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A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

The CCAAT-binding factor CBF/NF-Y is needed for cell proliferation and early embryonic development. NF-Y can regulate the expression of different cell type-specific genes that are activated by various physiological signaling pathways. Dysregulation of NF-Y was observed in pathogenic conditions in humans such as scleroderma, neurodegenerative disease, and cancer. Conditional inactivation of the NF-YA gene in mice demonstrated that NF-Y activity is essential for normal tissue homeostasis, survival, and metabolic function. Altogether, NF-Y is an essential transcription factor that plays a critical role in mammalian development, from the early stages to adulthood, and in human pathogenesis. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2cre/Osxf/f/SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osxf/f/SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa.

The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression.

Previously the analysis of promoters regulated by endoplasmic reticulum (ER) stress identified a composite promoter element, ERSE that interacts with both CBF/NF-Y (CBF) and ATF6(N) transcription factors. This prompted us to investigate the underlying mechanism by which CBF, a ubiquitously binding transcription factor, specifically controls transcription activation during ER stress. The in vitro DNA binding study performed using purified recombinant proteins revealed that CBF specifically recruits ATF6(N) to ERSE DNA but it does not interact with ATF6(N) in absence of DNA binding. Inhibition of CBF binding resulted in a significant reduction of optimal transcription activation of cellular genes during ER stress. Analysis of cellular promoters by ChIP demonstrated that CBF is needed for recruitment of both ATF6(N) and TBP but not for either acetylation of histone H3-K9 or trimethylation of histone H3-K4 during ER stress. Together these study results reveal that CBF controls ER stress-inducible transcription through recruitment of both ATF6(N) and TBP but not through chromatin modifications. Our observations are in agreement with the results of recently published studies that have shown that CBF controls transcription of varieties of inducible promoters through recruitment of general transcription factors but not through acetylation of histone H4. These findings provide a paradigm of the function of CBF in inducible transcription. J. Cell. Biochem. 104: 1708–1723, 2008. © 2008 Wiley-Liss, Inc.

Roles for SOX9 have been extensively studied in development and particular emphasis has been placed on SOX9 roles in cell lineage determination in a number of discrete tissues. Aberrant expression of SOX9 in many cancers, including colorectal cancer, suggests roles in these diseases as well and recent studies have suggested tissue- and context-specific roles of SOX9. Our genome wide approach by chromatin immunoprecipitation sequencing (ChIP-seq) in human colorectal cancer cells identified a number of physiological targets of SOX9, including ubiquitously expressed cell cycle regulatory genes, such as CCNB1 and CCNB2, CDK1, and TOP2A. These novel high affinity-SOX9 binding peaks precisely overlapped with binding sites for histone-fold NF-Y transcription factor. Furthermore, our data showed that SOX9 is recruited by NF-Y to these promoters of cell cycle regulatory genes and that SOX9 is critical for the full function of NF-Y in activation of the cell cycle genes. Mutagenesis analysis and in vitro binding assays provided additional evidence to show that SOX9 affinity is through NF-Y and that SOX9 DNA binding domain is not necessary for SOX9 affinity to those target genes. Collectively, our results reveal possibly a context-dependent, non-classical regulatory role for SOX9.

To understand the role of the CCAAT binding factor (CBF) in transcription during the cell cycle, we studied the mouse topoisomerase IIα (topo IIα) promoter, which is activated during the late S and G2/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 IIα 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 vitroreconstituted transcription system shows that CBF activates transcription of the topo IIα 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 IIα 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 IIα 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 IIα 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 IIα 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 IIα promoter is a major function of CBF by which it regulates the cell cycle-dependent transcription of the topo IIα promoter and possibly many other cell cycle-regulated promoters containing CBF binding sites.

Androgen deprivation is the standard treatment for advanced prostate cancer (PCa), but most patients ultimately develop resistance and tumor recurrence. We found that MYB is transcriptionally activated by androgen deprivation therapy or genetic silencing of the androgen receptor (AR). MYB silencing inhibited PCa growth in culture and xenografts in mice. Microarray data revealed that c-Myb and AR shared a subset of target genes that encode DNA damage response (DDR) proteins, suggesting that c-Myb may supplant AR as the dominant regulator of their common DDR target genes in AR inhibition-resistant or AR-negative PCa. Gene signatures including AR, MYB, and their common DDR-associated target genes positively correlated with metastasis, castration resistance, tumor recurrence, and decreased survival in PCa patients. In culture and in xenograft-bearing mice, a combination strategy involving the knockdown of MYB, BRCA1, or TOPBP1 or the abrogation of cell cycle checkpoint arrest with AZD7762, an inhibitor of the checkpoint kinase Chk1, increased the cytotoxicity of the poly[adenosine 5'-diphosphate (ADP)-ribose] polymerase (PARP) inhibitor olaparib in PCa cells. Our results reveal new mechanism-based therapeutic approaches for PCa by targeting PARP and the DDR pathway involving c-Myb, TopBP1, ataxia telangiectasia mutated- and Rad3-related (ATR), and Chk1.

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 IIα (topo IIα) and α2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIα 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 IIα promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIα 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 IIα promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse α2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in anin 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 IIα 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 IIα promoter template.

The heterotrimeric CCAAT-binding factor CBF specifically interacts with the CCAAT motif present in the proximal promoters of numerous mammalian genes. To understand the in vivo function of CBF, a dominant negative mutant of CBF-B subunit that inhibits DNA binding of wild type CBF was stably expressed in mouse fibroblast cells under control of tetracycline-responsive promoter. Expression of the mutant CBF-B but not the wild-type CBF-B resulted in retardation of fibroblast cell growth. The analysis of cell growth using bromodeoxyuridine labeling showed that expression of the mutant CBF-B decreased the number of cells entering into S phase, and also delayed induction of S phase in the quiescent cells after serum stimulation, thus indicating that the inhibition of CBF binding prolonged the progression of S phase in fibroblasts. These results provide direct evidence for the first time that CBF is an important regulator of fibroblast growth. The inhibition of CBF binding reduced expression of various cellular genes including the alpha2(1) collagen, E2F1, and topoisomerase IIalpha genes which promoters contain the CBF-binding site. This result implied that expression of many other genes which promoters contain CBF-binding site was also decreased by the inhibition of CBF binding, and that the decreased expression of multiple cellular genes possibly caused the retardation of fibroblast cell growth.

We report that the heterotrimeric transcription factor NFY or “CAAT-binding factor” binds the −60 region of the human H ferritin promoter, the B site. DNA binding analysis with specific antibodies demonstrates that NFY/B/C subunits tightly bind this site and that NFY/C subunit is masked in vivo by binding with other protein(s). NFY binds the co-activator p300. Specifically, the NFY/B subunit interacts with the central segment of p300 in vivo and in vitro. cAMP substantially increases the formation of the NFY·p300 complex. Taken together these data provide a general model of cAMP induction of non-CRE-containing promoters and suggest that the NFY-B·p300 complex is located at the 5′ end of the promoter and the NFY-B·C·TFIIB on the 3′ end toward the transcription start site.

In this study, we used a photocross-linking method to identify specific contact of CCAAT-binding factor (CBF) subunits in a CBF-DNA complex. The analysis showed that all three subunits in the CBF-DNA complex were cross-linked to DNA and that CBF-B and CBF-C were cross-linked more strongly than CBF-A. None of the CBF-A and CBF-C subunits, which together formed a CBF-A/CBF-C heterodimer, were cross-linked without CBF-B; in contrast, CBF-B was cross-linked in the absence of CBF-A/CBF-C. No subunit of heterotrimeric CBF containing DNA-binding domain mutant of either CBF-B or CBF-C was cross-linked to DNA, and interestingly, cross-linking of CBF-B that occurred without CBF-A/CBF-C was inhibited in presence of mutant CBF-C/CBF-A heterodimer. Altogether, these results indicated that the specific DNA contact surface of each CBF subunit is generated as a result of interaction between CBF-B and CBF-A/CBF-C heterodimer and that the three CBF subunits interact interdependently with DNA to form a CBF-DNA complex. Equilibrium interactions among the three CBF subunits and between CBF subunits and DNA were studied by electrophoretic mobility shift assay. This showed that at equilibrium DNA-binding conditions, the CBF-A/CBF-C heterodimer is very stable, but association between CBF-B and CBF-A/CBF-C is very weak. The nature of the association of CBF-B with CBF-A/CBF-C was also revealed by studying the inhibition of CBF-DNA complex formation by the mutant CBF-B. This study indicated that the association between CBF-B and CBF-A/CBF-C is stabilized upon interaction with DNA, a process likely to favor formation of a high-affinity CBF-DNA complex.

Nuclear transcription factor-Y (NF-Y), a key regulator of cell-cycle progression, often loses its activity during differentiation into nonproliferative cells. In contrast, NF-Y is still active in mature, differentiated neurons, although its neuronal significance remains obscure. Here we show that conditional deletion of the subunit NF-YA in postmitotic mouse neurons induces progressive neurodegeneration with distinctive ubiquitin/p62 pathology; these proteins are not incorporated into filamentous inclusion but co-accumulated with insoluble membrane proteins broadly on endoplasmic reticulum (ER). The degeneration also accompanies drastic ER disorganization, that is, an aberrant increase in ribosome-free ER in the perinuclear region, without inducing ER stress response. We further perform chromatin immunoprecipitation and identify several NF-Y physiological targets including Grp94 potentially involved in ER disorganization. We propose that NF-Y is involved in a unique regulation mechanism of ER organization in mature neurons and its disruption causes previously undescribed novel neuropathology accompanying abnormal ubiquitin/p62 accumulation.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.

The CCAAT binding factor CBF is a heteromeric transcription factor, which binds to functional CCAAT motifs in many eukaryotic promoters. cDNAs for the A and B subunits of CBF (CBF-A and CBF-B) and for their yeast homologues HAP3 and HAP2 have been previously isolated, but the purified recombinant CBF-A and CBF-B together are unable to bind to CCAAT motifs in DNA. Here we report the isolation of a cDNA coding for rat CBF-C, demonstrate that recombinant CBF-C is required together with CBF-A and CBF-B to form a CBF-DNA complex, and show that CBF-C is present in this protein-DNA complex together with the other two subunits. We further show that CBF-C allows formation of a complex between the purified recombinant yeast HAP2 and HAP3 polypeptides and a CCAAT-containing DNA and is present in this complex, implying the existence of a CBF-C homologue in yeast. We show that CBF-A and CBF-C interact with each other to form a CBF-A-CBF-C complex and that CBF-B does not interact with CBF-A or CBF-C individually but that it associates with the CBF-A-CBF-C complex. Our results indicate that CBF is a unique evolutionarily conserved DNA binding protein.

Mutations in SOX9, a gene essential for chondrocyte differentiation cause the human disease campomelic dysplasia (CD). To understand how SOX9 activates transcription, we characterized the DNA binding and cell-free transcription ability of wild-type SOX9 and a dimerization domain SOX9 mutant. Whereas formation of monomeric mutant SOX9–DNA complex increased linearly with increasing SOX9 concentrations, formation of a wild-type SOX9–DNA dimeric complex increased more slowly suggesting a more sigmoidal-type progression. Stability of SOX9–DNA complexes, however, was unaffected by the dimerization mutation. Both wild-type and mutant SOX9 activated transcription of a naked Col2a1 DNA template. However, after nucleosomal assembly, only wild-type and not the mutant was able to remodel chromatin and activate transcription of this template. Using a cell line, in which the Col2a1 vector was stably integrated, no differences were seen in the interactions of wild-type and mutant SOX9 with the chromatin of the Col2a1 vector using ChIP. However, the mutant was unable to activate transcription in agreement with in vitro results. We hypothesize that the SOX9 dimerization domain is necessary to remodel the Col2a1 chromatin in order to allow transcription to take place. These results further clarify the mechanism that accounts for CD in patients harboring SOX9 dimerization domain mutations.

