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-γ (IFN-γ) 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-γ for 24 hr, suggesting that a posttranscriptional mechanism downregulates TAP gene expression following activation. Treatment of cells with both IFN-γ and lipopolysaccharide (LPS) altered the kinetics and amount of Tap-1 mRNA and protein expression, compared to those with stimulation with IFN-γ alone. These data suggest that LPS enhances the ability of macrophages stimulated with IFN-γ to initiate a cellular immune response by altering the kinetics of TAP gene expression.
The ETS oncogene family member PU.1 is a transcriptional activator that is dysregulated by Friend erythroleukemia virus insertion. Northern analysis found that PU.1 is highly expressed in cells of ...myeloid and B-lymphoid origin, but not expressed at all in a number of nonhematopoietic tissues. Interferon-gamma and retinoic acid downregulated PU.1 expression in marrow macrophages. In situ immunohistochemistry found that PU.1 is expressed only in early granulocytic and erythroid cells and megakaryocytes, but not in mature erythroid cells, mature granulocytes, endothelial cells, or osteocytes. Thus, its expression pattern makes PU.1 a candidate for a genetic determinant of lineage commitment and stage progression in blood cell development. It also lends insight into how PU.1 might play a role in Friend virus erythroleukemia.
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.
Histone acetylation plays an important role in chromatin remodeling and transcription control. Acetylation of histones is regulated by histone acetyltransferases and histone deacetylases (HDACs). ...Human papillomavirus type 16 (HPV16) E7 can inactivate retinoblastoma protein (pRB), which recruits histone deacetylases, and also physically interacts with histone acetyltransferases and histone deacetylases, suggesting E7 may affect histone acetylation. To test this, we have analyzed the state of acetylation of histone H3 in human foreskin keratinocytes. HPV16 E7 increased acetylation of histone H3 on lysine 9, which is related to transcription activation. The ability to bind both pRB and histone deacetylase was required for HPV16 E7 to increase histone acetylation. Chromatin immunoprecipitations showed HPV16 E7 increases histone acetylation on the E2F1 and cdc25A promoters. Consistent with this, RT-PCR analysis showed an increase in the expression of E2F-responsive genes involved in cell cycle control. HPV16 E7 affected neither the steady-state levels of histone acetyltransferases or deacetylases nor histone deacetylase activity. However, HPV16 E7 did increase the level of methylation of histone H3 on lysine 4, which normally requires displacement of histone deacetylase. In contrast, sodium butyrate, a known inhibitor of histone deacetylases, caused an increase in acetylated but not methylated histone H3. These data suggest HPV16 E7, by increasing histone acetylation, may create a transcriptionally active chromatin structure to promote expression of genes vital for cell cycle progression.
Transcription factors belonging to the ets family regulate gene expression and share a conserved ETS DNA-binding domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The domain is similar to ...alpha+beta ("winged") helix-turn-helix DNA-binding proteins. The crystal structure of the PU.1 ETS domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ets proteins confirms this complex as a paradigm for ets DNA recognition. The second helix in the helix-turn-helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.
MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation. The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains ...containing zinc fingers 1 through 4 and, in the carboxy-terminus, 5 through 13. We previously identified the DNA consensus binding site recognized by the two DNA binding domains. To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4. The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3,293, K562, and Jurkat cell lines. MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293. In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat. The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter. MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines. Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays. MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines. As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines. Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites. The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function.
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.
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