CCAAT Motif Research Articles

P73 competes with co-activators and recruits histone deacetylase to NF-Y in the repression of PDGF beta-receptor.

We investigated mechanisms of the p73alpha-mediated repression of the platelet-derived growth factor beta-receptor (PDGFRB) promoter caused by its interaction with NF-Y. Treatment of cells with the histone deacetylase (HDAC) inhibitor, Trichostatin A, increases PDGFRB promoter activity through the CCAAT motif and counteracts the repression caused by p73alpha. Activation of the PDGFRB promoter by the co-activator p300 also occurs through the CCAAT motif. Expression of p73alpha counteracts both p300- and P/CAF-mediated activation of the PDGFRB promoter, and expression of p300 or P/CAF attenuates the p73alpha-mediated repression of the promoter activity. In concordance, p73alpha decreases the p300-mediated acetylation of NF-YC, p300 competes with p73alpha for binding NF-YB, and P/CAF competes with p73alpha for binding NF-YB and NF-YC. Furthermore, p73alpha, but not the oncogenic DeltaNp73alpha, binds directly to HDAC1. We performed chromatin immunoprecipitation with antibodies against p73, DeltaNp73, NFYB, p300 and HDAC1 at different periods after serum stimulation in serum-starved NIH3T3 cells. A marked decrease of DeltaNp73, NF-YB and p300 was detected 6 hours after serum stimulation when the expression of PDGFRB decreased. Conversely, HDAC1 was found bound at its maximum and the anti-p73 detecting both TAp73 and DeltaNp73 was found at all time points, indicating that p73, but not DeltaNp73, remains bound at this time. Double immunofluorescence staining of TAp73 and HDAC1 revealed that both of these molecules exist in the nucleus at this time point, supporting the presence of endogenous interaction. These results suggest that p73 and DeltaNp73 behave as physiological regulators for the transcription of the PDGFRB promoter.

Molecular cloning and characterization of the translationally controlled tumor protein gene in Bombyx mori

Translationally controlled tumor protein (Tctp/p23) is known to be synthesized preferentially in cells during the early growth phase of tumors, but is also expressed in normal cells. To elucidate its molecular basis of the expression and physiological significance, a cDNA encoding for the Bombyx mori Tctp (BmTctp) was deduced by editing the partial cDNA sequences registered in a Bombyx EST database. RT-PCR analyses indicated that the BmTCTP mRNA was transcribed in all larval organs examined and was present constantly during the cell cycle of BmN4 cells. A genomic clone of 4255 nucloetide residues produced by inverse PCR contained the 5′-flanking region, two introns and three exons of the BmTCTP gene. Sequence analysis of the 5′-flanking region indicated that a putative promoter region contains several canonical transcription elements such as GATA box, CCAAT motif, MEF2, E4BP4.01 and AP-1, but lacks a TATA box element. Luciferase reporter assay of the deletion constructs of the 5′-flanking region revealed that the −676 to +66 region enhanced the promoter activity the most markedly. In addition to this, there were at least two enhancer-like elements and several repressor elements.

Suppression of C/EBP α expression in biliary cell differentiation from hepatoblasts during mouse liver development

Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent mesenchyme, which leads to the suppression of mature hepatocyte marker expression. This study was undertaken to analyze C/EBP alpha and beta expression, which may govern transcription of mature hepatocyte marker genes, during mouse liver development with special attention given to biliary differentiation. Expression of C/EBP alpha and beta was immunohistochemically examined. Expression of alpha-fetoprotein, albumin and urea cycle enzymes, the genes of which have CCAAT motifs in their upstream regulatory sequences, was examined immunohistochemically or by using in situ hybridization. C/EBP alpha started to be expressed in endodermal cells of 9.5-day liver primordium, and continued to be expressed in hepatoblasts and hepatocytes throughout development. Although biliary cell progenitors transiently expressed mature hepatocyte markers, their expression of C/EBP alpha was weak or totally absent. The signals of C/EBP beta in hepatocytes were weak in fetal liver, but became stronger with postnatal development. Differentiated epithelial cells of intrahepatic biliary structures did not express C/EBP alpha. These data suggest that the suppression of C/EBP alpha expression may be prerequisite to biliary cell differentiation in the hepatoblast population and one of its earliest signs.

