, imperfect CCAAT box and an adjacent GC box in the proximal region. These sites are bound in Ii-ositive cell lines and upon interferon-gamma induction of Ii transcription. In contrast, both sites are unoccupied in Ii-egative cell lines and in inducible cell lines prior to interferon-gamma treatment. Together these two sites synergize to stimulate transcription. Independently, the transcription factor NF-Y binds poorly to the imperfect CCAAT box with a rapid off rate, while Sp1 binds to the GC box. Stabilization of NF-Y binding occurs upon Sp1 binding to DNA. In addition, the half-life of Sp1 binding also increased in the presence of NF-Y binding. These findings suggest a mechanism for the complete functional synergy of the GC and CCAAT elements observed in Ii transcription. Furthermore, this report defines a CCAAT box of imperfect sequence which binds NF-Y and activates transcription only when stabilized by an adjacent factor, Sp1.

Regulation of the Mouse Histone H2A.X Gene Promoter by the Transcription Factor E2F and CCAAT Binding Protein

We have molecularly cloned the genomic gene encoding the mouse histone variant H2A.X and characterized the promoter. The promoter region of the H2A.X gene was characterized by chloramphenicol acetyltransferase analysis using Balb/c 3T3 cells. Maximal promoter activity was found in the construct containing up to -282 base pairs H2A.X upstream region. Within this region, we found two sequences regulating the promoter activation; one was an E2F site and another was a CCAAT box. These sequences were also required for the DNA/protein binding activities. Thus, these activities corresponded to the promoter activities, implying that the promoter activity H2A.X gene was controlled by both the transcription factor E2F and H1TF2 through the E2F and CCAAT element. The CCAAT box binding activity was constitutive when cell cycle was progressed by release from G1 arrest, but transiently transfected chloramphenicol acetyltransferase activity slightly increased when cells entered S phase. Similarly, the level of the smallest form of E2F (free E2F) became higher when cells reentered the cell cycle, indicating that the free E2F was one capable of inducing the promoter activation. Thus, the free E2F and CCAAT DNA binding activity correlated with regulation of the promoter activity.