A multiplicity of CCAAT box-binding proteins

NF-Y is a sequence-specific DNA-binding protein that recognizes the Y box, a promoter element common to all major histocompatibility complex class II genes. Since the 14-base Y element harbors a CCAAT box in reverse, we were prompted to ask whether NF-Y is actually a CCAAT box-binding protein and whether it is related to the previously described CCAAT-binding factors CBP and CTF/NF-I. Data from gel retardation, methylation interference, saturation mutagenesis, and cross-competition experiments establish definitively that NF-Y is an entirely distinct CCAAT box-binding entity. Moreover, these experiments have uncovered a fourth CCAAT-binding protein, NF-Y(star) that interacts with the thymidine kinase promoter. Clearly, then, there exists a multiplicity of factors that recognize CCAAT sequences; it now becomes imperative to understand the functional significance of this multiplicity.     

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.     

The -117 mutation in Greek HPFH affects the binding of three nuclear factors to the CCAAT region of the gamma-globin gene.

The Greek form of hereditary persistence of fetal hemoglobin (HPFH) is associated with a point mutation immediately upstream of the distal of the two CCAAT elements of the A gamma-globin gene. Three proteins present in nuclear extracts of erythroleukemia cells bind to this CCAAT region and contact the nucleotide mutated in Greek HPFH. The ubiquitous CCAAT-binding factor CP1 interacts preferentially with the proximal CCAAT sequence. An erythroid cell-specific factor, referred to as NF-E, binds with a higher affinity to the distal CCAAT region and interacts only with sequences flanking the CCAAT motif. The third protein is the vertebrate homologue of the sea urchin CCAAT displacement protein and recognizes sequences in both CCAAT elements and their flanking sequences. While the point mutation in Greek HPFH slightly strengthens the binding of CP1 and the CCAAT displacement protein, the same base change strongly reduces the binding of NF-E to the distal CCAAT region, suggesting a possible role of NF-E in the repression of gamma-globin genes in adult erythroid cells.     

Purification of nuclear factor I by DNA recognition site affinity chromatography

Nuclear factor I (NF-I) is a cellular protein that enhances the initiation of adenovirus DNA replication in vitro. The enhancement of initiation correlates with the ability of NF-I to bind a specific nucleotide sequence within the viral origin of replication. We have developed a method for the purification of NF-I which is based upon the high affinity interaction between the protein and its recognition site. This approach may be generally applicable to the purification of other site-specific DNA binding proteins. The essential feature of the method is a two-step column chromatographic procedure in which proteins are first fractionated on an affinity matrix consisting of nonspecific (Escherichia coli) DNA and then on a matrix that is highly enriched in the specific DNA sequence that is recognized by NF-I. During the first step NF-I coelutes with proteins that have similar general affinity for DNA. During the second step NF-I elutes at a much higher ionic strength than the contaminating nonspecific DNA binding proteins. The DNA recognition site affinity matrix used in the second step is prepared from a plasmid (pKB67-88) that contains 88 copies of the NF-I binding site. This plasmid was constructed by means of a novel cloning strategy that generates concatenated NF-I binding sites arranged exclusively in a direct head to tail configuration. Using this purification scheme, we have obtained a 2400-fold purification of NF-I from crude HeLa nuclear extract with a 57% recovery of specific DNA binding activity. Throughout the purification procedure NF-I retained the ability to enhance the efficiency of initiation of adenovirus DNA replication in vitro. Electrophoresis of the purified fraction on sodium dodecyl sulfate-polyacrylamide gels revealed a population of related polypeptides that ranged in apparent molecular weight from 66,000 to 52,000. The native molecular weight of NF-I deduced from gel filtration and glycerol sedimentation studies is 55,000 and the frictional ratio is 1.3. These results suggest that NF-I exists as a globular monomer in solution.     

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

Peroxisome proliferator-activated receptor gamma suppresses proximal alpha1(I) collagen promoter via inhibition of p300-facilitated NF-I binding to DNA in hepatic stellate cells

Depletion of peroxisome proliferator-activated receptor gamma (PPARgamma) represents one of the key molecular changes that underlie transdifferentiation (activation) of hepatic stellate cells in the genesis of liver fibrosis (Miyahara, T., Schrum, L., Rippe, R., Xiong, S., Yee, H. F., Jr., Motomura, K., Anania, F. A., Willson, T. M., and Tsukamoto, H. (2000) J. Biol. Chem. 275, 35715-35722; Hazra, S., Xiong, S., Wang, J., Rippe, R. A., Krishna, V., Chatterjee, K., and Tsukamoto, H. (2004) J. Biol. Chem. 279, 11392-11401). In support of this notion, ectopic expression of PPARgamma suppresses hepatic stellate cells activation markers, most notably expression of alpha1(I) procollagen. However, the mechanisms underlying this antifibrotic effect are largely unknown. The present study utilized deletion-reporter gene constructs of proximal 2.2-kb alpha1(I) procollagen promoter to demonstrate that a region proximal to -133 bp is where PPARgamma exerts its inhibitory effect. Within this region, two DNase footprints with Sp1 and reverse CCAAT box sites exist. NF-I, but not CCAAT DNA-binding factor/NF-Y, binds to the proximal CCAAT box in hepatic stellate cells. A mutation of this site almost completely abrogates the promoter activity. NF-I mildly but independently stimulates the promoter activity and synergistically promotes Sp1-induced activity. PPARgamma inhibits NF-I binding to the most proximal footprint (-97/-85 bp) and inhibits its transactivity. The former effect is mediated by the ability of PPARgamma to inhibit p300-facilitated NF-I binding to DNA as demonstrated by chromatin immunoprecipitation assay.     

High affinity binding site for nuclear factor I next to the hepatitis B virus S gene promoter.

The hepatitis B virus (HBV) surface antigen (HBsAG) is encoded by the S gene under the regulation of a promoter in the pre-S1 region. The S gene promoter does not contain a 'TATA' box-like sequence, but there is a sequence resembling, in part, the late promoter of Simian virus 40 (SV40). In an attempt to study the regulation of the S gene promoter we looked for cellular proteins which bind to this region. We report here that a nuclear protein is tightly bound to the HBV genome at a position approximately 190 bases upstream from the S gene promoter. Evidence is provided to show that (a) this nuclear protein is the nuclear factor I (NF-I) that was previously found to be bound to the inverted terminal repeat of the adenovirus (Ad) DNA and to enhance Ad DNA replication in vitro and (b) this NF-I binding site is required for optimal activity of the S gene promoter.     

Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain.

We describe a detailed genetic analysis of the DNA-binding regions in the HAP2/HAP3 CCAAT-binding heteromeric complex. The DNA-binding domain of HAP2 is shown to be a 21 residue region containing three critical histidines and three critical arginines. Mutation of an arginine at position 199 to leucine alters the DNA-binding specificity of the complex to favor CCAAC over CCAAT. Residues in HAP3 that are critical for DNA-binding comprise a short, seven amino acid region. Three different mutations in the HAP2 DNA-binding domain are suppressed by a mutation in the HAP3 DNA-binding domain. This HAP3 mutation also suppresses mutations in a different region of HAP2 which promotes subunit assembly of the complex. These findings suggest that short regions of HAP2 and HAP3 comprise a hybrid DNA-binding domain and that this domain can help hold the two subunits together in the CCAAT-binding complex.