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.

Full-length cDNA clones for the CBF-A and CBF-B subunits of the CCAAT binding mammalian heteromeric transcription factor (CBF) have previously been isolated from both rat and mouse. Whereas recombinant CBF-B binds to DNA after complementation with a highly purified CBF-A fraction, recombinant CBF-A was unable to bind to DNA after complementation with either purified CBF-B or recombinant CBF-B. However, when recombinant CBF-A, synthesized as a fusion protein with glutathione S-transferase was denatured together with a highly purified fraction containing CBF-A in the presence of 5.5 M guanidine hydrochloride and subsequently renatured, the recombinant CBF-A bound to DNA after complementation with CBF-B. This binding of recombinant CBF-A could not be detected if recombinant CBF-A was not mixed during the denaturation-renaturation process together with the purified fraction containing the 32-kDa CBF-A. Using a Southwestern blot we demonstrated that a polypeptide of approximately 40 kDa, present in the purified CBF-A fraction, bound to DNA after complementation with both recombinant CBF-A and CBF-B. After fractionation of the purified CBF-A preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a species of approximately 40 kDa was eluted from the gel and shown to have DNA binding activity after complementation with both recombinant CBF-A and CBF-B. Our results indicate that a third polypeptide, designated CBF-C, forms a tight complex with CBF-A. Together with CBF-A and CBF-B, CBF-C is required for the DNA binding activity of CBF.

CCAAT-binding factors A (CBF-A) and B (CBF-B) are two subunits of the heteromeric CCAAT-binding factor. Portions of CBF-A and CBF-B have a high degree of amino acid sequence identity to segments of the HAP3 and HAP2 subunits of a yeast multimeric transcription factor. We show here that the subunits of CBF interact with each other in the absence of DNA binding. This interaction was revealed by cross-linking and coimmunoprecipitation studies. Both the DNA binding and subunit interaction functions of CBF-B have been examined by mutational analysis. A segment of 83 amino acids from residues 252 to 334, which corresponds to the evolutionarily conserved portion of CBF-B, is necessary and sufficient for CBF-A-dependent DNA binding. Carboxyl-terminal deletions of this segment (or mutations in arginine residues in this carboxyl-terminal part) abolish DNA binding, but do not alter subunit interactions between CBF-A and CBF-B. Mutations in hydrophobic amino acids within the amino-terminal part of the evolutionarily conserved sequence at positions 252-334 result in loss of both DNA binding and subunit interaction activities. Our results indicate that the evolutionarily conserved segment of CBF-B contains both DNA-binding and subunit interaction domains and that the integrity of both domains is essential for DNA binding.

CCAAT-binding factor (CBF) is a heteromeric mammalian transcription factor which binds to sequences containing a CCAAT motif in a number of promoters such as those for type I collagen, albumin, MHC Class II, beta-actin, and others. It consists of two different components that are both needed for DNA binding. We have purified the "A" chain of CBF to apparent homogeneity by sequence-specific DNA affinity chromatography followed by Mono S and Mono Q ion-exchange chromatography and obtained the amino acid sequences of tryptic peptides of this polypeptide. Amino acid sequences of two of these tryptic peptides were used to synthesize oligonucleotide primers. The primers served to obtain a small cDNA by the polymerase chain reaction method, which was then further used to obtain larger cDNA clones. DNA sequence analysis of a representative cDNA clone revealed the presence of an open reading frame of 207 amino acids coding for a putative polypeptide of 25 kDa. Transcription of these cDNAs in vitro followed by translation in a reticulocyte lysate produced a polypeptide that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility as the native A chain. The deduced amino acid sequence of the A chain showed a remarkable identity over a length of 90-amino acid residues with a sequence of the Hap3 polypeptide, a component of a heteromeric multisubunit yeast transcription 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.

