Cognate DNA binding specificity retained after leucine zipper exchange between GCN4 and C/EBP.

Both C/EBP and GCN4 are sequence-specific DNA binding proteins that control gene expression. Recent evidence implicates C/EBP as a transcriptional regulator of genes involved in lipid and carbohydrate metabolism. The C/EBP protein binds avidly to the dyad symmetric sequence 5'-ATTGCGCAAT-3'; GCN4 regulates the transcription of genes that control amino acid biosynthesis in yeast, and binds avidly to the dyad symmetric sequence 5'-ATGA(G/C)TCAT-3'. Both C/EBP and GCN4 bind DNA via the same structural motif. This motif has been predicted to be bipartite, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." Specificity of DNA binding has been predicted to be imparted by the basic region. As a test of this hypothesis, recombinant proteins were created wherein the basic regions and leucine zippers of GCN4 and C/EBP were reciprocally exchanged. In both of the recombinant polypeptides, DNA binding specificity is shown to track with the basic region.

Cloning of yeast transfer RNA genes in Escherichia coli.

Four thousand Escherichia coli clones containing yeast DNA inserted into the plasmid pBR313 have been isolated. Of these, 175 clones were identified as carrying yeast transfer RNA genes. The initial analysis of the inserted transfer RNA genes via the colony hybridization technique with individual radioactive transfer RNA species is reported. The data indicate that yeast transfer RNA genes are not highly clustered, although some clustering exists. In addition, it was observed that the reiteration number of different transfer RNA genes may vary extensively.

The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins.

A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.

The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite.

C/EBP is a rat liver nuclear protein capable of sequence-specific interaction with DNA. The DNA sequences to which C/EBP binds in vitro have been implicated in the control of messenger RNA synthesis. It has therefore been predicted that C/EBP will play a role in regulating gene expression in mammalian cells. The region of the C/EBP polypeptide required for direct interaction with DNA has been identified and shown to bear amino acid sequence relatedness with the product of the myc, fos, and jun proto-oncogenes. The arrangement of these related amino acid sequences led to the prediction of a new structural motif, termed the "leucine zipper," that plays a role in facilitating sequence-specific interaction between protein and DNA. Experimental tests now provide support for the leucine zipper hypothesis.

Directed deletion of a yeast transfer RNA intervening sequence.

Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA (tRNA Tyr) suppressor gene was deleted in order to test its role in the expression of the gene. The altered gene and its parent were introduced into yeast by transformation. Both genes exhibited suppressor function, showing that the intervening sequence is not absolutely essential for the expression of this gene.

Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference.

C/EBP and GCN4 are basic region-leucine zipper (bZIP) DNA-binding proteins that recognize the dyad-symmetric sequences ATTGCGCAAT and ATGAGTCAT, respectively. The sequence specificities of these and other bZIP proteins are determined by their alpha-helical basic regions, which are related at the primary sequence level. To identify amino acids that are responsible for the different DNA sequence specificities of C/EBP and GCN4, two kinds of hybrid proteins were constructed: GCN4-C/EBP chimeras fused at various positions in the basic region and substitution mutants in which GCN4 basic region amino acids were replaced by the corresponding residues from C/EBP. On the basis of the DNA-binding characteristics of these hybrid proteins, three residues that contribute significantly to the differences in C/EBP and GCN4 binding specificity were defined. These residues are clustered along one face of the basic region alpha helix. Two of these specificity residues were not identified as DNA-contacting amino acids in a recently reported crystal structure of a GCN4-DNA complex, suggesting that the residues used by C/EBP and GCN4 to make base contacts are not identical. A random binding site selection procedure also was used to define the optimal recognition sequences for three of the GCN4-C/EBP fusion proteins. These experiments identify an element spanning the hinge region between the basic region and leucine zipper domains that dictates optimal half-site spacing (either directly abutted for C/EBP or overlapping by one base pair for GCN4) in high-affinity binding sites for these two proteins.

Interleukin-6-specific activation of the C/EBPdelta gene in hepatocytes is mediated by Stat3 and Sp1.

C/EBPdelta (CCAAT/enhancer binding protein delta) has been implicated as a regulator of acute-phase response (APR) genes in hepatocytes. Its expression increases dramatically in liver during the APR and can be induced in hepatic cell lines by interleukin-6 (IL-6), an acute-phase mediator that activates transcription of many APR genes. Here we have investigated the mechanism by which C/EBPdelta expression is regulated by IL-6 in hepatoma cells. C/EBPdelta promoter sequences to -125 bp are sufficient for IL-6 inducibility of a reporter gene and include an APR element (APRE) that is essential for IL-6 responsiveness. DNA binding experiments and transactivation assays demonstrate that Stat3, but not Stat1, interacts with this APRE. Two Sp1 sites, one of which is adjacent to the APRE, are required for IL-6 induction and transactivation by Stat3. Thus, Stat3 and Sp1 function cooperatively to activate the C/EBPdelta promoter. Replacement of the APRE with Stat binding elements (SBEs) from the ICAM-1 or C/EBPbeta promoter, both of which recognize both Stat1 and Stat3, confers responsiveness to gamma interferon, a cytokine that selectively activates Stat1. Sequence comparisons suggest that the distinct Stat binding specificities of the C/EBPdelta and C/EBPbeta SBEs are determined primarily by a single base pair difference. Our findings indicate that the cytokine specificity of C/EBPdelta gene expression is governed by the APRE sequence.

