Binding Of CCAAT-binding Factor Research Articles

CCAAT box is required for the induction of human thrombospondin‐1 gene by trichostatin A

Histone deacetylase (HDAC) inhibitors have been reported to inhibit angiogenesis as well as tumor growth. Thrombospondin-1 (TSP1) has been recognized as a potent inhibitor of angiogenesis. Such an action of TSP1 may account for the effect of HDAC inhibitors. In the present study, we investigated the molecular mechanism by which trichostatin A, a HDAC inhibitor, induces the expression of TSP1 gene. Trichostatin A increased both mRNA and protein levels of TSP1 in HeLa cells. Promoter and actinomycin D chase assays showed that trichostatin A-induced TSP1 expression was regulated at the transcriptional level without changing mRNA stability. CCAAT box on the TSP1 promoter was found to primarily mediate the trichostatin A response by deletion and mutation analyses of the TSP1 promoter. Electrophoretic mobility shift assay indicated that CCAAT-binding factor (CBF) was specifically bound to the CCAAT box of TSP1 promoter. Moreover, chromatin immunoprecipitation assay showed that trichostatin A increased the binding of acetylated form of histone H3 to the CCAAT box region of TSP1 promoter. Taken together, these results strongly suggest that trichostatin A activates the transcription of TSP1 gene through the binding of transcription factor CBF to CCAAT box and the enhanced histone acetylation. Thus, the present study provides the clue that the inhibition of angiogenesis by trichostatin A is accomplished through the upregulation of TSP1, the anti-angiogenic factor.

Abstract 725: Protein Kinase C delta Inhibits Type I Collagen Basal Expression through CCAAT-binding Factor and c-Krox in Vascular Smooth Muscle Cells

Protein Kinase C delta (PKCδ) regulates multiple functions in vascular smooth muscle cells (VSMCs) including proliferation, migration, and apoptosis. In previous studies, we have demonstrated that PKCδ activity is necessary for TGFβ-induced fibronectin production in VSMCs. The purpose of the current study is to understand the role of this kinase in regulation of type I collagen synthesis. Using A10 VSMCs derived from rat thoracic aorta, we showed that TGFβ (5ng/ml x 48 hours) increased type I collagen expression. Pretreatment of A10 with Rottlerin (2uM, 1 hour), a selective inhibitor of PKCδ, dramatically increased type I collagen basal expression independent of TGFβ. To understand the mechanism involved with PKCδ, we used a Luciferase reporter that contains the human COL1A2 promoter (−772 to +58). Rottlerin stimulated the Luciferase reporter activity by 3.53±0.13 fold (n=3, p<0.01). Moreover, inhibition of PKCδ with a dominant negative mutant led to upregulation of reporter activity (1.77±0.15 fold induction, n=3, p<0.01), while activation of PKCδ with a constitutive active mutant reduced collagen reporters (0.19±0.02 fold reduction, n=3, p<0.01). Using a series of 5′ deletions of COL1A2/Luciferase constructs (−772, −353, and −108), we narrowed the location of the PKCδ-response element to the -108/+58 region of the COL1A2 promoter, which is distinct from the TGFβ response element that is further upstream. This region contains a CCAAT motif and a c-Krox motif that have been previously implicated in collagen expression. Using a gel shift assay, we found that inhibition of PKCδ with Rottlerin or the dominant negative mutant increased the binding of CCAAT-binding Factor (CBF) to the CCAAT motif but decreased the binding at the c-Krox site. Taken together, our data indicate that PKCδ inhibits type I collagen basal expression by affecting its gene transcription through a mechanism involving CBF and c-Krox in VSMCs. We believe that PKCδ is one of the key players in mediating extracellular matrix production and could contribute to pathogenesis of vascular diseases.

Regulation of human methylthioadenosine phosphorylase gene by the CBF (CCAAT binding factor)/NF-Y (nuclear factor-Y)

hMTAP (human 5'-deoxy-5'-methylthioadenosine phosphorylase) is a key enzyme in the methionine salvage pathway and is frequently inactivated in human tumour cells. To understand the mechanism of the transcriptional regulation of the MTAP gene, we have cloned the 1.29 kb fragment of the hMTAP promoter and identified cis-acting regulatory sequences using a luciferase reporter gene assay. Maximal promoter activity was associated with sequences between -446 and -152, where two CCAAT elements were located. Electrophoretic mobility-shift assay reveals binding of specific complexes at both CCAAT motifs within the MTAP promoter, although more prominent bands were associated with the distal motif (-372 to -368). Supershift experiments and chromatin immunoprecipitation assays indicate that both the proximal and distal complexes bind CBF (CCAAT-binding factor; also known as nuclear factor-Y), and that the distal CCAAT motif has increased levels of CBF binding. We have mapped seven different transcriptional start sites between -135 and -58. Our results show that the hMTAP expression is regulated by a CBF and that the distal one of two CCAAT motifs plays a major role in the transcriptional activation of hMTAP gene.

