NF-BETA-A, A FACTOR REQUIRED FOR MAXIMAL INTERLEUKIN-1-BETA GENE-EXPRESSION IS IDENTICAL TO THE ETS FAMILY MEMBER PU.1 - EVIDENCE FOR STRUCTURAL ALTERATION FOLLOWING LPS ACTIVATION

We have previously identified and characterized the macrophage-, neutr ophil- and B cell-specific nuclear factor beta A (NF beta A), which is involved in transcriptional regulation of the interleukin-1 beta (IL- 1 beta) gene. NF beta A binds to a highly conserved sequence element l ocated 6 bp upstream of the TATA motif within the IL-1 beta promoter a nd is required for maximal expression of the IL-1 beta gene. Here we s how that NF beta A is identical to the previously identified ets gene family member PU.1. The NF beta A binding element shares 100% sequence identity with a novel PU.1 binding element recently found in the immu noglobulin J-chain promoter. Methylation interference DNA footprinting data demonstrated that NF beta A and PU.1 make identical protein/DNA contacts. In vitro synthesized PU.1 possesses a mobility and binding s pecificity identical to NF beta A as determined by electrophoretic mob ility shift analysis (EMSA). Antisera directed against amino acids 39- 55 of PU.1 recognizes NF beta A in a manner indistinguishable from PU. 1 in EMSA 'supershift' studies. NF beta A and PU.1 also possess simili ar protein structure as determined by proteolytic clipping bandshift a nalysis. Furthermore, we show that PU.1 is able to transactivate an NF beta A-dependent promoter when co-transfected into HeLa cells which l ack PU.1/NF beta A. EMSA studies using recombinant TATA binding protei n (TBP) and PU.1 suggest that PU.1 may induce assembly of a distinct T BP-dependent complex on the IL-1 beta promoter. Finally, immunohistoch emical confocal laser scanning microscopy studies suggest that LPS sti mulation of RAW macrophages induces a structural change in the N-termi nal transcriptional activation domain of PU.1.