Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity.
We have previously identified a tonicity-responsive enhancer (TonE) in the promoter region of the canine BGT1 gene. TonE mediates hypertonicity-induced stimulation of transcription. Here, we characterize TonE and TonE binding proteins (TonEBPs) to provide a biochemical basis for cloning of the TonEBPs. Mutational analysis applied to both hypertonicity-induced stimulation of transcription and TonEBP binding reveals that TonE is 11 base pairs in length, with the consensus sequence of (C/T)GGAAnnn(C/T)n(C/T). Activity of the TonEBPs increases in response to hypertonicity with a time course similar to that of transcription of the BGT1 gene. Studies with inhibitors indicate that translation, but not transcription, is required for activation of the TonEBPs. Phosphorylation is required for the stimulation of transcription but not for activation of DNA binding by the TonEBPs. In vivo methylation by dimethyl sulfate reveals that the TonE site of the BGT1 gene is protected with a time course like that of activity of the TonEBPs and activation of transcription. Ultraviolet cross-linking indicates that the TonEBPs share a DNA binding subunit of 200 kDa.
Miyakawa
H
H
Division of Nephrology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Woo
S K
SK
Chen
C P
CP
Dahl
S C
SC
Handler
J S
JS
Kwon
H M
HM
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D004285:Dogs; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D009154:Mutation; D013237:Stereoisomerism; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects* / Q000502:physiology*
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D006982:Hypertonic Solutions; D014157:Transcription Factors; C072207:betaine plasma membrane transport proteins