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M J Klemsz

Publications and source records attributed to M J Klemsz.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of the PU.1 gene by distal elements.

The transcription factor PU.1 (also known as Spi-1) plays a critical role in the development of the myeloid lineages, and myeloid cells derived from PU.1(-/-) animals are blocked at the earliest stage of myeloid differentiation. Expression of the PU.1 gene is tightly regulated during normal hematopoietic development, and dysregulation of PU.1 expression can lead to erythroleukemia. However, relatively little is known about how the PU.1 gene is regulated in vivo. Here it is shown that myeloid cell type-specific expression of PU.1 in stable cell lines and transgenic animals is conferred by a 91-kilobase (kb) murine genomic DNA fragment that consists of the entire PU.1 gene (20 kb) plus approximately 35 kb of upstream and downstream sequences, respectively. To further map the important transcriptional regulatory elements, deoxyribonuclease I hypersensitive site mapping studies revealed at least 3 clusters in the PU.1 gene. A 3.5-kb fragment containing one of these deoxyribonuclease I hypersensitive sites, located -14 kb 5' of the transcriptional start site, conferred myeloid cell type-specific expression in stably transfected cell lines, suggesting that within this region is an element important for myeloid specific expression of PU.1. Further analysis of this myeloid-specific regulatory element will provide insight into the regulation of this key transcriptional regulator and may be useful as a tool for targeting expression to the myeloid lineage.

Animals↗
PubMed2001

Loss of PU.1 expression following inhibition of histone deacetylases.

Altering chromatin structure by blocking histone deacetylase activity with specific inhibitors such as trichostatin A can result in an up-regulation of gene expression. In this report, however, we show that expression of the ETS domain transcription factor PU.1 is down-regulated in cells following the addition of trichostatin A. The loss of PU.1 is seen at both the mRNA and protein levels in multiple cell lines and is reversible following removal of the drug. More importantly, we show that the loss of PU.1 results in a loss of PU.1 target gene expression, including CD11b, c-fms, Toll-like receptor 4, and scavenger receptor. Chromatin immunoprecipitation analysis of cells treated with trichostatin A showed a significant increase in the acetylation of histone H4, but not histone H3, across approximately 650 bp of the PU.1 promoter region. Our data suggest that the consequences of using drugs that inhibit histone deacetylase activity may be a loss of blood cell development and/or function due to a block in PU.1 gene expression.

Acetylation↗
PubMed2000

PU.1 is a lineage-specific regulator of tyrosine phosphatase CD45.

The hematopoietic cell-specific ets family transcription factor PU.1 regulates many lymphoid and myeloid genes. We have determined that PU.1 is critical for lineage-specific expression of the tyrosine phosphatase CD45. CD45 is expressed exclusively in hematopoietic cells at all stages of development, except for mature red cells and platelets. Although CD45 is normally expressed in all leukocyte lineages, it is critically regulated by PU.1 only in myeloid cells. Whereas myeloid cells from PU.1 null mice failed to express CD45, lymphoid cells were CD45(+) by flow cytometry. Additionally, mRNA for CD45 was absent from PU.1-deficient myeloid cells. To understand the molecular basis for these observations, we characterized a transcriptional regulatory region of the murine CD45 gene containing exons 1a, 1b, and 2. Distinct transcriptional initiation sites for CD45 were demonstrated in T and B cells versus myeloid cells. A transcriptional initiation site in exon 1b (P1b) was principally utilized by myeloid cells. A PU.1 binding site was identified upstream of exon 1b by sequence analysis and DNA binding assays. Using this region of the CD45 locus we demonstrated that PU.1 directly transactivated reporter gene expression. Finally, retrovirus-mediated restoration of PU.1 expression to PU.1-deficient myeloid cells resulted in expression of cell surface CD45 and restored phosphatase activity, confirming the role of PU.1 in the positive regulation of this well known signaling molecule. We conclude that CD45 is regulated differentially in myeloid and lymphoid cells and that sequences critical to direct myeloid expression include a PU.1 binding site upstream of the P1b transcriptional initiation site.

Animals↗
PubMed2000

Synergistic induction of the Tap-1 gene by IFN-gamma and lipopolysaccharide in macrophages is regulated by STAT1.

