Inverted CCAAT Element Research Articles

A key role of nuclear factor Y in the refeeding response of fatty acid synthase in adipocytes.

Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipocytes. Using the invivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around -100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepatocytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes.

Indirubin derivatives alter DNA binding activity of the transcription factor NF-Y and inhibit MDR1 gene promoter

Indirubin derivatives exert antitumor activity. However, their effects on the expression of multidrug resistance gene 1 (MDR1) have not been investigated. Here we found three derivatives that inhibit the MDR1 gene promoter. To investigate the effects of indirubins on the DNA binding of NF-Y, a major MDR1 gene transcription factor that recognizes an inverted CCAAT element in the promoter, gel mobility shift assay was performed using the element as a probe with nuclear extracts from NG108-15, MCF7, HepG2, C2C12, and SK-N-SH cells. Among 17 compounds, 5-methoxyindirubin inhibited the DNA binding of NF-Y significantly, whereas indirubin-3′-oxime and 7-methoxyindirubin 3′-oxime increased the binding considerably. After evaluating a suitable concentration of each compound for transcription analysis using living tumor cells, we performed a reporter gene assay using a reporter DNA plasmid containing EGFP cDNA fused to the MDR1 gene promoter region. Indirubin-3′-oxime exerted a significant inhibitory effect on the MDR1 promoter activity in MCF7 and HepG2 cells, and 5-methoxyindirubin inhibited the activity only in MCF7 cells; 7-methoxyindirubin 3′-oxime suppressed the activity in all of the cell lines. We further confirmed that the compounds reduced endogenous MDR1 transcription without any inhibitory effect on NF-Y expression. Moreover, each compound increased the doxorubicin sensitivity of MCF7 cells. These results indicate that each indirubin derivative acts on the DNA binding of NF-Y and represses the MDR1 gene promoter with tumor cell-type specificity.

Recruitment of Nuclear Factor Y to the Inverted CCAAT Element (ICE) by c-Jun and E1A Stimulates Basal Transcription of the Bone Sialoprotein Gene in Osteosarcoma Cells

Bone sialoprotein (BSP), a major protein in the extracellular matrix of bone, is expressed almost exclusively by bone cells and by cancer cells that have a propensity to metastasize to bone. Previous studies have shown that v-src stimulates basal transcription of bsp in osteosarcoma (ROS 17/2.8) cells by targeting the inverted CCAAT element (ICE) in the proximal promoter. To identify possible downstream effectors of Src we studied the effects of the proto-oncogene c-jun, which functions downstream of Src, on basal transcription of bsp using transient transfection assays. Increased expression of endogenous c-Jun induced by the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate and ectopic expression of c-Jun increased basal transcription of chimeric reporter constructs encompassing the proximal promoter by 1.5-3-fold in ROS 17/2.8 osteosarcoma cells, with more modest effects in a normal bone cell line, RBMC-D8. The effects of c-Jun were abrogated by mutations in the ICE box and by co-expression of dominant negative nuclear factor Y, subunit A (NF-YA). The increase in bsp transcription did not require phosphorylation of c-Jun and was not altered by trichostatin treatment or by ectopic expression of p300/CREB-binding protein (CBP) or mutated forms lacking histone acetyltransferase (HAT) activity. Similarly, ectopic expression of p300/CBP-associated factor (P/CAF), which transduces p300/CBP effects, or of HAT-defective P/CAF did not influence the c-jun effects. Surprisingly, E1A, which competes with P/CAF binding to p300/CBP, also stimulated BSP transcription through NF-Y independently of c-jun, p300/CBP, and P/CAF. Collectively, these studies show that c-Jun and E1A regulate basal transcription of bsp in osteosarcoma cells by recruiting the NF-Y transcriptional complex to the ICE box in a mechanism that is independent of p300/CBP and P/CAF HAT activities.