PRb, Myc and p53 are critically involved in SV40 large T antigen repression of PDGF beta-receptor transcription.

The expression of the PDGF beta-receptor is tightly regulated during a normal cell cycle. c-Myc and p73alpha repress transcription of the receptor through interaction with NF-Y. In ST15A cells which stably express the temperature-sensitive SV40 large T antigen (LT) the receptor expression and ligand binding decreased under the permissive condition. Transient expression of the LT, but not small t, decreased the endogenous receptor expression at mRNA and protein levels in NIH3T3 cells but not in the myc-null HO15.19 cells. The wild-type LT, but not the various pRb or p53 binding defective LT mutants, represses the PDGF beta-receptor promoter activity. Moreover, the inability of the LT-mediated repression in the myc-null cells, the Rb-null 3T3 cells, and the Saos-2 cells lacking pRb and p53, indicates that Myc, pRb and p53 are all necessary elements. PDGF beta-receptor promoter-luciferase assays revealed that the CCAAT motif is important for the repression. Furthermore, p53 was found to increase the promoter activity mainly via the upstream Sp1 binding sites together with the CCAAT motif in the NIH 3T3 cells. This was confirmed by Schneider's Drosophila line (SL2) cells deficient in both endogenous NF-Y and Sp1. Chromatin immunoprecipitation using ST15A cells revealed that both LT and p53 bound the PDGF beta-receptor promoter and the binding of p53 diminished when LT was expressed in the permissive condition. However, LT binds the promoter in the absence of pRb and p53 in Saos-2 cells stably expressing LT. These results suggest that LT binds the promoter and interferes with NF-Y and Sp1 to repress it in the presence of Myc, pRb and p53.

Transcription Factors NF-YA Regulate the Induction of Human OGG1 Following DNA-alkylating Agent Methylmethane Sulfonate (MMS) Treatment

A human 8-oxoguanine-DNA glycosylase (hOGG1) is the main enzyme that repairs 8-oxoG, which is a critical mutagenic lesion. There is a great deal of interest in the up- or down-regulation of OGG1 expression after DNA damage. In this study, we investigated the effect of a DNA-alkylating agent, methylmethane sulfonate (MMS), on hOGG1 expression level and found that MMS treatment resulted in an increase in the functional hOGG1 expression in HCT116 cells. A region between -121 and -61 of the hOGG1 promoter was found to be crucial for this induction by MMS. Site-directed mutations of the two inverted CCAAT motifs substantially abrogated the induction of the hOGG1 promoter as a result of MMS treatment. In addition, the NF-YA protein (binding to the inverted CCAAT box) was induced after exposing cells to MMS. Moreover, gel shift and supershift analyses with the nuclear extracts prepared from HCT116 cells identified NF-YA as the transcription factor interacting with the inverted CCAAT box. Mutations of the inverted CCAAT box either prevented the binding of this factor or abolished its activation as a result of MMS treatment. Finally, this study showed that hOGG1-expressing HCT116 cells exhibited increased hOGG1 repair activity and resistance to MMS. Overall, these results demonstrate that MMS can up-regulate hOGG1 expression through the induction of the transcription factor, NF-YA, and increased transcription level of the hOGG1 gene correlates with an increase in enzyme activity providing functional protection from MMS.

