Identification and characterization of a critical CP2-binding element in the human interleukin-4 promoter.

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

The pentanucleotide ATTGG, the "inverted CCAAT," is an essential element for HLA class I gene transcription.

The HLA class I genes, HLA-A, -B, and -C, contain an inverted CCAAT sequence (ATTGG) located 20 bp upstream of the canonical CCAAT and approximately 70 bp upstream of the transcription initiation site. We have investigated the transcriptional function of the class I inverted CCAAT sequence using the HLA-normal cell line, HeLa. Deletion, mutation, or inversion of the inverted CCAAT sequence abrogated or reduced the activity of the class I promoter, as assessed by luciferase reporter gene assays in transient gene expression experiments. This activity coincided with occupancy of the inverted CCAAT motif, as tested by electrophoretic mobility shift assays using the wild-type sequence and mutated variants of the sequence. The ATTGG-binding protein was not CP2, NF-1, or other known CCAAT-binding proteins, but the complex may contain a CP1/NF-Y-like protein. Our results indicate that this inverted CCAAT sequence is an essential element for the expression of HLA class I genes and that its transcriptional activity depends upon the sequence, position, and orientation of the pentanucleotide.

Evidence that levels of the dimeric cellular transcription factor CP2 play little role in the activation of the HIV-1 long terminal repeat in vivo or following superinfection with herpes simplex virus type 1.

The dimeric transcription factor CP2 binds a sequence element found near the transcription start site of the human immunodeficiency virus (HIV-1) long terminal repeat. Several groups have suggested that cellular factors binding this element might play a role in modulating HIV-1 promoter activity in vivo. For example, induction of latent HIV-1 gene expression in response to superinfection by herpes simplex virus type 1 (HSV-1) or cytomegalovirus is thought to be mediated, in part, by factors binding the CP2 site. In this report we began to examine directly the relationship between CP2 and expression of the HIV-1 promoter. First, we tested what effect HSV-1 infection of T cells had on the cellular levels of CP2. The results showed that HSV-1 infection led to a significant reduction in the level of CP2 DNA binding activity and protein within 20 h. Next, we tested the effect of overexpressing either the wild-type factor or a dominant negative variant of CP2 on HIV-1 promoter activity in vivo. The results showed that CP2 had little effect or slightly repressed HIV-1 promoter activity in vivo. In addition, these expression constructs had little effect on the induction of HIV-1 promoter activity elicited by HSV-1 infection.

PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter.

Expression of the low-affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type-specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis-acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3' purine tract (-50 to -39) within the MRR binds the macrophage and B cell-specific factor, PU.1, and a second E box-like element, termed MyE, upstream of the PU.1 box (-88 to -78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid-specific activity of the Fc gamma RIIIA promoter.

Characterization of the genomic structure, chromosomal location, promoter, and development expression of the alpha-globin transcription factor CP2.

We recently cloned murine and human cDNAs that encode CP2, a cellular transcription factor that interacts with the alpha-globin promoter as well as with additional cellular and viral promoter elements. We have now characterized the genomic structure, chromosome location, promoter, and expression pattern of the factor. Genes for the murine and human mRNAs contained 16 and 15 exons, respectively. Both genes spanned approximately 30 kilobases of chromosomal DNA, and among coding exons, all exon/intron boundaries were conserved. The human gene for CP2 was found to reside on chromosome 12 while the murine gene mapped to the distal end of chromosome 15, near Gdc-1, Wnt-1, and Rarg, a region syntenic with human chromosome 12. The murine and human promoters initiated mRNAs at multiple start sites in a conserved region that spanned more than 450 nucleotides. Lastly, a study of the pattern of CP2 gene expression showed that the factor was expressed in all adult and fetal murine tissues examined from at least day 9.5 of development.

DNA-binding proteins for transcription enhancing region of HLA class I gene.

Class I regulatory complex (CRC) located in the 5'-upstream region of MHC class I gene contains transcriptional enhancing sequences, called Enh A. This Enh A region contains tandem-arranged kappa B-like sites, one of which has a well-conserved perfect palindromic sequence. The second kappa B-like site, juxtaposed to the perfect palindrome, contains an imperfect palindromic sequence. In B-cell nuclear extracts, we have identified at least four sequence-specific protein complexes; three shared the repeated kappa B enhancer as their binding motifs. The perfect palindromic sequence facilities the binding of a complex termed BI, while the imperfect palindrome provides the binding sites for two other complexes, BII and BIII. The BII and BIII complexes exhibited binding crossreactivity with other kappa B-related motifs and recognized both the perfect and imperfect palindromic sequences, whereas the BI complex was specific for the perfect palindromic sequence which is unique to the class I promoters. A DNA segment outside the repeated kappa B enhancers probably binds the fourth complex, BIV. These complexes, except for the perfect palindrome-binding complex, differ from those described for the murine class I promoter. The binding characteristics of these factors suggest that the mechanism controlling the class I transcription may be quite complex.