Reconstitution of TIMP-2 expression in SH-SY5Y neuroblastoma cells by 5-azacytidine is mediated transcriptionally by NF-Y through an inverted CCAAT site

Advanced stage neuroblastomas (NB) exhibit a tissue inhibitor of metalloproteinase (TIMP)-2/matrix metalloproteinase (MMP) imbalance, considered a prerequisite for MMP involvement in tumor progression in vivo. Human SH-SY5Y NB cells exhibit a similar TIMP-2/MMP imbalance that promotes in vitro invasive behavior that is inhibited by exogenous TIMP-2. The DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) redresses this TIMP-2/MMP imbalance, reconstituting TIMP-2 expression, without effecting that of MMP-2, by stimulating TIMP-2 transcription and inhibiting in vitro invasivity of SH-SY5Y cells. 5-AzaC stimulated transcription from a nonmethylated TIMP-2 promoter reporter gene construct consistent with regulation of a TIMP-2 transactivator. Promoter deletion and point-mutation analysis localized this effect to an inverted CCAAT element at position −73. This element bound specific complexes containing NF-YA and NF-YB proteins in SH-SY5Y nuclear extracts, the binding of which was augmented by 5-AzaC in association with enhanced levels of NF-YB protein and the function of which was confirmed by inhibition using dominant-negative NF-YA. The data highlight a novel indirect methylation-mediated mechanism for regulating the TIMP/MMP equilibrium in NB cells, involving repression of TIMP-2 relative to MMP-2 expression, dependent upon suboptimal NF-Y transcription factor function, which can be reversed by methyltransferase inhibition.

Promoter structure and transcriptional activity of human complement receptor type I (CR1) gene

Until recently, interest in human complement receptor type I (CR1) has focused on immune complex processing, which contributed to our understanding of regulatory mechanism of complement activation. However, the promoter structure and transcriptional regulation of human CR1 gene has not been clear. To study the unique regulation of human CR1 gene expression, we assessed promoter activity of the 5'‐flanking region of human CR1 gene using transient transfection and gel mobility shift assays. In this study we demonstrated that NF‐Y binds to the inverted CCAAT element and that the functional interaction with protein(s) which bind to the GC‐rich motif may be necessary for optimal transcription of human CR1 gene. We also show that sequence elements which located at ‐95/‐58 and +45/ +50 are important for optimal transcription of CR1 gene.

The inverted CCAAT motif is an indispensable element of the enhancer B of the mouse major histocompatibility I H2-Kb gene.

We have identified a strong binding of nuclear proteins derived from Ltk(-) fibroblasts to the enhancer B of the mouse MHC class I H2-K(b) gene. The inverted CCAAT motif and its adjacent upstream sequences have been revealed as protein-binding sites by electrophoretic mobility-shift, methylation interference, and DNase I footprint assays. Specific mutations in the inverted CCAAT motif as well as in the 5'-flanking cytosine pentanucleotide abrogated the formation of the major DNA-protein complex. Transcription of the chloramphenicol acetyltransferase (CAT) reporter gene driven by the H2-K(b) promoter in the Ltk(-) cell line was reduced substantially when a two-nucleotide mutation was introduced into the CCAAT element (CCAATCgcAT). The indicated two-nucleotide mutation decreased transcription initiated from both the homologous and a heterologous promoter. Furthermore, cotransfected MHC class II transactivator (CIITA) elevated the transcription of the reporter gene under the control of the H2-K(b) upstream sequences in the NIH 3T3 cell line. The intact enhancer B involving both the inverted CCAAT motif and the site alpha was found to play an indispensable role in the CIITA-mediated gene transactivation. The band-shift assay with the enhancer B probe revealed forming of a protein complex in a cooperative manner, which was again prevented by mutations in either element. Our results suggest an essential role of the inverted CCAAT element in the constitutive as well as inducible transcription of the mouse MHC class I genes.

The FIRE3-mediated sterol response of the FAS promoter requires NF-Y/CBF as a coactivator.

The transcription of the fatty acid synthase (FAS) gene is regulated by the sterol status of the cell via cleavage of the sterol regulatory element-binding protein (SREBP). When human HepG2 hepatoma cells were cotransfected with an expression plasmid for mature SREBP-1a together with FAS promoter/reporter constructs significant increases in reporter activity were observed. Deletion analysis of the FAS promoter between -151 and -52 relative to the transcription start site pinpoint two cis-elements important in sterol regulation of the FAS gene. One element, FIRE3, between -71 and -52 can bind in vitro translated and transcribed SREBP-1a whereas the other element, the inverted CCAAT element ICE(-97/-92), binds the trimeric transcription factor NF-Y/CBF as shown with rat liver extract and reconstituted, recombinant NF-Y. The results clearly show that the coactivator for SREBP-1a in this cell line is NF-Y. This finding was confirmed by using a dominant negative form of NF-YA, NF-YAm29, which interferes with the effect of ectopically expressed SREBP-1a on FAS reporter activity.