Disruption of the c/ebp alpha gene in adult mouse liver.

The liver-enriched transcription factor C/EBP alpha has been implicated in the regulation of numerous liver-specific genes. It was previously reported that mice carrying a homozygous null mutation at the c/ebp alpha locus died as neonates due to the absence of hepatic glycogen and the resulting hypoglycemia. However, the lethal phenotype precluded further analysis of the role of C/EBP alpha in hepatic gene regulation in adult mice. To circumvent this problem, we constructed a conditional knockout allele of c/ebp alpha by using the Cre/loxP recombination system. Homozygous c/ebp-loxP mice, (c/ebp alpha(fl/fl);fl, flanked by loxP sites) were found to be indistinguishable from their wild-type counterparts. However, when Cre recombinase was delivered to hepatocytes of adult c/ebp alpha(fl/fl) mice by infusion of a recombinant adenovirus carrying the cre gene, more than 80% of the c/ebp alpha(fl/fl) genes were deleted specifically in liver and C/EBP alpha expression was reduced by 90%. This condition resulted in a reduced level of bilirubin UDP-glucuronosyltransferase expression in the liver. After several days, the knockout mice developed severe jaundice due to an increase in unconjugated serum bilirubin. The expression of genes encoding phosphoenolpyruvate carboxykinase, glycogen synthase, and factor IX was also strongly reduced in adult conditional-knockout animals, while the expression of transferrin, apolipoprotein B, and insulin-like growth factor I genes was not affected. These results establish C/EBP alpha as an essential transcriptional regulator of genes encoding enzymes involved in bilirubin detoxification and gluconeogenesis in adult mouse liver.

DNA-binding specificity of the PAR basic leucine zipper protein VBP partially overlaps those of the C/EBP and CREB/ATF families and is influenced by domains that flank the core basic region.

The PAR subfamily of basic leucine zipper (bZIP) factors comprises three proteins (VBP/TEF, DBP, and HLF) that have conserved basic regions flanked by proline- and acidic-amino-acid-rich (PAR) domains and functionally compatible leucine zipper dimerization domains. We show that VBP preferentially binds to sequences that consist of abutted GTAAY half-sites (which we refer to as PAR sites) as well as to sequences that contain either a C/EBP half-site (GCAAT) or a CREB/ATF half-site (GTCAT) in place of one of the PAR half-sites. Since the sequences that we describe as PAR sites and PAR-CREB/ATF chimeric sites, respectively, were both previously described as high-affinity binding sites for the E4BP4 transcriptional repressor, we infer that these sequences may be targets for positive and negative regulation. Similarly, since the sequences that we describe as PAR-C/EBP and PAR-CREB/ATF chimeric sites are known to be high-affinity binding sites for C/EBP and CREB/ATF factors, respectively, we infer that these sites may each be targets for multiple subfamilies of bZIP factors. To gain insights regarding the molecular basis for the binding-site specificity of PAR factors, we also carried out an extensive mutational analysis of VBP. By substituting five amino acid residues that differ between the Drosophila giant bZIP factor and the vertebrate PAR bZIP factors, we show that the fork region, which bridges the basic and leucine zipper domains, contributes to half-site sequence specificity. In addition, we report that at least two domains amino terminal to the core basic region are required for VBP to bind to the full spectrum of PAR target sites. Thus, whereas direct base contacts may be restricted to basic-region residues (as indicated by GCN4-DNA crystal structures), several other domains also influence the DNA-binding specificity of PAR bZIP proteins.

The ability of C/EBP beta but not C/EBP alpha to synergize with an Sp1 protein is specified by the leucine zipper and activation domain.

The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. We previously showed that liver-specific transcription of the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5, that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBP alpha and C/EBP beta are expressed abundantly in liver, only C/EBP beta is capable of stimulating the 2D5 promoter in HepG2 hepatocarcinoma cells. In addition, activation of the 2D5 promoter by C/EBP beta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBP beta proteins containing either the leucine zipper or the activation domain of C/EBP alpha are unable to stimulate the 2D5 promoter yet are fully capable of transactivating an artificial promoter bearing a high-affinity C/EBP site. Thus, the leucine zipper and the activation domain of C/EBP beta are absolutely required to support transactivation of the 2D5 promoter. Using Drosophila cells that lack endogenous Sp1 activity, we show that the serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperatively with C/EBP beta. Furthermore, analysis of c/ebp beta-deficient mice shows that mutant animals are defective in expression of a murine CYP2D5 homolog in hepatic cells, confirming the selective ability of C/EBP beta to activate this liver-specific P-450 gene in vivo. Our findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein.

A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-kappaB p50.

Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-alpha. This activity, termed TNF-alpha-inhibiting factor (TIF), suppressed the induction of TNF-alpha expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-alpha expression by macrophage conditioned medium was associated with selective induction of the NF-kappaB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-alpha promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-alpha gene. Repression of the TNF-alpha promoter by TIF required a distal region that includes three NF-kappaB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-alpha promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-alpha expression in activated macrophages. TIF is distinct from the known TNF-alpha-inhibiting factors IL-4, IL-10, and transforming growth factor beta and may represent a novel cytokine.

C/EBPbeta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression.

The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPbeta within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPbeta in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPbeta-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPbeta-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPbeta-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPbeta plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

C/EBPbeta, when expressed from the C/ebpalpha gene locus, can functionally replace C/EBPalpha in liver but not in adipose tissue.

Knockout of C/EBPalpha causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBPalpha-null mouse strain in which C/EBPbeta, in addition to its own expression, substituted for C/EBPalpha expression in tissues. The homozygous mutant mice C/ebpalpha(beta/beta) are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPalpha-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebpalpha(beta/beta) and wild-type mice. However, despite their normal growth rate, C/ebpalpha(beta/beta) mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebpalpha(beta/beta) mice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. Our study demonstrates that when expressed from the C/ebpalpha gene locus, C/EBPbeta can act for C/EBPalpha to maintain liver functions during development. Moreover, our studies with the C/ebpalpha(beta/beta) mice provide new insights into the nonredundant functions of C/EBPalpha and C/EBPbeta on gene regulation in WAT.

Role of the transcription factor C/EBP beta in expression of a rat pregnancy-specific glycoprotein gene.

Pregnancy-specific glycoproteins (PSGs), which are the major placental proteins, and the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. To understand the molecular mechanisms underlying the control of PSG expression, we characterized the promoter elements of a rodent PSG gene, rnCGM3, and showed that DNA elements at nucleotides -326 to -185 (PI) relative to the translation start site of rnCGM3 function as a promoter. The rnCGM3 PI promoter contains two placental factor binding sites, PISI and PISII. Both are transcription activation elements. In the present report, we screened a placental expression cDNA library with a rnCGM3-PISII probe (nucleotides -263 to -233) encompassing two overlapping palindromes (TGTTGCTCAACATGTTG) and demonstrated that the PISII-binding factor is C/EBP beta, a leucine zipper family of transcription factor. Gel mobility-shift and transient expression analyses showed that C/EBP beta and C/EBP isoforms, C/EBP alpha and C/EBP delta, bind to the PISII element and trans-activate rnCGM3 gene expression. Deletion of PISII from the rnCGM3 PI promoter greatly reduced the basal as well as the C/EBP-activated rnCGM3 expression. Gel supershift assays demonstrated that C/EBP beta is the placental isoform that binds to the PISII site rnCGM3. Moreover, C/EBP beta is expressed in high levels in the placenta, ovary, liver, lung, heart, and spleen, in contrast to C/EBP alpha, which is expressed primarily in the liver and only low levels in the placenta. Our results demonstrate that C/EBP beta is one of the transcription factors that positively regulate rnCGM3 expression during pregnancy.

C/EBP proteins contain nuclear localization signals imbedded in their basic regions.

The C/EBP-related proteins (C/EBPalpha, CRP1, C/EBPbeta, and C/EBPdelta) form a subfamily of bZIP (basic region/leucine zipper) transcription factors that display sequence homology within the bZIP domain. The conserved basic region contains two motifs that exhibit significant homology to the bipartite nuclear localization signal (NLS) first described in nucleoplasmin. CRP1 and C/EBPbeta proteins bearing deletions of the basic region accumulate in the cytoplasm, in contrast to their normal nuclear location. Analysis of chimeric proteins consisting of CRP1 basic region sequences fused to beta-galactosidase revealed that the CRP1 basic region contains a single NLS that differs from conventional bipartite signals in two ways. First, mutation of a pair of arginine residues at the N-terminus of the proposed NLS does not disrupt its function. Second, the CRP1 NLS requires additional nonbasic residues at its C-terminus. A basic residue within the CRP1 NLS that is not conserved within the C/EBP family is occupied instead by an uncharged residue in C/EBPalpha and C/EBPbeta. When this nonconserved arginine residue was changed to alanine the CRP1 NLS behaved as a classical bipartite signal, suggesting that bipartite NLSs are present in all family members but that NLSs of the individual members differ slightly. Additionally, mutation of critical NLS residues in the intact CRP1 and C/EBPbeta proteins showed that these elements exhibit more bipartite-like characteristics when present in their normal sequence context. Finally, we observed that a C/EBPbeta protein lacking its NLS can be localized to the nucleus when coexpressed with C/EBPalpha, indicating that a single NLS is sufficient to promote nuclear transport of a bZIP dimer.

Prosthodontics. Diagnostic, treatment planning, and prognostic considerations.

The prosthodontic rehabilitation of a patient is usually the culmination of a patient's dental treatment. In order to maximize the prognosis, it is necessary to understand the patient, to make a thorough diagnosis, to coordinate the restoration with the other disciplines of dentistry, and to be knowledgeable of the spectrum of treatment modalities available.