Characterization of the molecularly cloned murine alpha-globin transcription factor CP2.

We recently cloned human and murine cDNAs that encode CP2, a transcription factor that interacts with the murine alpha-globin promoter. In this report, we exploited our ability to express CP2 in bacteria and eukaryotic cells to further investigate factor activities in vitro and in vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif that spans one DNA helix turn. In addition, the enriched bacterial protein activated transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. We then tested the effect of elevating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of the intact murine alpha-globin promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into these overexpressing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo.

Molecular cloning of the alpha-globin transcription factor CP2.

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.

Promoter elements and erythroid cell nuclear factors that regulate alpha-globin gene transcription in vitro.

We have previously purified four factors (alpha-IRP, alpha-CP1, alpha-CP2, and NF-E1) that interact with the promoter of the alpha-globin gene. One of these (NF-E1) is a tissue-restricted factor that has recently been cloned. The binding sites of these factors identify DNA sequence elements that might mediate the tissue-specific and inducible transcription of the alpha-globin gene. This possibility was tested in a series of in vitro transcription experiments. An examination of 5' truncated templates and synthetic promoters constituted from individual factor-binding sites apposed to the alpha-TATAA box showed that the binding elements of three factors (alpha-CP1, alpha-IRP, and NF-E1) mediate four- to sixfold activation of transcription in vitro. In contrast, one element (alpha-CP2) stimulated transcription less than twofold. The 5- to 10-fold stimulation of these latter templates upon addition of a DNA sequence affinity-purified factor suggests that alpha-CP2 is functionally limiting in nuclear extracts. Additional experiments further tested the effect of supplementing extracts with factors purified from erythroid cell nuclear extracts or, in the case of NF-E1, enriched from a bacterial cDNA expression system. Each factor tested stimulated transcription in vitro in a binding-site-dependent manner. Our results provide a comprehensive functional view of the murine alpha-globin promoter and suggest possible mechanisms for activation of alpha-globin gene transcription during induced differentiation of murine erythroleukemia cells.

Physical characterization of the purified CCAAT transcription factor, alpha-CP1.

We have used DNA sequence affinity chromatography previously to purify a murine erythroid cell nuclear factor termed alpha-CP1. This promoter selective transcription factor is a heterotypic CCAAT factor composed of at least seven polypeptides with Mr values that range from 27,000 to 38,000. Peptide mapping experiments reported here show that these seven polypeptides fall into three distinct classes (alpha, beta, and gamma). In addition chemical cross-linking, sedimentation, and gel filtration studies suggest that alpha-CP1 is a heterotrimeric factor composed of one polypeptide from each class. A core component of the factor (alpha beta) is stable at moderately high ionic strengths, whereas the gamma polypeptides are more weakly associated with the particle. The native factor binds tightly to the alpha-globin CCAAT box (Kd = 5.71 x 10(-11 M), and mutational studies show that the DNA recognition site resides in a sequence decamer. DNA binding is significantly stabilized, however, by apparently nonspecific sequences 3' of the CCAAT recognition motif. Finally, the DNA binding domain of purified alpha-CP1 is moderately stable to protease digestion, a feature characteristic of heterotypic CCAAT factors. The proteolyzed factor has a slightly higher affinity for the CCAAT box (Kd = 2.8 x 10(-11) M), and its footprint cannot be distinguished from that of the intact factor. In contrast protease treatment abolishes the ability of alpha-CP1 to activate alpha-globin gene transcription in vitro. These latter results show that the DNA binding domain of alpha-CP1 is readily distinguished from the domains required to mediate activation of gene transcription.

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene.

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity.

Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene.

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

The expression of integrated plasmid DNA depends on copy number.

The effect of copy number, integration site, and enhancers on the expression of stably integrated exogenous DNA was examined in Chinese hamster cells. Three similar plasmids were constructed with the mouse beta maj-globin promoter fused to the galK gene either with no enhancer or with the SV40 or Harvey sarcoma virus (HaSV) enhancer. Eighteen stable cell lines were obtained and characterized with respect to plasmid copy number and galactokinase activity. At copy numbers of four or less, the enhancers showed detectable activity and a DNase I hypersensitive site was present. Above four copies, gene activity decreased as the copy number increased, the enhancer sequences were apparently inactive, and the DNase I hypersensitive site disappeared. These data suggest that, at least in this model system, when exogenous DNA is integrated as multiple head-to-tail copies, the entire multigene unit expresses poorly and inappropriately. When the same exogenous DNA integrates as a single (or low number) copy, expression appears to be relatively normal as judged by enhancer stimulation and DNase I hypersensitivity.

Changes in gene expression during hexamethylene bisacetamide induced erythroleukemia differentiation.