Nuclear factor Y regulation and promoter transactivation of human ribonucleotide reductase subunit M2 gene in a Gemcitabine resistant KB clone

Overexpression of human ribonucleotide reductase subunit M2 ( hRRM2) has been shown as a potential factor causing Gemcitabine (Gem) resistance. We hypothesized the nuclear factor Y (NF-Y) would transcriptionally regulate hRRM2 and contribute to overexpression of hRRM2 in a Gem resistant clone. A luciferase and gel shift assay, and a Southwestern blot were employed to analyze the promoter activity of hRRM2. The data exhibited the hRRM2 promoter was upregulated almost 5-fold in the Gem resistant KB clone (KBGem) via three sequential CCAAT boxes located in the proximal promoter region. Nuclear extracts from KB and KBGem could interact with the CCAAT motif of the hRRM2 proximal promoter region, and could form DNA–protein complexes with different binding patterns. The complexes could be further recognized with antibodies against NF-Y subunits A and B. Histone deacetylases (HDAC) involvement in NF-Y transcription repression in the KBGem clone was examined. A HDAC activity assay revealed a 3-fold decrease of HDAC activity in the KBGem clone compared to KB cells. Parental cells were treated with trichostatin A (TSA), a HDAC inhibitor. NF-Y transactivation was induced, resulting in an increase of hRRM2 expression. This led to an expanded dCTP pool and an abrogated [ 3 H ]Gemcitabine incorporation. In addition, microarray analysis results showed most of the proliferation-related genes were upregulated in KBGem. This finding was consistent with enhanced NF-Y transactivation in KBGem. In summary, upregulation of NF-Y transactivation increased hRRM2 transcription, which played a pivotal role in the Gem resistant KB clone.

Genomic structure, organisation, and promoter analysis of the bovine (Bos taurus) Mx1 gene.

Some MX proteins are known to confer a specific resistance against a panel of single-stranded RNA viruses. Many diseases due to such viruses are known to affect cattle worldwide, raising the possibility that the identification of an antiviral isoform of a bovine MX protein would allow the implementation of genetic selection programs aimed at improving innate resistance of cattle. With this potential application in mind, the present study was designed to isolate the bovine Mx1 gene including its promoter region and to investigate its genomic organisation and promoter reactivity. The bovine Mx1 gene is made up of 15 exons. All exon-intron boundaries conformed to the consensus sequences. A PCR product that contained a approximately 1-kb, 5'-flanking region upstream from the putative transcription start site was sequenced. Unexpectedly, this DNA region did not contain TATA or CCAAT motifs. A computer scan of the region disclosed a series of putative binding sites for known cytokines and transcription factors. There was a GAAAN(1-2)GAAA(C/G) motif, typical of an interferon-sensitive responsive element, between -118 and -107 from the putative transcription start site. There were also a NF-kappaB, two interleukin-6 binding sites, two Sp1 sites and five GC-rich boxes. The region also contained 12 stretches of the GAAA type, as described in all IFN-inducible genes. Bovine Mx1 expression was assessed by Northern blotting and immunofluorescence in the Madin Darby bovine kidney cells (MDBK) cell line treated with several stimuli. In conclusion, the bovine Mx1 gene and promoter region share the major structural and functional characteristics displayed by their homologs described in the rainbow trout, chicken, mouse and man.

Isolation and characterization of the human syncytin gene promoter.

Syncytin, a protein encoded by an envelope gene of a human endogenous retrovirus-W (HERV-W), plays a critical role in trophoblast differentiation. We isolated the 5'-flanking region of the syncytin gene from human genomic DNA by PCR and identified cis-acting elements on the promoter that are important for transcription. The major transcription initiation site identified by mung bean nuclease protection assays is 56 base pairs (bp) downstream from a putative CCAAT box. Deletion analysis of the 5'-flanking region of the syncytin gene indicated that the proximal 148 bp are essential for minimal promoter activity and that regions of the promoter from nt -1519 to -984 and nt -294 to -148 are required for maximal expression in normal trophoblast cells. DNase I footprint analysis of the region between nt -252 and +110 revealed three protected regions, FP1-FP3. Mutagenesis of a hepatocyte-specific nuclear protein-1 (HAPF1) binding site in FP1 and a TATA box in FP3 had no effects on basal promoter activity. However, mutation of the CCAAT motif and the octamer protein (Oct) binding site in FP2 decreased promoter activity by 88% and 76%, respectively. Mutation of the ecdysone receptor (EcR) response element in FP2, which may bind a nuclear hormone receptor, increased basal promoter activity by 2-fold. Gel shift and supershift assays indicated that CCAAT-binding factor (CBF) binds to the CCAAT motif and that Oct binds to the Oct binding site. Taken together, these findings indicate that the syncytin promoter is located in the 5' long terminal repeat (LTR) of the HERV-W gene and that binding sites for CBF and Oct in the proximal promoter are critical for transcriptional regulation of the gene in trophoblast cells.