Promoter methylation inhibits APC gene expression by causing changes in chromatin conformation and interfering with the binding of transcription factor CCAAT-binding factor.

As an important regulator in Wnt-signaling pathway, the APC gene is involved in apoptosis and cell cycle arrest. The loss of APC function is observed in most familial adenomatous polyposis-associated and sporadic colorectal cancer. APC gene is frequently inactivated by DNA mutations. However, hypermethylation in APC gene promoter was also observed in different cancers. In this study, by analyzing the methylation status of APC promoter in 22 colorectal cancer cell lines with different APC expression levels, we identified Regions A and B in the promoter, where the methylation of CpG sites was invariably correlated with the loss of gene expression. By nuclease accessibility assay, we also observed a correlation between the closed chromatin conformation in APC promoter and loss of gene expression. When the nonexpressing cell lines were treated with a DNA methyltransferase inhibitor, 5-Aza-2'-Deoxycytidine, the APC expression in these cells was induced, CpG sites were demethylated, and closed chromatin conformation was opened. However, when these cell lines were treated with a histone deacetylase inhibitor, Trichostatin A, no significant changes in APC expression, methylation status, and chromatin conformation were observed. Using transient transfection assay, a CCAAT box located in Region B was identified, which was involved in up-regulation of APC expression. Methylation of CpG sites around the CCAAT box resulted in a significant inhibition in the gene expression. The specific binding of a transcription factor CCAAT-binding factor (CBF) to the CCAAT box was determined by electrophoretic mobility shift analysis. The binding was inhibited after CpG sites close to the CCAAT box were methylated, indicating that DNA methylation can silence gene expression through interfering with the binding of transcription factors to the promoter. The biological function of CBF in APC gene regulation was further indicated by the decrease of luciferase activities in cells cotransfected with a plasmid carrying APC promoter/luciferase gene and a plasmid expressing dominant negative CBF mutant. In summary, methylation of CpG sites around CCAAT box in APC promoter inhibits the gene expression by changing the chromatin conformation and interfering with the binding of transcription factor CBF to CCAAT box.

B-Myb acts as a repressor of human COL1A1 collagen gene expression by interacting with Sp1 and CBF factors in scleroderma fibroblasts

We investigated the role of B-Myb, a cell-cycle-regulated transcription factor, in the expression of the alpha1 (I) pro-collagen gene (COL1A1) in scleroderma fibroblasts. Scleroderma or SSc (systemic sclerosis) is a fibrotic disease characterized by excessive production of extracellular matrix components, especially type I collagen. Northern-blot analysis showed an inverse relationship between COL1A1 mRNA expression and that of B-Myb during exponential cell growth and during quiescence in human SSc fibroblasts. Overexpression of B-Myb in SSc fibroblasts was correlated with decreased COL1A1 mRNA expression. Transient transfections localized the down-regulatory effect of B-Myb to a region containing the proximal 174 bp of the COL1A1 promoter that does not contain B-Myb consensus binding sites. Gel-shift analysis, using nuclear extracts from normal and SSc fibroblasts transfected with B-Myb, showed no differences in DNA-protein complex formation when compared with the nuclear extracts from mock-transfected cells. However, we found that B-Myb decreases Sp1 (specificity protein 1) and CBF (CCAAT-binding factor) binding for their specific sites localized in the 174 bp COL1A1 proximal promoter. These results were also confirmed using B-Myb-immunodepleted nuclear extracts. Furthermore, immunoprecipitation assays using SSc nuclear extracts demonstrated a physical interaction of B-Myb with Sp1 and CBF transcription factors, and also an interaction between Sp1 and CBF. In addition, by employing full-length or deleted B-Myb cDNA construct, we found that B-Myb down-regulates the COL1A1 proximal promoter through its C-terminal domain. Thus these results suggest that B-Myb may be an important factor in the pathway(s) regulating collagen production in SSc fibroblasts.

Transcriptional Inhibition of Type I Collagen Gene Expression in Scleroderma Fibroblasts by the Antineoplastic Drug Ecteinascidin 743

