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-, neutrophil- 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 located 6 bp upstream of the TATA motif within the IL-1 beta promoter and is required for maximal expression of the IL-1 beta gene. Here we show 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 immunoglobulin 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 specificity identical to NF beta A as determined by electrophoretic mobility 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 similar protein structure as determined by proteolytic clipping bandshift analysis. Furthermore, we show that PU.1 is able to transactivate an NF beta A-dependent promoter when co-transfected into HeLa cells which lack PU.1/NF beta A. EMSA studies using recombinant TATA binding protein (TBP) and PU.1 suggest that PU.1 may induce assembly of a distinct TBP-dependent complex on the IL-1 beta promoter. Finally, immunohistochemical confocal laser scanning microscopy studies suggest that LPS stimulation of RAW macrophages induces a structural change in the N-terminal transcriptional activation domain of PU.1.
Buras
J A
JA
Evans Department of Clinical Research, Boston, MA 02118, USA.
Reenstra
W R
WR
Fenton
M J
MJ
D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000737:chemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D015682:Retroviridae Proteins, Oncogenic; D012689:Sequence Homology, Nucleic Acid; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D007106:Immune Sera; D007375:Interleukin-1; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D015682:Retroviridae Proteins, Oncogenic; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D014157:Transcription Factors; C062022:v-Spi-1 protein, Friend spleen focus-forming virus