Rat hepatonuclear factor PS-1 regulates tissue-specific activity of the S14 promoter in vitro

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.