FIRE3 in the promoter of the rat fatty acid synthase (FAS) gene binds the ubiquitous transcription factors CBF and USF but does not mediate an insulin response in a rat hepatoma cell line.

Several putative insulin-responsive elements (IRE) in the fatty acid synthase (FAS) promoter have been identified and shown to be functional in adipocytes and hepatocytes. Here we report on the insulin-responsiveness in the rat hepatoma cell line H4IIE of four cis-elements in the FAS promoter: the FAS insulin-responsive elements, FIRE2 and FIRE3; the inverted CCAAT element, ICE; and the insulin/glucose-binding element, designated hepatic FIRE element, hFIRE, originally identified in rat hepatocytes. Using electrophoretic mobility shift assay (EMSA) competition experiments together with supershifts and in vitro transcription/translation we show that FIRE3 (-68/-58) binds not only the upstream stimulatory factors USF-1/USF-2 but also the CCAAT-binding factor CBF, also known as the nuclear factor Y, NF-Y. The putative IRE FIRE2, which shows sequence similarity to FIRE3, is located between -267 and -249. Gel retardation experiments indicate that USF-1 and USF-2 also bind to this element, which contains an imperfect E-box motif. Using the same approach we have shown that hFIRE binds the stimulatory proteins Sp1 and Sp3 in addition to CBF. Transient transfection experiments using FAS promoter constructs deleted for FIRE2 and FIRE3 demonstrate that neither of these elements mediates the insulin response of the FAS promoter in the rat hepatoma cell line H4IIE, however, ICE at -103/-87 is responsible for mediating the effect of the insulin antagonist cAMP. The hFIRE element located at -57/-34, in spite of its role in the glucose/insulin response in primary rat hepatocytes, is apparently not involved in the insulin regulation of the rat FAS promoter in H4IIE cells. The fact that the topology of the promoters of the FAS genes in rat, human, goose and chicken is conserved regarding CBF-binding sites and USF-binding sites implies an important role for these ubiquitously expressed transcription factors in the regulation of the FAS promoter.

The Role of an Inverted CCAAT Element in Transcriptional Activation of the Human DNA Topoisomerase IIα Gene by Heat Shock

Expression of the DNA topoisomerase IIalpha (topoIIalpha) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIalpha mRNA was increased 1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 degreesC for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIalpha gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) -295 to +85) and of three deletion constructs (nt -197 to +85, -154 to +85, and -74 to +85) was increased approximately 3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt -20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt -74 and -21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3-24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIalpha gene promoter. Finally, in vivo footprint analysis of the topoIIalpha gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIalpha gene by heat shock requires the release of a negative regulatory factor from ICE1.

Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora

A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I–P–X–P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein ( Cs- mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned S a cI fragment of 2.5 kb contained the complete sequence of the Cs-mnp1 gene, including 162 bp and 770 bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT–AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs- mnp1 gene further, a fragment of 1.9 kb was amplified using inverse PCR. A putative TATAA element was identified 5′ of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.

FIRE (fatty acid synthase insulin-responsive element) and ICE (inverted CCAAT element) regulate fatty acid synthase.

Conference Article| November 01 1997 FIRE (fatty acid synthase insulin-responsive element) and ICE (inverted CCAAT element) regulate fatty acid synthase M. Schweizer; M. Schweizer 1 1Genetics and Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. 1To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar S. S. Wolf; S. S. Wolf 1Genetics and Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. Search for other works by this author on: This Site PubMed Google Scholar K. F. Roder K. F. Roder 1Genetics and Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1997) 25 (4): 1220–1224. https://doi.org/10.1042/bst0251220 Article history Received: June 26 1997 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation M. Schweizer, S. S. Wolf, K. F. Roder; FIRE (fatty acid synthase insulin-responsive element) and ICE (inverted CCAAT element) regulate fatty acid synthase. Biochem Soc Trans 1 November 1997; 25 (4): 1220–1224. doi: https://doi.org/10.1042/bst0251220 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: FAS, fatty acid synthase, FIRE, FAS insulin-responsive element, IRE, insulin-responsive element, CAT, chloramphenicol acetyltransferase, RA, retinoic acid, SRE, sterol regulatory element, SREBP, SRE-binding protein, h, hepatocyte, NF, nuclear factor, HMG, 3-hydroxy-3-methylglutaryl This content is only available as a PDF. © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

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.

