J. Biol. Chem., Vol. 267, Issue 35, 25167-25173, 12, 1992
Rat hepatonuclear factor PS-1 regulates tissue-specific activity of the S14 promoter in vitro
BJ Deschamps, DE Lawless, FE Carr and NC Wong
Department of Medicine, University of Calgary, Alberta, Canada.
We have previously identified a rat hepatonuclear factor, PS-1 that binds
to the thyroid hormone responsive gene, S14. To determine whether PS-1 is
involved in regulating tissue-specific expression of the S14 gene, we have
correlated the DNA binding activity of PS-1 with mRNA-S14 expression in a
variety of tissues. Gel retardation analysis revealed a pattern of binding
to the recognition site that was characteristic of tissues with high levels
of mRNA-S14, a different pattern was found in tissues which do not express
the gene. Competition studies using mutant oligonucleotides showed that the
first 4 nucleotides and the CAAT motif contained within the PS-1
recognition sequence are essential for protein binding. C/EBP, a
CCAAT-transcription factor binds to the PS-1 recognition site thus raising
the possibility that both C/EBP and PS-1 may belong to the same family of
proteins. Next we used a cell-free transcription assay to measure activity
of a template, pS14-GFC(-72), that contained the PS-1 sequence. The
pS14-GFC(-72) template was active in hepatonuclear extracts but deletion of
or competition with the PS-1 binding sequence rendered the construct
inactive. A template containing three PS-1 binding sequences increased S14
promoter activity by 12- to 13-fold. In nuclear extracts from spleen and
testis, relative S14 promoter activity was only 2% of that in the liver,
this observation mimicked closely in vivo expression of the gene. Mixing
together extracts from liver and spleen in varying proportions, prior to
incubation with S14 template, yielded a linear increase in S14 promoter
activity that correlated with the amount of liver extract in the reaction.
This finding is consistent with the absence of an essential factor or
factors in spleen that is/are required for S14 promoter activity in vitro.
In summary, PS-1 binds to a DNA sequence that contains a CAAT motif and
appears to play a critical role in determining tissue-specific activity of
the S14 promoter in vitro.
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