Proper regulation of the Tap-1 gene is critical for the initiation and continuation of a cellular immune response. Analysis of the Tap-1/low molecular mass polypeptide 2 bidirectional promoter showed that the IFN-gamma activation site element is critical for the rapid induction of the promoter by IFN-gamma following transfection into the human macrophage cell line THP-1. Furthermore, activation of STAT1 binding to this site was important for the synergistic response seen following the stimulation with both IFN-gamma and LPS. Mutation of an IFN-stimulated regulatory element that binds IFN regulatory factor 1 appeared to enhance the response to IFN-gamma and LPS. These data show that STAT1 is necessary for the activation of Tap-1 gene expression in APCs and initiation of cellular immune responses. Furthermore, our data suggest that bacterial products such as LPS may enhance cellular immune responses through augmenting the ability of STAT1 to regulate IFN-gamma-inducible genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

DNA methylation and chromatin structure regulate PU.1 expression.

Knockout studies have shown that PU.1 is required for the normal development of many blood cell lineages, yet overexpression of this transcription factor in erythroid cells can lead to erythroleukemia. Thus, how the tissue-specific expression of PU.1 is regulated is important to our understanding of hematopoiesis. In this study, we showed that B and macrophage cell lines expressing PU.1 contained DNase I-hypersensitive sites in intron 1 and were hypomethylated at three MspI sites flanking exon 1. Results from studies using several T-cell lines suggested that the pattern of methylation changed as these cells matured. A pre-T cell line that expresses PU.1 contained DNase I-hypersensitive sites in intron 1 and was also hypomethylated at both MspI sites. Other immature T-cell lines had methylated at least one of the MspI sites and displayed no hypersensitive sites. Mature T-cell lines had a methylation pattern more similar to that of fibroblasts. Treatment of an immature T-cell line with 5-azacytidine resulted in the expression of PU.1 transcripts. These data suggest that the tissue-specific expression of PU.1 is controlled by chromatin structure and DNA methylation and that this may be a mechanism used to shut off PU.1 expression in specific cell lineages during hematopoiesis.

Animals↗
PubMed1997

Altered kinetics of Tap-1 gene expression in macrophages following stimulation with both IFN-gamma and LPS.

With recent studies suggesting a key role for professional antigen presenting cells in the induction of major histocompatibility class I cellular immune responses, we initiated studies on the regulation of Tap-1 and Tap-2 gene expression in macrophages. Stimulation of the human macrophage cell line THP-1 with interferon-gamma (IFN-gamma) resulted in maximal induction of both Tap-1 and Tap-2 mRNA within 24 hr. Nuclear run-on analyses showed that the increased expression of Tap-1 and Tap-2 was controlled at the level of transcription. Half-life studies demonstrated that mRNAs for both genes became destabilized after stimulation of THP-1 cells with IFN-gamma for 24 hr, suggesting that a posttranscriptional mechanism down-regulates TAP gene expression following activation. Treatment of cells with both IFN-gamma and lipopolysaccharide (LPS) altered the kinetics and amount of Tap-1 mRNA and protein expression, compared to those with stimulation with IFN-gamma alone. These data suggest that LPS enhances the ability of macrophages stimulated with IFN-gamma to initiate a cellular immune response by altering the kinetics of TAP gene expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Cloning and characterization of exodus, a novel beta-chemokine.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue. Some chemokines such as MIP-1 alpha also inhibit hematopoietic progenitor cell proliferation. Recently, three chemokines, MIP-1 alpha, MIP-1 beta, and RANTES, have been found to significantly decrease human immunodeficiency virus production from infected T cells. We report here the cloning and characterization of a novel human chemokine termed Exodus for its chemotactic properties. This novel chemokine is distantly related to other chemokines (28% homology with MIP-1 alpha) and shares several biological activities. Exodus is expressed preferentially in lymphocytes and monocytes, and its expression is markedly upregulated by mediators of inflammation such as tumor necrosis factor or lipopolysaccharide. Purified synthetic Exodus was found to inhibit proliferation of myeloid progenitors in colony formation assays. Exodus also stimulated chemotaxis of peripheral blood mononuclear cells. The sequence homology, expression, and biological activity indicate that Exodus represents a novel divergent beta-chemokine.

Amino Acid Sequence↗
PubMed1997

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.

Animals↗
PubMed1996

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.

Acids↗
PubMed1993

Characterization of the ets oncogene family member, fli-1.

The recently cloned fli-1 gene is a member of the ets oncogene family that is preferentially expressed in hematopoietic cells. It is a target of dysregulation by Friend leukemia virus insertion and translocation in Ewing's sarcoma and neuroepithelioma. In this report, we have studied the function and regulation of both murine and human fli-1. Analysis of the human and mouse fli-1 proteins showed that fli-1 binds to specific DNA sequences highly related to m-ets-2 binding sites. Methylation protection experiments showed that fli-1 and m-ets-2 contacted the same nucleotides in two different binding sites. The fli-1 protein was shown to be a transcriptional activator in co-transfection studies. Stimulation of murine bone marrow macrophages by mediators of inflammation, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, interleukin-1, and interferon-gamma resulted in the reduced expression of fli-1 mRNA. fli-1 was only expressed in a defined subset of human erythroleukemia cell lines.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1992

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).

Base Sequence↗
PubMed1990

The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene.

We have isolated a cDNA clone, PU.1, that codes for a new tissue-specific DNA binding protein. Analysis of the binding site by methylation interference and DNAase 1 protection revealed that the PU.1 protein recognized a purine-rich sequence, 5'-GAGGAA-3' (PU box). The PU.1 protein was shown to be a transcriptional activator that is expressed in macrophages and B cells. cDNA constructions used to generate proteins lacking portions of either the amino- or carboxy-terminal ends of the PU.1 protein placed the DNA binding domain in the highly basic carboxy-terminal domain of the protein. The amino acid sequence in the binding domain of PU.1 has considerable identity with proteins belonging to the ets oncogene family.

Amino Acid Sequence↗
PubMed1989

Induction of c-fos and c-myc expression during B cell activation by IL-4 and immunoglobulin binding ligands.

The data presented here indicated that both membrane Ig and IL-4 receptors transduce signals across the plasma membrane of quiescent B cells, which results in the induction of c-fos and c-myc proto-oncogene mRNA expression. Monoclonal anti-Ig antibodies with specificity for mu, delta, or kappa chains, regardless of mitogenicity, induced increased c-fos and c-myc mRNA expression with kinetics and magnitude similar to that observed following stimulation of B cells with IL-4. Maximal levels of c-fos mRNA, approximately 30-fold over background, were observed 30 min after stimulation. Maximal levels of c-myc mRNA, approximately 10-fold over background, were observed 60 min after stimulation. Phorbol myristate acetate alone induced expression of these two oncogenes in a similar fashion, suggesting that protein kinase C may be involved in the regulation of their expression following anti-Ig crosslinking. Ionomycin induced only a small increase in c-myc and c-fos message (three- to four-fold), and did not synergize with phorbol myristate acetate, suggesting that the membrane Ig-mediated calcium mobilization may not play a major role in regulation of c-myc or c-fos expression in mouse B cells. In vitro nuclear run-on analyses indicate that c-myc expression is primarily regulated post-transcriptionally, whereas c-fos expression is regulated at the level of transcription. Anti-sense transcription was found to be constitutive for both the c-myc and C-fos loci and was further induced by anti-Ig and IL-4, suggesting an additional mechanism for regulation of these genes. The observation that both anti-Ig and IL-4 regulate the expression of c-fos and c-myc suggests that multiple second messenger generating systems regulate the expression of these oncogenes in normal B cells and that their expression may be necessary, but is not sufficient to drive quiescent B cells into cell cycle.

Animals↗
PubMed1989

Interferon-gamma activates multiple pathways to regulate the expression of the genes for major histocompatibility class II I-A beta, tumor necrosis factor and complement component C3 in mouse macrophages.

The purpose of this study was to obtain additional information on the mechanism by which interferon-gamma (IFN-gamma) is able to regulate gene expression in macrophages. The expression of the genes for class II histocompatibility I-A beta, tumor necrosis factor (TNF) and complement component C3 was assayed after treating bone marrow macrophages with IFN-gamma. Each gene displayed a characteristic pattern of regulation. First, the increase in the level of RNA for each gene followed different kinetics. The level of TNF RNA increased within 15 min after IFN-gamma treatment and reached a plateau after 4 h. In contrast, there was a lag of about 4 h before the level of I-A beta RNA began to rise and a plateau was not reached until 48 h after the IFN-gamma treatment began. C3 gene expression followed an intermediate time course between that for TNF and I-A beta. Second, the expression of I-A beta was inhibited when cells were treated with both IFN-gamma and cycloheximide, while the expression of TNF and C3 was not. Interestingly, the sensitivity to cycloheximide only lasted 30 min following the addition of IFN-gamma, after which cycloheximide had no effect on the expression of I-A beta. Third, lipopolysaccharide abolished the IFN-gamma-induced expression of I-A beta, but enhanced the expression of TNF. Based on these observations, we conclude that IFN-gamma must activate multiple pathways to regulate gene expression in macrophages.

Animals↗