Michael J. Klemsz

Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities.

PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3–5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic‐treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1.

EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewings sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-1 has been shown to be a potent transforming gene, suggesting that it plays an important role in the genesis of these human tumors. We now demonstrate that EWS/FLI-1 has the characteristics of an aberrant transcription factor. Subcellular fractionation experiments localized the EWS/FLI-1 protein to the nucleus of primitive neuroectodermal tumor cells. EWS/FLI-1 specifically bound in vitro an ets-2 consensus sequence similarly to normal FLI-1. When coupled to a GAL4 DNA-binding domain, the amino-terminal EWS/FLI-1 region was a much more potent transcriptional activator than the corresponding amino-terminal domain of FLI-1. Finally, EWS/FLI-1 efficiently transformed NIH 3T3 cells, but FLI-1 did not. These data suggest that EWS/FLI-1, functioning as a transcription factor, leads to a phenotype dramatically different from that of cells expressing FLI-1. EWS/FLI-1 could disrupt normal growth and differentiation either by more efficiently activating FLI-1 target genes or by inappropriately modulating genes normally not responsive to FLI-1.

Hematopoietic transcriptional regulation by the myeloid zinc finger gene, MZF-1.

Transcriptional regulators control much of hematopoiesis. One such transcriptional regulator is the myeloid zinc finger gene MZF-1. MZF-1 has been localized to the telomere of chromosome 19q, where a large number of related zinc finger genes reside. It has been found to be essential in granulopoiesis. It is a bi-functional transcriptional regulator, repressing transcription in non-hematopoietic cells, and activating transcription in cells of hematopoietic origins. Its consensus DNA binding site has been isolated, and sites in several promoters of myeloid-specific genes, such as CD34, lactoferrin, and myeloperoxidase, have been defined. In co-transfection experiments MZF-1 has been found to regulate transcription from the CD34 promoter.

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.

Cathepsin E: A Novel Target for Regulation by Class II Transactivator

The aspartic proteinase cathepsin E (CatE) has been implicated in Ag processing. In this study we report that CatE expression is negatively regulated by the MHC class II transactivator (CIITA). CIITA-deficient murine and human B cells expressed greater CatE than wild-type B cells, whereas overexpression of CIITA in a human gastric carcinoma cell line, AGS, resulted in decreased CatE mRNA and protein. AGS cells expressing CIITA also exhibited decreased processing of OVA Ag. Inhibition of CatE expression is specific to the type III CIITA isoform and maps to the acidic and proline/serine/threonine-rich (PST) protein domains of CIITA. We found that CatE expression is inducible by PU.1 and p300, and that this induction can be reversed by CIITA. These findings demonstrate a novel phenomenon: regulation of CatE Ag processing by CIITA in an isoform-dependent manner.

Cutting Edge: Induction of the Antigen-Processing Enzyme IFN-γ-Inducible Lysosomal Thiol Reductase in Melanoma Cells Is STAT1-Dependent but CIITA-Independent

Presentation and CD4+ T cell responses to Ag in the context of MHC class II molecules require processing of native proteins into short peptide fragments. Within this pathway, IFN-γ-inducible lysosomal thiol reductase (GILT) functions to catalyze thiol bond reduction, thus unfolding native protein Ag and facilitating further processing via cellular proteases. In contrast with professional APCs such as B cells, class II-positive human melanomas expressed relatively little to no GILT protein or mRNA. Tumor cell GILT expression was partially restored with IFN-γ treatment but unlike other genes required for class II Ag presentation, GILT was not regulated by CIITA. Rather, studies revealed STAT1 plays a direct role in IFN-γ-inducible GILT expression. These results define a molecular mechanism for the uncoupled regulation of MHC class II genes and the processing enzyme GILT in human melanomas.

Vaccinia Virus Blocks Stat1-Dependent and Stat1-Independent Gene Expression Induced by Type I and Type II Interferons

Blocking the function of Stat (signal transducer and activator of transcription) proteins, which are critical for antiviral responses, has evolved as a common mechanism for pathogen immune evasion. The poxvirus-encoded phosphatase H1 is critical for viral replication, and may play an additional role in the evasion of host defense by dephosphorylating Stat1 and blocking interferon (IFN)-stimulated innate immune responses. Vaccinia virus (VACV) H1 can inhibit the phosphorylation of the transcription factor Stat1 after IFN-gamma stimulation of epithelial cells, greatly attenuating IFN-induced biological functions. In this study, we demonstrate that VACV infection is capable of inhibiting the phosphorylation of Stat1 and Stat2 after stimulation of fibroblasts or bone marrow-derived macrophages with either type I or type II IFNs, but did not inhibit the activation of Stat3 or Stat5 in either cell type. By using recombinant proteins for in vitro assays, we observe that variola virus H1 is more active than VACV H1, although it has similar selectivity for Stat targets. Differential effects of VACV infection were observed on the induction of IFN-stimulated genes, with complete inhibition of some genes by VACV infection, while others were less affected. Despite the IFN-gamma-induced expression of some genes in VACV-infected cells, IFN-gamma was unable to rescue the VACV-mediated inhibition of MHC class II antigen presentation. Moreover, VACV infection can affect the IFN-induced expression of Stat1-dependent and Stat1-independent genes, suggesting that the virus may target additional IFN-activated pathways. Thus, VACV targets multiple signaling pathways in the evasion of antiviral immune responses.

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.

Synergistic Induction of the Tap-1 Gene by IFN-γ 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-γ activation site element is critical for the rapid induction of the promoter by IFN-γ 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-γ and LPS. Mutation of an IFN-stimulated regulatory element that binds IFN regulatory factor 1 appeared to enhance the response to IFN-γ 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-γ-inducible genes.

PU.1 Is a Lineage-specific Regulator of Tyrosine Phosphatase CD45

The hematopoietic cell-specific etsfamily 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 cellsversus 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.