Scaffold molecules interact with multiple effectors to elicit specific signal transduction pathways. CIITA, a non-DNA-binding regulator of class II major histocompatibility complex (MHC) gene transcription, may serve as a transcriptional scaffold. Regulation of the class II MHC promoter by CIITA requires strict spatial-helical arrangements of the X and Y promoter elements. The X element binds RFX (RFX5/RFXANK-RFXB/RFXAP) and CREB, while Y binds NF-Y/CBF (NF-YA, NF-YB, and NF-YC). CIITA interacts with all three. In vivo analysis using both N-terminal and C-terminal deletion constructs identified critical domains of CIITA that are required for interaction with NF-YB, NF-YC, RFX5, RFXANK/RFXB, and CREB. We propose that binding of NF-Y/CBF, RFX, and CREB by CIITA results in a macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.

CBF is a heterotrimeric protein that binds to DNA containing CCAAT motifs. Here we have analyzed interactions of recombinant CBF with DNA using hydroxyl radical footprinting and methylation interference assays. In the CBF-DNA complex, three separate DNA regions are protected from hydroxyl radical cleavage, one located over and immediately adjacent to the CCAAT motif itself and the other two located on both sides of the CCAAT motif. The methylation interference assay showed, however, that only in the CCAAT motif region methylation of bases was able to interfere with the formation of a CBF-DNA complex, suggesting that CBF makes sequence-specific contacts only in the CCAAT motif region. To further determine the specific DNA sequences necessary for CBF binding, we employed a polymerase chain reaction-mediated random binding site selection method. This analysis showed that CBF binding to DNA requires the CCAAT sequence and other specific sequences immediately flanking both ends of the CCAAT motif. We also showed that the nature of the flanking nucleotide sequences affects the affinity of CBF for DNA. Interestingly, most of the CCAAT motifs present in various higher eukaryotic promoters correspond to the CBF binding sites that were selected, consistent with the hypothesis that these motifs are binding sites for CBF and, hence, that CBF could regulate transcription of numerous eukaryotic genes.

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

CBF-A, CBF-B, and CBF-C together form the heterotrimeric mammalian CCAAT-binding factor, CBF, which binds to DNA to form a CBF-DNA complex. Here we examined the transcription activation function of CBF in an in vitro reconstituted system using the three purified recombinant CBF subunits expressed in Escherichia coli. Two of the subunits, CBF-A and CBF-C, were coexpressed and purified as a CBF-A/CBF-C complex. Addition of the three wild-type recombinant CBF subunits to EL4 cell nuclear extracts depleted of CBF stimulated transcription 5-20-fold from proalpha2(1) collagen promoters and 10-fold from the Rous sarcoma virus long terminal repeat. Two CBF deletion mutants, one containing full-length CBF-A and CBF-C and a CBF-B lacking the NH2-terminal residues 1-224, and the other containing full- length CBF-A and CBF-B and a CBF-C lacking the COOH-terminal residues 114-309, also stimulated transcription from these promoters, but the level of activation was reduced to half that obtained with the full-length CBF subunits. In contrast, a CBF deletion mutant protein containing full-length CBF-A and deleted forms of both CBF-B and CBF-C showed very little transcription activation from these promoters. Hence, this study demonstrates that the heterotrimeric CBF protein consists of two transcription activation domains, one present in CBF-B and the other in CBF-C, and that the two domains act additively in the in vitro assay. The activation domains of both CBF-B and CBF-C, which are rich in glutamine and hydrophobic residues, showed amino acid sequence similarities with each other and with the glutamine-rich activation domain of transcription factor Sp1.

The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex. In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit. This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals. Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex. The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C. Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2. We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.