Transactivation of human cdc2 promoter by adenovirus E1A.

Expression of the adenovirus oncoprotein E1A 12S induces the heterotrimeric transcription factor, NF-Y. NF-Y binds to the two CCAAT motifs upstream of the transcriptional start site of the human cdc2 promoter and is required for activation of the promoter by E1A 12S in cycling cells. The observations that a number of eukaryotic cell cycle regulatory genes also contain the CCAAT motifs and NF-Y binds to them support the notion that E1A 12S could play an important role in deregulated expression of these genes through activation of NF-Y gene in cycling cells.

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.

CCAAT Binding Factor (CBF) Binding Mediates Cell Cycle Activation of Topoisomerase IIα: CONVENTIONAL CBF ACTIVATION DOMAINS ARE NOT REQUIRED

To understand the role of the CCAAT binding factor (CBF) in transcription during the cell cycle, we studied the mouse topoisomerase II alpha (topo II alpha) promoter, which is activated during the late S and G(2)/M phases of the cell cycle and contains multiple CBF binding sites. Mutational analysis of the promoter shows that CBF binding to an inverted orientation of the CCAAT motif in the topo II alpha promoter, but not to a direct orientation, is required for transcription activation during the cell cycle. In contrast, analysis of the promoter in an in vitro reconstituted transcription system shows that CBF activates transcription of the topo II alpha promoter irrespective of the orientation of the CBF binding sites. This analysis demonstrates that only one of the three transcription start sites of the topo II alpha promoter is stimulated by CBF, indicating that transcription activation by CBF is dependent on basal promoter structure. Interestingly, mutations of the start site that abolish CBF-dependent transcription activation in vitro do not inhibit activation of the promoter during the cell cycle. Consistent with this observation, expression of a truncated CBF-B subunit lacking a transcription activation domain, which inhibits activity of a collagen promoter, does not affect activity of the topo II alpha promoter in fibroblast cells. In contrast, expression of an allele-specific CBF-B mutant that binds high affinity to a mutant CBF binding site containing a CCAAC motif revives transcription activation of an inactive mutant topo II alpha promoter containing CCAAC during the cell cycle. Altogether, this study indicates that CBF binding, but not conventional CBF activation domains, are required for activation of the topo II alpha promoter during the cell cycle. Considering these results together with results of another recent study, we hypothesize that binding of CBF that disrupts the nucleosomal structure in the topo II alpha promoter is a major function of CBF by which it regulates the cell cycle-dependent transcription of the topo II alpha promoter and possibly many other cell cycle-regulated promoters containing CBF binding sites.

Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA.

A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.

Ectoderm gene activation in sea urchin embryos mediated by the CCAAT-binding factor

