Cell-free conversion of a ubiquitous nuclear protein into a killer-cell-specific form that binds to the NF-P enhancer element of the mouse perforin gene.
Two nuclear factors, designated NF-PI and NF-P2, have been shown to bind to an enhancer 9-base motif (5'-ACAGGAAGT-3', NF-P motif) present within the 5'-flanking region of the mouse perforin gene. Our previous studies have shown that, although NF-P1 and NF-P2 differ in cell-type distribution and molecular mass, with NF-P2 being killer-cell-specific and smaller, the two factors appear to share common DNA-binding subunit(s). We have postulated that the biochemical event involved in the induction of NF-P2 could be the dissociation of a non-DNA-binding subunit from NF-P1, rendering the newly formed NF-P2 transcriptionally active. By using a cell-free system in the present study, we have demonstrated that a variety of chemical agents capable of denaturing or dissociating protein complexes, including guanidinium/HCl, detergents (SDS plus Nonidet P-40) and high-salt solutions, could convert NF-P1 into NF-P2. Unlike in intact cells, where induction of NF-P2 is restricted to killer lymphocytes, this conversion occurred in nuclear extracts derived from both cytotoxic lymphocytes and non-cytotoxic cells. Although the mechanism that restricts the induction of NF-P2 to killer- lymphocytes in vivo remains unresolved, these results support the hypothetical 'dissociation' model for the generation of NF-P2. The results also imply that the absence of perforin expression in non-cytotoxic cells may be due to the suppression of the induction of the killer-cell-specific trans-acting factor NF-P2.
Horta
M F
MF
Laboratory of Molecular Immunology and Cell Biology, The Rockfeller University, New York, USA.
Fu
K C
KC
Koizumi
H
H
Young
J D
JD
Liu
C C
CC
D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts / Q000737:chemistry*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D007694:Killer Cells, Natural / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011489:Protein Denaturation; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry* / Q000187:drug effects; D015854:Up-Regulation
D002457:Cell Extracts; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin