Growth state-dependent binding of USF-1 to a proximal promoter E box element in the rat plasminogen activator inhibitor type 1 gene.
Induced PAI-1 gene expression in renal epithelial (NRK-52E, clone EC-1) cells occurs as part of the immediate-early response to serum. PAI-1 transcripts are maximally expressed early in G(1) (within 4 h of serum addition to quiescent EC-1 cells) and then subsequently decline to basal levels prior to entry into DNA synthetic phase. Comparative analysis of PAI-1 mRNA abundance and de novo-synthesized thiolated RNA in quiescent cells, as well as at 4 h (early G(1)) and 20 h (late G(2)) postserum addition, in conjunction with RNA decay measurements indicated that PAI-1 gene regulation upon growth activation was predominantly transcriptional. An E box motif (CACGTG), important in the induced expression of some growth state-dependent genes, mapped to nucleotides -160 to -165 upstream of the transcription start site in the PAI-1 proximal promoter. Mobility-shift assessments, using a 18-bp deoxyoligonucleotide construct containing the E box within the context of PAI-1-specific flanking sequences, confirmed binding of EC-1 nuclear protein(s) to this probe and, specifically, to the E box hexanucleotide site. The specificity of this protein-probe interaction was verified by competition analyses with double-stranded DNA constructs that included E box deoxyoligonucleotides with non-PAI-1 flanking bases, mutant E box sequences incapable of binding NRK nuclear proteins, and unrelated (i.e., AP-1) target motifs. Extract immunodepletion and supershift/complex-blocking experiments identified one PAI-1 E box-binding protein to be upstream stimulatory factor-1 (USF-1), a member of the HLH family of transcription factors. Mutation of the CACGTG site to TCCGTG in an 18-bp PAI-1 probe inhibited the formation of USF-1-containing complexes confirming that an intact E box motif at -160 to -165 bp in the PAI-1 promoter and, in particular, the CA residues at -165 and -164 are essential for USF-1 binding. Incorporation of this 2 bp change into a reporter construct containing 764 bp of the proximal PAI-1 "promoter" ligated to a CAT gene effectively reduced (by 74%) CAT activity in cycling cells. An intact E box motif at nucleotides -160 to -165 in the PAI-1 promoter, thus, is an important functional element in the regulation of PAI-1 transcriptional activity in renal cells.
White
L A
LA
Center for Cell Biology & Cancer Research, Albany Medical College, Albany, New York 12208, USA.
Bruzdzinski
C
C
Kutz
S M
SM
Gelehrter
T D
TD
Higgins
P J
PJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D017931:DNA Primers; D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase