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

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Abstract
Two nuclear factors, designated NF-P1 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 in vivo may be due to the suppression of the induction of the killer-cell-specific trans-acting factor NF-P2.