Transcriptional enhancers are short stretches of DNA that function to achieve highly specific patterns of gene expression. To identify the mechanisms by which enhancers achieve their specificity, we made use of an enhancer from the aboral ectoderm-specific spec2a gene of the sea urchin Strongylocentrotus purpuratus. The spec2a enhancer contains five cis-regulatory elements within 78 base pairs that interact with five distinct DNA-binding proteins to confer aboral ectoderm expression. Here, we present an analysis of the sea urchin CCAAT binding factor (CBF), which binds to a CCAAT motif within the spec2a enhancer. S. purpuratus CBF and SpOtx, a ubiquitously expressed factor, act together at closely placed cis-regulatory elements to mediate spec2a transcription in the ectoderm. SpCBF was the sole factor that bound to the spec2a CCAAT element, and two of the three subunits that make up the CBF holoprotein were cloned and shown to have high sequence conservation with their vertebrate orthologs. Based on its involvement in the regulation of several other sea urchin genes, SpCBF appears to be a major transcription factor in the sea urchin embryo for positive regulation of ectoderm gene expression. In addition to its role in vertebrate cell growth and proliferation, our results indicate that CBF also functions at the early stages of germ layer formation, namely ectoderm differentiation.

Identification of the N-acetyl-D-glucosamine-inducible element in the promoter of the Trichoderma atroviride nag1 gene encoding N-acetyl-glucosaminidase.

We have investigated the regulation by N-acetyl-glucosamine of the nag1 gene of the mycoparasitic biocontrol fungus Trichoderma atroviride (= T. harzianum P1), which encodes a 73-kDa N-acetyl-beta-D-glucosaminidase. The use of translational fusions revealed that a 290-bp fragment of the 5' regulatory region of nag1 is sufficient to confer inducibility on the Aspergillus niger goxA gene. The region between positions -150 and -290, upstream of the nag1 coding region, was investigated using in vivo methylation protection analysis and electrophoretic mobility shift assays (EMSAs). Two neighbouring regions that interacted with regulatory proteins were identified, and bases essential for these interactions were determined in vitro. These data reveal protein binding to a CCCCT element at -240, a CCAGN(13)CTGG motif at -284, and a CCAAT-box which is present in the spacer of the latter motif. Evidence for the binding of a Hap2/3/5 complex to this CCAAT motif is presented. Protein binding to all three motifs was constitutive, and no differences were observed between induced and non-induced cultures. Mutation of either the CCAGN(13)CTGG or the AGGGG motif resulted in loss of inducibility of nag1 expression by N-acetyl-D-glucosamine in vivo.

CDP and AP-2 mediated repression mechanism of the replication-dependent hamster histone H3.2 promoter.

The replication-dependent hamster histone H3.2 promoter contains two tandem CCAAT repeats located upstream of the TATA element. It has been shown that the NF-Y/CBF complex binds to a single CCAAT motif with high affinity, whereas the CCAAT displacement protein (CDP) binds to at least two CCAAT motifs in close proximity. Here, we report that the two CCAAT motifs within the H3.2 promoter confer transcriptional repression of the promoter during the cell cycle. While we cannot detect direct association of CDP with Rb in vitro, we discover that CDP can bind AP-2, a ubiquitous factor that interacts with Rb. The interaction domains between CDP and AP-2 are mapped to the highly conserved cut repeats of CDP as well as the basic and dimerization region of AP-2. Further, in transfection assays, CDP and AP-2 act synergistically to suppress the H3.2 promoter. Together, these data support a repression mechanism mediated by CDP and AP-2 that regulates H3.2 gene expression during the mammalian cell cycle.

Characterization of human cathepsin L promoter and identification of binding sites for NF-Y, Sp1 and Sp3 that are essential for its activity.

Cathepsin L is a cysteine protease whose overexpression in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic. Regulation of the transcription of the gene encoding human cathepsin L has not been yet studied and only preliminary data exist on the promoter regulation of the gene encoding rodent cathepsin L. In the present study we identified molecular elements involved in the transcriptional regulation of human cathepsin L in melanoma cells. The sequence of the 5'-flanking region of the gene encoding human cathepsin L was determined up to 3263 bp upstream of the translation start site. The major transcription intiation site was located. Three mRNA splice variants, differing in their 5' untranslated ends, were identified. Regulatory regions crucial for cathepsin L promoter activity were characterized between -1489 and -1646 bp. In this region, two GC boxes (-1590/-1595 and -1545/-1550) and a CCAAT motif (-1571/-1575) were involved in specific DNA-protein interactions. An electrophoretic mobility-shift assay demonstrated that Sp1 and Sp3 transcription factors bound to these GC boxes, and only the transcription factor nuclear factor Y (NF-Y) bound to the CCAAT motif. Mutagenesis studies demonstrated that these binding sites contributed at least 85% of cathepsin L promoter activity. Thus structural and functional analysis demonstrated that binding sites for NF-Y, Sp1 and Sp3 are essential for transcription of the gene encoding human cathepsin L in melanoma cells.

Characterization of human cathepsin L promoter and identification of binding sites for NF-Y, Sp1 and Sp3 that are essential for its activity

Cathepsin L is a cysteine protease whose overexpression in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic. Regulation of the transcription of the gene encoding human cathepsin L has not been yet studied and only preliminary data exist on the promoter regulation of the gene encoding rodent cathepsin L. In the present study we identified molecular elements involved in the transcriptional regulation of human cathepsin L in melanoma cells. The sequence of the 5′-flanking region of the gene encoding human cathepsin L was determined up to 3263bp upstream of the translation start site. The major transcription intiation site was located. Three mRNA splice variants, differing in their 5′ untranslated ends, were identified. Regulatory regions crucial for cathepsin L promoter activity were characterized between −1489 and −1646bp. In this region, two GC boxes (−1590/−1595 and −1545/−1550) and a CCAAT motif (−1571/−1575) were involved in specific DNA–protein interactions. An electrophoretic mobility-shift assay demonstrated that Sp1 and Sp3 transcription factors bound to these GC boxes, and only the transcription factor nuclear factor Y (NF-Y) bound to the CCAAT motif. Mutagenesis studies demonstrated that these binding sites contributed at least 85% of cathepsin L promoter activity. Thus structural and functional analysis demonstrated that binding sites for NF-Y, Sp1 and Sp3 are essential for transcription of the gene encoding human cathepsin L in melanoma cells.

NGF-dependent and tissue-specific transcription of vgf is regulated by a CREB–p300 and bHLH factor interaction

Neurotrophins support neuronal survival, development, and plasticity through processes requiring gene expression. We studied how vgf target gene transcription is mediated by a critical promoter region containing E-box, CCAAT and cAMP response element (CRE) sites. The p300 acetylase was present in two distinct protein complexes bound to this region. One complex, containing HEB (ubiquitous basic helix–loop–helix (bHLH)), bound the promoter in non-neuronal cells and was involved in repressing vgf expression. Neurotrophin-dependent transcription was mediated by the second complex, specific for neuronal cells, which included CRE binding protein and MASH1 (neuro-specific bHLH), bound the CCAAT motif, and was target of neurotrophin signalling. The interaction, mediated by p300, of different transcription factors may add specificity to the neurotrophin response.

TAK1 activation of the mouse JunB promoter is mediated through a CCAAT box and NF-Y

The JunB gene is activated by many stimuli including transforming growth factor β (TGFβ) family members and interleukin-6 (IL-6). Here the effect of TGFβ activated kinase 1 (TAK1), a mitogen activated protein kinase kinase kinase (MAPKKK) implicated in TGFβ, bone morphogenetic protein (BMP) and interleukin-1 (IL-1) signaling, on JunB promoter activity was investigated. Promoter analysis led to the identification of a CCAAT motif in the JunB gene, essential for activation by TAK1. Transfer of this CCAAT element to a heterologous minimal promoter conferred TAK1-responsiveness. The CCAAT-binding transcription factor, nuclear factor Y (NF-Y), activated the JunB promoter and a dominant negative NF-YA construct inhibited TAK1 activation of JunB. Our results demonstrate that JunB gene activation by TAK1 is mediated by the CCAAT-binding factor NF-Y.

CBF/NF-Y functions both in nucleosomal disruption and transcription activation of the chromatin-assembled topoisomerase IIalpha promoter. Transcription activation by CBF/NF-Y in chromatin is dependent on the promoter structure.

To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.