Publisher Summary Several proteins have been isolated that bind to CCAAT motifs. The CCAAT-binding factor (CBF; also designated NF-Y and CP1) is one of these and is capable of forming a specific complex with a number of promoter DNAs containing a CCAAT motif. Point mutations in each base of the CCAAT motif of these DNAs resulted in either loss or decrease in formation of the DNA-CBF complex, indicating that DNA binding of CBF requires a high degree of conservation of the CCAAT sequence. Moreover, mutations in the CCAAT motif that inhibit formation of the CBF-DNA complex also resulted in reduced transcription activity. These and other results indicated that CBF is a transcription factor that can activate various eukaryotic genes. Other CCAAT-binding polypeptides include CTF/NF1 and C/EBP, but the DNA sequences in the binding sites for these two proteins often do not contain a complete CCAAT motif. Hence, CBF can be distinguished from these two proteins on the basis of the sequence requirements in the DNA-binding sites.

The CCAAT-binding factor CBF is a heteromeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. CBF consists of three subunits, CBF-A, CBF-B, and CBF-C, all three of which are necessary for DNA binding. In this study we examined the transcription activation function of CBF by two different approaches. We first used a heterologous system in which a series of deletion mutations of CBF-B, fused to the bacterial LexA DNA binding domain, were transfected into HeLa cells together with a reporter gene driven by a minimal promoter containing LexA binding sites. These experiments showed that CBF-B needed both a glutamine-rich domain and an adjacent serine/threonine-rich domain to activate the reporter gene optimally. The glutamine-rich domain by itself activated transcription only modestly. We also set up an in vitro transcription reconstituted system in which trans-activation by CBF occurred through a physiological CCAAT motif. Nuclear extracts from NIH 3T3 cells were first depleted of CBF and then complemented with recombinant CBF-B and a highly purified fraction containing native CBF-A and CBF-C. Recombinant full-length CBF-B together with CBF-A and CBF-C activated transcription of several alpha 2(I) collagen gene promoter constructs. We then tested whether in this system the glutamine- and serine/threonine-rich domains of CBF-B were needed for trans-activation by CBF. We generated a truncated form of CBF-B that was still able to bind DNA in the presence of CBF-A and CBF-C. Even in the absence of the glutamine- and serine/threonine-rich domains of CBF-B, reconstituted CBF did activate transcription, suggesting that CBF transcriptional activation can also be mediated by the other subunits of CBF or by another transcription factor present in the nuclear extracts that interacts with CBF. Taken together our results suggest a model in which CBF has the potential to activate transcription either through the glutamine- and serine/threonine-rich domains of CBF-B or through the other subunits of CBF or through another component recruited by CBF.

Absorption spectra of colchicine and its analogues are affected by the presence of the B-ring, although it is not part of the chromophore (C-ring). Thus, 2-methoxy-5-(2′,3′4′-trimethoxyphenyl)tropone has absorption maxima at 341 nm, whereas that of desacetamidocolchicine is at 353 nm. A similar red shift in the λmax of colchicine, desacetamidocolchicine and 2-methoxy-(2′,3′,4′-trimethoxyphenyl)tropone also occurs when they are immobilized in the binding site to tubulin or in pure glycerol. We also observed that the B-ring of colchicine alone or with substituent does not affect the UV-induced rearrangement of colchicine to lumicolchicine. However, in the absence of the B-ring, as in the case of 2-methoxy-5-(2′,3′4′-trimethoxyphenyl)tropone, the rearrangement reaction of the C-ring slows down significantly.

Taxol-induced assembly of purified tubulin is not inhibited by the colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone. Colchicine analogues having intact A, C and B-rings (without NH-CO-CH3) such as desacetamidocolchicine have also been found to be inactive. It has been observed that these two colchicine analogues are incorporated into polymers when incubated in the presence of taxol. Furthermore, preformed taxol-induced polymers of tubulin have been found to bind these two colchicine analogues. These results suggest that colchicine-binding domains on the tubulin molecule are mostly (if not completely) exposed in the taxol-induced polymers.