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.

Role of the CCAAT-binding protein CBF/NF-Y in transcription.

The CCAAT motif is one of the common promoter elements present in the proximal promoter of numerous mammalian genes transcribed by RNA polymerase II. CBF (also called NF-Y and CP1) consists of three different subunits and interacts specifically with the CCAAT motif. In each CBF subunit, the segment needed for formation of the CBF-DNA complex is conserved from yeast to human and, interestingly, the conserved segment of two CBF subunits, CBF-A and CBF-C, are homologous to the histone-fold motif of eukaryotic histones and archaebacterial histone-like protein HMf-2. The histone fold motifs of CBF-A and CBF-C interact with each other to form a heterodimer that associates with CBF-B to form a heterotrimeric CBF molecule, which then binds to DNA.

Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule.

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.

Transcriptional scaffold: CIITA interacts with NF-Y, RFX, and CREB to cause stereospecific regulation of the class II major histocompatibility complex promoter.

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.

Three classes of mutations in the A subunit of the CCAAT-binding factor CBF delineate functional domains involved in the three-step assembly of the CBF-DNA complex.

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.

An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

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.

Inhibition of FOXC2 restores epithelial phenotype and drug sensitivity in prostate cancer cells with stem-cell properties.

Advanced prostate adenocarcinomas enriched in stem-cell features, as well as variant androgen receptor (AR)-negative neuroendocrine (NE)/small-cell prostate cancers are difficult to treat, and account for up to 30% of prostate cancer-related deaths every year. While existing therapies for prostate cancer such as androgen deprivation therapy (ADT), destroy the bulk of the AR-positive cells within the tumor, eradicating this population eventually leads to castration-resistance, owing to the continued survival of AR-/lo stem-like cells. In this study, we identified a critical nexus between p38MAPK signaling, and the transcription factor Forkhead Box Protein C2 (FOXC2) known to promote cancer stem-cells and metastasis. We demonstrate that prostate cancer cells that are insensitive to ADT, as well as high-grade/NE prostate tumors, are characterized by elevated FOXC2, and that targeting FOXC2 using a well-tolerated p38 inhibitor restores epithelial attributes and ADT-sensitivity, and reduces the shedding of circulating tumor cells in vivo with significant shrinkage in the tumor mass. This study thus specifies a tangible mechanism to target the AR-/lo population of prostate cancer cells with stem-cell properties.

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-amino-acid (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.

Properties of B-ring analogues of colchicine.

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 lambda max 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.

Biochemical analysis of the B subunit of the heteromeric CCAAT-binding factor. A DNA-binding domain and a subunit interaction domain are specified by two separate segments.

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.

Pathway of complex formation between DNA and three subunits of CBF/NF-Y. Photocross-linking analysis of DNA-protein interaction and characterization of equilibrium steps of subunit interaction and dna binding.

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.

Stable expression of a dominant negative mutant of CCAAT binding factor/NF-Y in mouse fibroblast cells resulting in retardation of cell growth and inhibition of transcription of various cellular genes.

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.

The B subunit of a rat heteromeric CCAAT-binding transcription factor shows a striking sequence identity with the yeast Hap2 transcription factor.

CBF is a heteromeric mammalian transcription factor that binds to CCAAT sequences in a number of promoters such as the two type I collagen promoters, the albumin promoter, the major histocompatibility complex class II promoter, and others. It is composed of two components, A and B, that are both needed for DNA binding. We have isolated a rat cDNA containing the complete 341-amino acid coding sequence of the B component of CBF. Expression of this cDNA in vitro generates a polypeptide that shows the same dependency on the A component as the native B component in the formation of a complex with a CCAAT-containing DNA. The C-terminal portion of the B component from residue 260 to residue 312 shows a 75% sequence identity with a portion of the Hap2 protein, a component of a heteromeric CCAAT-binding protein in yeast. In contrast, the rest of the protein shows little sequence homology with Hap2, although both proteins contain glutamine-rich domains. In the B component of CBF this domain spans the amino-terminal 60% of the protein, whereas in Hap2 this domain is much smaller. Hence, only a few changes in one domain of this protein were tolerated during evolution between yeast and mammals, whereas the rest of the protein diverged much more extensively.

Purification and molecular cloning of the "A" chain of a rat heteromeric CCAAT-binding protein. Sequence identity with the yeast HAP3 transcription factor.

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.

Studies on transcription activation by the multimeric CCAAT-binding factor CBF.

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.

The two activation domains of the CCAAT-binding factor CBF interact with the dTAFII110 component of the Drosophila TFIID complex.

The CCAAT-binding factor CBF is a heterotrimeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. Previous studies have shown that CBF contains two transcription activation domains: a glutamine-rich, serine-threonine-rich domain present in the CBF-B subunit and a glutamine-rich domain in the CBF-C subunit. In this study, by using a series of deletion mutations of CBF-B and CBF-C in transcription assay in vitro, we further delineated smaller segments in these domains that were sufficient to support transcriptional activation by CBF. To test whether transcription activation by CBF requires co-activators, we examined the interaction between CBF and dTAF110, a component of the Drosophila TFIID complex. Recent work has demonstrated that glutamine-rich domains of the Sp1 transcription factor interact with dTAF110 and that this interaction has an important role in mediating transcription activation. Here we first demonstrate in a direct interaction assay in vitro that CBF binds dTAF110. By using a yeast two-hybrid system we show that both of the transcription activation domains of CBF interact with dTAF110. A deletion analysis suggests that a segment of CBF-B needed for transcription activation is also involved in interaction with dTAF110. In CBF-C the C-terminal portion of the molecule seems to be needed for these two activities. Our results suggest that TAF110 might represent one of the co-activators that mediate transcriptional activation by CBF.

Three different polypeptides are necessary for DNA binding of the mammalian heteromeric CCAAT binding factor.

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.

Chromosomal assignment and tissue expression of CBF-C/NFY-C, the third subunit of the mammalian CCAAT-binding factor.

The mammalian CCAAT-binding factor CBF (NFY) consists of three subunits, CBF-A, CBF-B, and CBF-C. All three subunits are evolutionarily conserved and are essential for DNA binding of CBF. In this study we report the identification of human and plant homologs of CBF-C. Northern analysis revealed that, like the other two subunits, CBF-C was produced at equal levels in all rat tissues that were examined. We assigned the mouse CBF-C gene (designated Nfyc) to chromosome 4 with tight linkage to Lmyc. Our mouse linkage data suggest that the human NFYC homolog will map to 1p32.