We previously showed that COL1A1 expression is up-regulated at the transcriptional level in systemic sclerosis (SSc) fibroblasts and that the CCAAT-binding factor (CBF) is involved in this increased expression. Ecteinascidin 743 (ET-743) is a chemotherapeutic agent that binds with sequence specificity to the minor groove of DNA and inhibits CBF-mediated transcriptional activation of numerous genes. Therefore, we examined the effects of ET-743 on the increased COL1A1 expression in SSc fibroblasts. The drug caused a potent and dose-dependent inhibition of type I collagen biosynthesis, which reached 70-90% at 700 pM without affecting cell viability. The same drug concentration caused 60-80% reduction in COL1A1 mRNA levels. The stability of the corresponding transcripts was not affected. In vitro nuclear transcription assays demonstrated a 54% down-regulation of COL1A1 transcription. Transient transfections with COL1A1 promoter constructs containing the specific CBF binding sequence into SSc cells previously treated with 700 pM ET-743 failed to show an effect on COL1A1 promoter activity. Furthermore, ET-743 did not affect the binding of CBF or Sp1 transcription factors to their cognate COL1A1 elements. However, treatment with 700 pM ET-743 of stably transfected NIH 3T3 cells expressing a human type II procollagen gene under the control of the human COL1A1 promoter caused a greater than 50% reduction in the production of type II procollagen and a similar decrease in the corresponding type II procollagen transcripts. These results indicate that ET-743 is a potent inhibitor of COL1A1 transcription. However, this effect cannot be explained by a direct effect on CBF binding to the COL1A1 promoter. Although the exact mechanisms responsible for the transcriptional inhibition of COL1A1 by ET-743 are not apparent, our observations suggest that the drug may be an effective agent to decrease collagen overproduction in SSc and other fibrotic diseases.

CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line.

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

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.

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.

SpSoxB1 serves an essential architectural function in the promoter SpAN, a tolloid/BMP1-related gene.

Transcription of SpAN, which encodes a secreted protease related to tolloid and BMP 1, is differentially regulated along the animal-vegetal axis of the sea urchin embryo by a maternally initiated mechanism. Regulatory sites that bind SpSoxB1 and CBF (CCAAT binding factor) are essential for strong transcriptional activity because mutations of these elements reduce promoter activity in vivo 20- and 10-fold, respectively. Here we show that multimerized SpSoxB1 elements cannot activate transcription from the SpAN basal promoter in vivo. However, like other factors containing HMG-class DNA binding domains, SpSoxB1 does induce strong bending of DNA. The CBF binding site lies abnormally far from the transcriptional start site at -200 bp. We show that the SpSoxB1 site is not required if the CCAAT element is moved 100 bp closer to the transcriptional start site, replacing the SpSoxB1 site. This supports a model in which the bending of SpAN promoter DNA by SpSoxB1 facilitates interactions between factors binding to upstream and downstream regulatory elements.

Studies on the structure of the mouse CBF-A gene and properties of a truncated CBF-A isoform generated from an alternatively spliced RNA

CCAAT-binding factor (CBF), a heteromeric transcription factor that binds to sequences containing a CCAAT motif, is composed of three subunits. A, B and C, which are all required for DNA binding. The mouse CBF-A gene contains seven coding exons, which span 12 kb. Evidence is also presented for an additional 5′ untranslated exon. The 90-aminoacid (aa) segment of CBF-A, which shows a high degree of sequence identity with the yeast transcription factor, HAP3, is split into exons 3 and 4. An alternatively spliced RNA that lacks exon 3 was identified by polymerase chain reaction. Although removal of exon 3 interrupts the CBF-A reading frame, a potential start codon at the 3′ end of exon 2 is in the same reading frame as the reading frame encoding CBF-A in exons 4 to 7. A CBF-A polypeptide of the predicted 17-kDa, size, was indeed identified after in vitro transcription and translation of the DNA complementary to RNA (cDNA) corresponding to the alternatively spliced CBF-A mRNA. In contrast to full-length CBF-A, this truncated CBFA did not bind to a DNA sequence containing the CCAAT motif in the presence of the other two components of CBF. This result indicates that the segment corresponding to the exons missing in the truncated isoform of CBF-A is essential for the binding of CBF to DNA.

Purification and functional characterization of a DNA-binding protein that interacts with a negative element in the mouse alpha 1(I) collagen promoter.

In an effort to understand the regulation of expression of the mouse type I collagen genes, we have purified to homogeneity a transcription factor, called inhibitory factor 2 (IF-2), that binds to the mouse alpha 1(I) collagen promoter upstream of the proximal CCAAT motif. IF-2 was purified to homogeneity from mouse lymphocyte nuclear extracts by ion-exchange chromatography and two different steps of DNA affinity chromatography, one using the wild-type IF-2-binding site as affinity ligand and one using a mutated IF-2-binding site that has a higher affinity for the factor. Renaturation of active DNA-binding proteins purified through several chromatography steps from sodium dodecyl sulfate-polyacrylamide gels identified two polypeptides of 120 and 100 kDa, respectively, capable of binding specifically to an IF-2-binding site. DNA transfection experiments of NIH 3T3 fibroblasts using an alpha 1(I) promoter-CAT chimeric gene in which mutations were introduced that either improved or decreased the binding affinity of IF-2 for its recognition site, strongly suggest that IF-2 acts as a transcriptional inhibitor of the mouse alpha 1(I) collagen gene. DNA-binding studies with similar concentrations of purified IF-2 and CBF (CCAAT-binding factor) indicate that CBF inhibits binding of IF-2, whereas IF-2 inhibits CBF binding more weakly.

Selective Activation of Transcription by a Novel CCAAT Binding Factor

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the alpha 2(I) collagen, the alpha 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.