HMBA induces MELC to terminal erythroid differentiation. The mechanism of HMBA action is not known. Culture with HMBA causes changes in gene expression which occur during the early "latent period", that is, prior to commitment to terminal differentiation. The inducer causes a decrease in diacylglycerol concentration, a decrease in Ca+2 and a decrease in phospholipid-dependent protein kinase C activity (within 2 hr) (Figure 2). There is an early suppression (within 1-2 hrs) of c-myb and c-myc gene transcription and an increase in c-fos mRNA (within 4 hrs). HMBA-induced commitment to terminal differentiation is detected by 12 hrs and over 95% become committed cells by 48 to 60 hrs. Commitment is associated with persistent suppression of c-myb gene transcription and elevated levels of c-fos mRNA whereas the level of c-myc mRNA returns to that of uninduced cells. By 36 to 48 hrs, transcription of alpha 1 and beta maj globin genes is increased 10 to 30 fold, while that of rRNA genes is suppressed. It is not yet clear how the protein products of proto-oncogenes elicit or modify cellular responses. Changes in expression of c-myb, c-myc, c-fos and p53 genes which occur during HMBA-induced differentiation, as well as in several other systems, suggest that products of these genes may have a role in regulating expression of multiple genes. One possible application of the established pattern of HMBA-induced modulation of gene expression during MELC differentiation may be in following the effects of cyto-differentiation agents during treatment of cancers. Phase I and Phase II chemical trials have been initiated to evaluate HMBA as a cytodifferentiation agent in human neoplasms (65). For most human tumors, assay for cytologic evidence of induced differentiation is difficult at best. Following the effects of a differentiation inducing agent by determining c-myc, or c-myb, mRNA levels may provide useful indicators of biological activity of HMBA and be a basis for evaluating whether continued administration of the agent is of interest in terms of potential clinical efficacy.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of transformed cells to terminal differentiation.

HMBA induces MEL cells to terminal erythroid differentiation. HMBA causes a decrease in diacylglycerol concentration, a decrease in Ca+2 and phospholipid-dependent protein kinase C activity (within 2 hr). There is an early (within 1-2 hrs) suppression of c-myb and c-myc gene transcription and an increase in c-fos mRNA (within 4 hrs). During the early or "latent" period there is no detectable commitment of MELC to terminal cell division or expression of differentiated genes such as alpha 1 or beta maj globin genes. HMBA-induced commitment to terminal differentiation is detected by 12 hrs and over 95% become committed cells by 48-60 hrs. Commitment is associated with persistent suppression of c-myb gene transcription and elevated levels of c-fos mRNA, whereas the level of c-myc mRNA returns to that of uninduced cells. By 36-48 hrs, transcription of the alpha 1 and beta maj globin genes increases 10-30 fold, and that of rRNA genes is suppressed. Changes in expression of c-myb, c-myc, c-fos and p53 genes that occur early during HMBA-induced differentiation may be important in the multistep process involved in commitment of MEL cells to terminal differentiation. Continued suppression of c-myb gene expression may be required for terminal differentiation of these cells.

Induced erythroleukemia differentiation: cellular and molecular aspects.

MELC may be induced to terminal erythroid differentiation by HMBA and other agents. Although the mechanism is not known, changes in cell function and gene expression can be identified during an early "latent" period, prior to commitment to terminal differentiation. These include a decrease in diacylglycerol concentration and in Ca+2 and phospholipid-dependent protein kinase C activity, accompanied by suppression of c-myb and c-myc gene transcription, a fall in p53 protein, and an increase in c-fos mRNA. Commitment is first detected by 12 hours and is associated with persistent suppression of c-myb gene transcription. Transcription of the erythroid-specific genes, alpha 1 and beta maj globin, is increased 10- to 30-fold, whereas synthesis of rRNA is suppressed, and there is activation or suppression of a number of additional genes that remain to be characterized. The potential regulatory roles of changes in protein kinase C activity and in proto-oncogene expression in initiating and sustaining the process of differentiation also remain to be elucidated.

Partial purification of a nuclear protein that binds to the CCAAT box of the mouse alpha 1-globin gene.

We enriched a fraction from nuclear extracts of murine erythroleukemia cells which contains a protein able to form stable complexes with the promoter region of the alpha 1-globin gene. Binding activity, which is present in mouse brain and a variety of cultured mouse and human cell lines, is not erythroid cell specific. Binding studies with alpha-globin gene promoter deletion mutants as well as DNase I footprinting and dimethyl sulfate protection studies showed that the factor bound specifically to the CCAAT box of the alpha 1 promoter. Enriched factor preparations exhibited weak binding to the promoter region of the beta maj-globin gene. This suggests that this protein could bind differentially to these two promoters in vivo. The enriched factor may be a ubiquitous nuclear protein involved in the differential regulation of the alpha 1- and beta maj-globin genes.