ABSTRACT
Systematic mutational analysis previously identified two primary regulatory elements within a minienhancer (-247 to -198) of the rat insulin I promoter that are critical for transcriptional activity. The Far box (-241 to -232) and the FLAT element (-222 to -208) synergistically upregulate transcription and, together, are sufficient to confer tissue-specific and glucose-responsive transcriptional activity on a heterologous promoter. Detailed analysis of the FLAT element further revealed that, in addition to the positive regulatory activity it mediates in tandem with the Far box, it is a site for negative regulatory control. A portion of the FLAT element bears considerable sequence similarity to the consensus binding site for hepatocyte nuclear factor 1 alpha (HNF1 alpha; LF-B1) a liver-enriched homeodomain-containing transcription factor. Here we show that the HNF1-like site within the FLAT element exhibited positive transcriptional activity in both HepG2 and HIT cells and bound similar, but distinguishable, nuclear protein complexes in the respective nuclear extracts. Screening of a hamster insulinoma cDNA library with a PCR-derived probe encompassing the DNA-binding domain of rat HNF1 alpha resulted in isolation of a hamster HNF1 alpha (hHNF1 alpha) cDNA homolog. Specific antiserum identified the HNF1 alpha protein as one component of a specific FLAT-binding complex in HIT nuclear extracts. Expression of the hHNF1 alpha cDNA in COS cells resulted in transactivation of reporter constructs containing multimerized segments of the rat insulin I minienhancer. Thus, HNF1 alpha, one component of a DNA-binding complex involved in transcriptional regulation of the rat insulin I gene, may play a significant role in nonhepatic as well as hepatic gene transcription.
Subject(s)
DNA-Binding Proteins , Insulin/genetics , Liver/physiology , Nuclear Proteins , Transcription Factors/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cricetinae , Gene Expression , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Humans , Insulinoma , Molecular Sequence Data , Pancreatic Neoplasms , Plasmids , Poly A/genetics , Poly A/metabolism , RNA/genetics , RNA/metabolism , RNA, Messenger , Rats , Sequence Homology, Nucleic Acid , Transcription Factors/genetics , Transcription Factors/isolation & purification , Tumor Cells, CulturedABSTRACT
A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two- to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5'-diphosphoethanolamine at concentrations above 100 microM. This effect appears to result from an increase in the accumulation of choline via the high-affinity, sodium-dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium-dependent high-affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.
