Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins.
The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.
Koizumi
H
H
Laboratory of Molecular Immunology and Cell Biology, Rockefeller University, New York, New York 10021.
Horta
M F
MF
Youn
B S
BS
Fu
K C
KC
Kwon
B S
BS
Young
J D
JD
Liu
C C
CC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects / Q000502:physiology; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D015458:Leukemia, T-Cell; D008213:Lymphocyte Activation; D012515:Mast-Cell Sarcoma; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013945:Thymoma; D013953:Thymus Neoplasms; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; C494042:Ets1 protein, mouse; D007376:Interleukin-2; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D052899:Pore Forming Cytotoxic Proteins; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins; D014157:Transcription Factors; D054353:Perforin; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate