Abstract
Insulin-like growth factor-I (IGF-I) gene transcription is mediated largely via exon 1. In an initial search for regulatory regions, rat hepatocytes were transfected with IGF-I constructs. Since omission of downstream sequences led to reduced expression, we then used in vitro transcription to evaluate potential metabolic regulation via downstream regions. With templates including 219 base pairs of downstream sequence, transcriptional activity was reduced 70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates lacking downstream sequences. The downstream region contained six DNase I footprints, and templates with deletion of either region III or V no longer provided reduced transcriptional activity with nuclear extracts from diabetic rats. Nuclear protein binding to regions III and V appeared to be metabolically regulated, as shown by reduced DNase I protection and activity in gel mobility shift assays with nuclear extracts from diabetic rats. Southwestern blotting probes corresponding to regions III and V recognized a approximately 65-kDa nuclear factor present at reduced levels in diabetic rats. These findings indicate that a downstream region in exon 1 may be important for both IGF-I expression and metabolic regulation. Altered concentration or activity of a transcription factor(s) binding to this region may contribute to reduced IGF-I gene transcription associated with diabetes mellitus.
Highlights
The insulin-like growth factors (IGFs)1 are polypeptides with sequence, structure, and biological actions similar to those of insulin (1)
We demonstrate that sequences downstream from the exon 1 major transcription initiation site are important both for hepatic Insulin-like growth factor-I (IGF-I) expression and metabolic regulation, we characterize nuclear protein binding to downstream sequences, and we identify two regions that may be involved in the decreased IGF-I gene transcription associated with diabetes mellitus
The IGF-I promoters analyzed to date have several common features, such as lack of a “TATA” box, presence of transcription “initiator” sequences (13, 32), and binding sites for well recognized transcription factors such as Sp1, C/EBP, and HNF-1 located upstream from the major transcription initiation sites (32)
Summary
The insulin-like growth factors (IGFs) are polypeptides with sequence, structure, and biological actions similar to those of insulin (1). Adamo et al (13) found that two initiation sites in exon 1 could account for 70 – 80% of IGF-I gene transcription in adult rat liver. Since relatively little is known about molecular regulation of IGF-I gene transcription, we have focused on the liver; while several laboratories have begun to study the basis of IGF-I gene transcription in different immortal cell lines (14, 17–19), there has been little evaluation of underlying mechanisms in the dominant source of IGF-I production (1, 3, 4). We demonstrate that sequences downstream from the exon 1 major transcription initiation site are important both for hepatic IGF-I expression and metabolic regulation, we characterize nuclear protein binding to downstream sequences, and we identify two regions that may be involved in the decreased IGF-I gene transcription associated with diabetes mellitus
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