Repression of major histocompatibility complex IA expression by glucocorticoids: the glucocorticoid receptor inhibits the DNA binding of the X box DNA binding protein.

Glucocorticoids are effective repressors of major histocompatibility complex (MHC) class II gene expression. The repression occurs in B cells, which constitutively express MHC class II, as well as in macrophages, which only express MHC class II after the cells are treated with interferon gamma. For the MHC class II gene IA beta, this negative regulation has been linked to the X box DNA sequence, located with the IA beta promoter. The addition of the glucocorticoid receptor was shown to inhibit the DNA binding of the X box DNA binding protein to the X box. The DNA binding of two other DNA binding proteins that recognize elements within this promoter was unaffected by the addition of glucocorticoid receptor. It is likely that the repression of IA beta gene expression by glucocorticoids occurs because the X box DNA binding protein is prevented from binding to the DNA and activating transcription.

The transcription factor PU.1 is involved in macrophage proliferation.

PU.1 is a tissue-specific transcription factor that is expressed in cells of the hematopoietic lineage including macrophages, granulocytes, and B lymphocytes. Bone marrow-derived macrophages transfected with an antisense PU.1 expression construct or treated with antisense oligonucleotides showed a decrease in proliferation compared with controls. In contrast, bone marrow macrophages transfected with a sense PU.1 expression construct displayed enhanced macrophage colony-stimulating factor (M-CSF)-dependent proliferation. Interestingly, there was no effect of sense or antisense constructs of PU.1 on the proliferation of the M-CSF-independent cell line, suggesting that the response was M-CSF dependent. This was further supported by the finding that macrophages transfected with a sense or an antisense PU.1 construct showed, respectively, an increased or a reduced level of surface expression of receptors for M-CSF. The enhancement of proliferation seems to be selective for PU.1, since transfections with several other members of the ets family, including ets-2 and fli-1, had no effect. Various mutants of PU.1 were also tested for their ability to affect macrophage proliferation. A reduction in macrophage proliferation was found when cells were transfected with a construct in which the DNA-binding domain of PU.1 was expressed. The PEST (proline-, glutamic acid-, serine-, and threonine-rich region) sequence of the PU.1 protein, which is an important domain for protein-protein interactions in B cells, was found to have no influence on PU.1-enhanced macrophage proliferation when an expression construct containing PU.1 minus the PEST domain was transfected into bone marrow-derived macrophages. In vivo, PU.1 is phosphorylated on several serine residues. The transfection of plasmids containing PU.1 with mutations at each of five serines showed that only positions 41 and 45 are critical for enhanced macrophage proliferation. We conclude that PU.1 is necessary for the M-CSF-dependent proliferation of macrophages. One of the proliferation-relevant targets of this transcription factor could be the M-CSF receptor.

Characterization of a cis-acting regulatory element which silences expression of the class II-A beta gene in epithelium.

Class II major histocompatibility complex (MHC) genes encode for alpha/beta chain pairs that are constitutively expressed principally on mature B cells and dendritic cells in mice. These gene products are easily induced on macrophages with cytokines, and may also aberrantly appear on the surface of epithelium during immune injury. The appearance of class II determinants in parenchymal tissue potentially renders these somatic cells capable of antigen presentation to circulating CD4+ T lymphocytes, and their absence may be protective for normal tissues expressing self-antigens. The low surface class II expression observed on parenchymal cells generally correlates with low levels of mRNA, suggesting that transcription rate is a major element in class II regulation. To understand the transcriptional mechanism maintaining low basal surface expression of class II in somatic cells, we transiently transfected mini-gene reporter constructs to study the regulation of the murine A beta promoter in a cultured renal epithelial cell line. We describe here a negative cis-acting regulatory region located between -552 and -489 bp upstream of the A beta cap site that silences the transcriptional activity of the A beta promoter in epithelial cells in an orientation-dependent manner, and is also able to silence a heterologous promoter. This region is not active in class II-expressing B cells (BAL-17) in culture, but is functional in two other murine class II-negative cell lines, fibroblasts and thymoma T cells. Using competition electrophoretic mobility shift assays, we have localized the core protein binding site within this region to an 8-10-bp response element, designated A beta NRE, at -543 to -534 bp. A nuclear extract from BAL-17 cells does not bind to this element. Mutation of this site abrogates the transcriptional silencing activity of the region. We conclude that the transcription of class II-A beta in parenchymal cells, and some lymphocytes, can be actively repressed by an upstream silencing element.

Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.

Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.

Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation.

PU.1 recruits the binding of a second B cell-restricted nuclear factor, NF-EM5, to a DNA site in the immunoglobulin kappa 3' enhancer. DNA binding by NF-EM5 requires a protein-protein interaction with PU.1 and specific DNA contacts. Dephosphorylated PU.1 bound to DNA but did not interact with NF-EM5. Analysis of serine-to-alanine mutations in PU.1 indicated that serine 148 (Ser148) is required for protein-protein interaction. PU.1 produced in bacteria did not interact with NF-EM5. Phosphorylation of bacterially produced PU.1 by purified casein kinase II modified it to a form that interacted with NF-EM5 and that recruited NF-EM5 to bind to DNA. Phosphopeptide analysis of bacterially produced PU.1 suggested that Ser148 is phosphorylated by casein kinase II. This site is also phosphorylated in vivo. Expression of wild-type PU.1 increased expression of a reporter construct containing the PU.1 and NF-EM5 binding sites nearly sixfold, whereas the Ser148 mutant form only weakly activated transcription. These results demonstrate that phosphorylation of PU.1 at Ser148 is necessary for interaction with NF-EM5 and suggest that this phosphorylation can regulate transcriptional activity.

Repression of major histocompatibility complex I-A beta gene expression by dbpA and dbpB (mYB-1) proteins.

The induction of major histocompatibility complex class II gene expression is mediated by three DNA elements in the promoters of these genes (W, X, and Y boxes). The Y box contains an inverted CCAAT box sequence, and the binding activity to the CAAT box is mediated by factor NF-Y, which is composed of subunits NF-YA and NF-YB. We have found that transfection of either dbpA or dbpB (mYB-1) or both inhibits I-A beta gene expression. Although the genes for some members of the Y-box family of binding proteins have been isolated by screening an expression library using the Y-box sequence, under our conditions no binding of dbpA or dbpB to the Y box of the I-A beta or I-E alpha promoter was detected. This suggested that repression of I-A beta gene expression by dbpA and dbpB was not due to competition for binding to the Y-box sequence. The results suggest two other mechanisms by which dbpA and dbpB can inhibit transcription from the I-A beta promoter. When dbpA was added, the binding of NF-YA to DNA increased, which could be explained by interaction between these two proteins whose purpose is to increase the binding affinity of NF-YA for DNA. However, this complex was unable to stimulate transcription from the I-A beta promoter. Thus, dbpA competed for the interaction between NF-YA and NF-YB by binding to NF-YA. When dbpB factor was added together with NF-YA and NF-YB, the binding of the NF-YA--NF-YB complex was reduced. This suggested that dbpB may complete with NF-YB for interaction with NF-YA. These results provide an example of how dbpA and dbpB may regulate transcription of promoters that utilize NF-Y as a transcription factor.

Activation of transcription by PU.1 requires both acidic and glutamine domains.

The B-lymphocyte- and macrophage-specific transcription factor PU.1 is a member of the ets family of proteins. To understand how PU.1 functions as a transcription factor, we initiated a series of experiments to define its activation domain. Using deletion analysis, we showed that the activation domain of PU.1 is located in the amino-terminal half of the protein. Within this region, we identified three acidic subdomains and one glutamine-rich subdomain. The deletion of any of these subdomains resulted in a significant loss in the ability of PU.1 to transactivate in cotransfection studies. Amino acid substitution analysis showed that the activation of transcription by PU.1 requires acidic residues between amino acids 7 and 74 and a group of glutamine residues between amino acids 75 and 84. These data show that PU.1 contains two types of known activation domains and that both are required for maximal transactivation.

DNA binding of the mouse class II major histocompatibility CCAAT factor depends on two components.

A CCAAT-binding factor that recognizes a CCAAT sequence (Y box) located upstream of the major histocompatibility class II gene I-A beta has been partially purified. This CCAAT-binding factor was found to consist of two components, designated factors A and B, both of which were required for efficient binding to the DNA. Factor A had an apparent molecular size of 34 kilodaltons, and factor B had an apparent molecular size of 42 to 46 kilodaltons.

Methylation of an ETS site in the intron enhancer of the keratin 18 gene participates in tissue-specific repression.

The activities of ETS transcription factors are modulated by posttranscriptional modifications and cooperation with other proteins. Another factor which could alter the regulation of genes by ETS transcription factors is DNA methylation of their cognate binding sites. The optimal activity of the keratin 18 (K18) gene is dependent upon an ETS binding site within an enhancer region located in the first intron. The methylation of the ETS binding site was correlated with the repression of the K18 gene in normal human tissues and in K18 transgenic mouse tissues. Neither recombinant ETS2 nor endogenous spleen ETS binding activities bound the methylated site effectively. Increased expression of the K18 gene in spleens of transgenic mice by use of an alternative, cryptic promoter 700 bp upstream of the enhancer resulted in modestly decreased methylation of the K18 ETS site and increased RNA expression. Expression in transgenic mice of a mutant K18 gene, which was still capable of activation by ETS factors but was no longer a substrate for DNA methylation of the ETS site, was fivefold higher in spleen and heart. However, expression in other organs such as liver and intestine was similar to that of the wild-type gene. This result suggests that DNA methylation of the K18 ETS site may be functionally important in the tissue-specific repression of the K18 gene. Epigenetic modification of the binding sites for some ETS transcription factors may result in a refractory transcriptional response even in the presence of necessary trans-acting activities.

PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3' enhancer activity.

PU.1 is a B-cell- and macrophage-specific transcription factor. By an electrophoretic mobility shift assay and dimethyl sulfate methylation interference assays, we show that PU.1 binds to DNA sequences within the immunoglobulin kappa 3' enhancer (kappa E3'). Binding of PU.1 to the kappa E3' enhancer assists the binding of a second tissue-restricted factor, NF-EM5, to an adjacent site. Binding of NF-EM5 to kappa E3' DNA sequences requires protein-protein interaction with PU.1 as well as specific protein-DNA interactions. This is the first known instance of PU.1 interacting with another cellular protein. NF-EM5 does not cofractionate with PU.1, suggesting that it is a distinct protein and is not a posttranslational modification of PU.1. UV-crosslinking studies and elution from sodium dodecyl sulfate-polyacrylamide gels indicate that NF-EM5 is a protein of approximately 46 kDa. Site-directed mutagenesis studies of the PU.1- and EM5-binding sites indicate that these sites play important roles in kappa E3' enhancer activity. By using a series of PU.1 deletion constructs, we have identified a region in PU.1 that is necessary for interaction with NF-EM5. This segment encompasses a 43-amino-acid region with PEST sequence homology, i.e., one that is rich in proline (P), glutamic acid (E), serine (S), and threonine (T).

Ras-mediated phosphorylation of a conserved threonine residue enhances the transactivation activities of c-Ets1 and c-Ets2.

The Ras oncogene products regulate the expression of genes in transformed cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are the targets of Ras signaling pathways, the abilities of several Ets factors to activate Ras-responsive enhancer (RRE) reporters in the presence of oncogenic Ras were examined. In transient transfection assay, reporters containing RREs composed of Ets-AP-1 binding sites could be activated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage-like line RAW264 by the combination of Ets1 or Ets2 and Ras but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. Phosphoamino acid analysis of radiolabeled Ets2 revealed that Ras induced normally absent threonine-specific phosphorylation of the protein. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors.

Isolation and characterization of mRNA from Paramecium aurelia.

Total cellular RNA was isolated from the ciliate protozoan Paramecium aurelia by pH 9.5 chloroform/octanol extraction. Passage of this RNA through an oligo(dT)-cellulose column in 0.5 M NaCl resulted in 2--3% binding, indicating the presence of polyadenylic acid sequences. These polyadenylic acid regions were estimated to be 250-500 nucleotides in length, based on their resistance to ribonuclease degradation. The oligo(dT)-cellulose bound RNA sedimented at 14--25 S in sodium dodecyl sulphate/sucrose gradients. The base composition of this RNA is similar to the base composition of the DNA. This RNA was also actively translated into protein by an in vitro protein synthesizing system isolated from wheat germ. Translation was optimal under conditions similar to those used for mammalian mRNA translation. In addition, translation of the P. aurelia oligo(dT)-cellulose bound RNA was inhibited 80% by the analog 7-methylguanosine-5'-phosphate, suggesting the presence of a 5'-capped terminus.

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.

Human FLI-1 localizes to chromosome 11Q24 and has an aberrant transcript in neuroepithelioma.

The v-ets oncogene family shares a conserved motif, termed the ETS-domain, that mediates sequence-specific DNA binding. This motif is unique among transcription factor families. Using partially degenerate oligonucleotides to highly conserved amino acids in this motif as primers for the polymerase chain reaction, a novel ETS-domain cDNA fragment was generated. This fragment was subsequently used to clone both mouse and human full length cDNAs for this gene. The amino acid sequence of the longest open reading frame showed that this gene was homologous to the mouse FLI-I gene, an ETS family gene activated by Friend erythroleukemia virus insertion. The gene is normally expressed only in hematopoietic cells. The gene was localized to chromosome 11q24, a region of aberrations in Ewing's sarcoma and neuroepithelioma. In the neuroepithelioma cell line TC-32 the FLI-1 transcript is present but has an aberrant structure, indicating that it may be rearranged in neuroepithelioma.

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach.

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.

Localization of agonist- and antagonist-binding domains of human corticotropin-releasing factor receptors.

The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the nonpeptide antagonist.

Induction of a prenylated 65-kd protein in macrophages by interferon or lipopolysaccharide.

Treatment of murine bone marrow-derived macrophages with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS) resulted in changes in the abundance of a number of prenylated proteins. The most significant change involved a protein of 65 kd (p65) that became one of the most abundant prenylated proteins following treatment. The 65-kd protein was induced by agents that stimulate macrophage activation (IFNs or LPS) but not by cytokines that promote macrophage proliferation, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, or interleukin-3. The majority of p65 was localized to subcellular fractions containing internal and plasma membranes but was not detected in nuclear membranes. The farnesyltransferase inhibitor BZA-5B caused a dramatic decrease in p65 prenylation, suggesting that this protein may be modified by the C15 isoprenoid farnesyl. These observations provide the first direct evidence that interferons and LPS cause changes in the abundance of specific isoprenoid-modified proteins in macrophages.

The transcription factor PU.1 does not regulate lineage commitment but has lineage-specific effects.

PU.1 is a transcription factor shown to regulate the expression of many important genes in myeloid and B cells. At birth, mice homozygous for the disruption of the PU.1 gene have erythrocytes, megakaryocytes, and T cells, but no mature myeloid or B cells. Cells with an inability to develop to maturity were found in this mouse for B cells, neutrophils, eosinophils, mast cells, and monocytes. Rescue of early monocytic cells by transfection with the PU.1 gene results in renewed development to macrophages. These results demonstrate that PU.1 is an important regulator in the development of cells in the hematopoietic system.

Thiocyanate supplementation decreases atherosclerotic plaque in mice expressing human myeloperoxidase.

Elevated levels of the heme enzyme myeloperoxidase (MPO) are associated with adverse cardiovascular outcomes. MPO predominantly catalyzes formation of the oxidants hypochlorous acid (HOCl) from Cl(-), and hypothiocyanous acid (HOSCN) from SCN(-), with these anions acting as competitive substrates. HOSCN is a less powerful and more specific oxidant than HOCl, and selectively targets thiols; such damage is largely reversible, unlike much HOCl-induced damage. We hypothesized that increased plasma SCN(-), and hence HOSCN formation instead of HOCl, may decrease artery wall damage. This was examined using high-fat fed atherosclerosis-prone LDLR(-/-) mice transgenic for human MPO, with and without SCN(-) (10 mM) added to drinking water. Serum samples, collected fortnightly, were analyzed for cholesterol, triglycerides, thiols, MPO, and SCN(-); study-long exposure was calculated by area under the curve (AUC). Mean serum SCN(-) concentrations were elevated in the supplemented mice (200-320 µM) relative to controls (< 120 µM). Normalized aortic root plaque areas at sacrifice were 26% lower in the SCN(-)-supplemented mice compared with controls (P = 0.0417), but plaque morphology was not appreciably altered. Serum MPO levels steadily increased in mice on the high-fat diet, however, comparison of SCN(-)-supplemented versus control mice showed no significant changes in MPO protein, cholesterol, or triglyceride levels; thiol levels were decreased in supplemented mice at one time-point. Plaque areas increased with higher cholesterol AUC (r = 0.4742; P = 0.0468), and decreased with increasing SCN(-) AUC (r = - 0.5693; P = 0.0134). These data suggest that increased serum SCN(-) levels, which can be achieved in humans by dietary manipulation, may decrease atherosclerosis burden.

Mutants of ETS domain PU.1 and GGAA/T recognition: free energies and kinetics.

The ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS-1 recognize different DNA sequences with a core element centered over 5'-GGAA-3' and 5'-GGAA/T-3', respectively. To understand the molecular basis of this recognition, we carried out site-directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein-DNA interaction with the 5'-GGAA-3' containing oligonucleotide caused by a faster release of the protein from the DNA (k(off) tenfold higher than the wild-type protein). With the double mutant A231S-N236Y, we obtained an increase in binding affinity and stability toward both 5'-GGAA-3' and 5'-GGAT-3' containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5'-GGAA-3' and 5'-GGAT-3' oligonucleotides. The free energy of dissociation for the double mutant A231S-N236Y with 5'-GGAA-3' (delta deltaG((A231S-N236Y) - (N236Y)) = -1.2 kcal mol confirm the stabilizing effect of this mutant in the protein-DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5'-GGAA-3' and 5'-GGAT-3' sequences, while A231S mutation modulates the degree of specificity toward 5'-GGAA-3' and 5'GGAT-3' sequences. This study explains why wild-type PU.1 does not recognize 5'-GGAT-3' sequences and in addition broadens our understanding of 5'-GGAA/T-3' recognition by ETS protein family members.