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.

A selective transcriptional induction system for mammalian cells based on Gal4-estrogen receptor fusion proteins.

Most mammalian cells neither express any Gal4-like activity nor endogenous estrogen receptor, thus rendering estrogen an inert signal for them. For these two reasons we have developed a selective induction system based on the estrogen-regulable transcription factor Gal-ER. Gal-ER consists of the DNA-binding domain of the yeast Gal4 protein fused to the hormone-binding domain of the human estrogen receptor and hence should exclusively regulate a transfected gene under the control of a Gal4-responsive promoter in mammalian cells. Two major improvements of this induction system were made. First, a synthetic Gal4-responsive promoter was constructed which consisted of four Gal4-binding sites, an inverted CCAAT element, a TATA box, and the adenovirus major late initiation region. This promoter showed extremely low basal activity in the absence and high inducibility in the presence of ligand-activated Gal-ER. Second, the transcription factor Gal-ER was rendered more potent and less susceptible to cell type-specific variation by fusing the strong activating domain of the herpesvirus protein VP16 onto its C terminus. In response to estrogen, Gal-ER-VP16 induced the Gal4-responsive promoter at least 100-fold in transiently transfected NIH 3T3 and P19 cells. Rat fibroblast cell lines expressing integrated Gal-ER and Gal4-responsive fos genes were transformed in a strictly estrogen-dependent manner. The exogenous fos gene was rapidly induced to maximal levels within 1-2 hr of estrogen addition. Elevated Fos activity in turn stimulated transcription of the endogenous fra-1 gene. These data demonstrate the utility of the Gal-ER induction system as a powerful genetic switch for regulating heterologous genes and, in particular, for identifying Fos targets in mammalian cells.

A specific CCAAT-binding protein, CBP/tk, may be involved in the regulation of thymidine kinase gene expression in human IMR-90 diploid fibroblasts during senescence.

The aging of IMR-90 human diploid fibroblasts in culture is accompanied by a 5-7 fold decrease in the level of thymidine kinase (TK) mRNA and TK activity (Chang, Z. F., and Chen, K. Y. (1988) J. Biol. Chem. 263, 11431-11435). We have employed a gel mobility shift analysis to investigate the molecular basis of the age-dependent attenuation of TK gene expression. Several cis-elements including two inverted CCAAT boxes, located at base pairs (bp) -36 and -67, and GC-rich Sp1 binding sites have been identified in the TK promoter. A 28-bp (-91 to -64) fragment containing the distal inverted CCAAT element was excised from the TK promoter to examine possible differences in nuclear protein binding between young and old IMR-90 cells. A prominent DNA-protein complex was identified in serum-stimulated young cells by a gel mobility shift assay. Competition analysis indicated that the binding was highly specific. The nuclear protein responsible for the complex formation was named CBP/tk (CCAAT Binding Protein for TK gene) since methylation interference assay showed that the inverted CCAAT box was involved in binding. The appearance of the CBP/tk-28-bp complex in IMR-90 cells was (i) serum-dependent, becoming prominent 12-24 h after serum stimulation, and (ii) age-dependent, prominent only in young but not in old IMR-90 cells. Similar serum- and age-dependent complex formations were also observed using a 67-bp fragment (-63 to +4) containing the proximal CCAAT element and a TATA box. In contrast, the binding activities for the Sp1 sequence were the same in young and old cells and appeared to be serum-independent. CBP/tk binding activity in nuclear extracts was abolished by heat (60 degrees C, 5 min) or treatment with proteinase K (0.1 microgram/ml) and sodium dodecyl sulfate (0.005%), but not by Nonidet P-40 or Triton X-100. Treatment of nuclear extracts with alkaline phosphatase or lectins (concanavalin A and wheat germ agglutinin) did not affect the binding activity. Metal chelators such as 1,10-ortho-phenanthroline (0.5 mM) inhibited the CBP/tk binding activity. Cycloheximide added to the serum-stimulated cultures at an early or mid-G1 phase inhibited the CBP/tk binding activity. The half-life of the serum-induced CBP/tk binding activity was estimated to be less than 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)