Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis

J D Haley,M D Waterfield

doi:10.1016/s0021-9258(18)52359-0

Abstract

Overexpression of the epidermal growth factor (EGF) receptor (c-erbB) proto-oncogene is a frequent occurrence in human carcinoma and appears to accompany autocrine or paracrine transforming growth factor-alpha expression, which in model systems can result in activation of EGF receptor tyrosine kinase activity and phenotypic transformation. Here we have investigated the transcriptional regulation of the EGF receptor gene, by run-on transcription in isolated nuclei derived from epithelioid tumor lines. The level of transcription was measured at various points on the 100-kilobase pair EGF receptor gene locus, on either sense or antisense DNA strands. We find the level of sense strand transcription along exon 1 is 8-fold higher than transcription in exons 2-26. Primary EGF receptor transcripts appear to pause or terminate prematurely between exons 1 and 2. Termination was mapped to a sequenced region approximately 2 kilobase pairs 3' of exon 1, proximal to a previously reported DNase I hypersensitive site and an enhancer-like activity. Transcription in the CpG-rich region surrounding exon 1 is bidirectional, with antisense transcripts initiating in intron 1 and extending through the coding first exon. Activation of protein kinase C results in a 5-fold induction of EGF receptor transcription, accompanied by a slow release in the block RNA elongation between exon 2 and exon 26, showing that EGF receptor RNA synthesis may be altered by changes in de novo transcription and by a block to RNA elongation.

Highlights

Contributory Effectsof de N o v o Transcription and Premature Transcript Termination in the Regulatioonf Human Epidermal Growth Factor Receptor Proto-oncogenReNA Synthesis*
MDA231 breast carcinoma lines and the HN-5 squamous carcinoma Transcription of EGFR RNA in Epithelioid Cell Lines Is line were cultured under similar conditions
EGFR gene was measured by nuclear run-on transcription

Summary

PTreermmaintuarteion of EGFR Transcription

RNA is attributable to both RNA stabilization (Clark et al, 1985)and toa lesser extent to de novo transcription (Fernandez-Pol et al, 1989; Hudson et al, 1989) in actively growing epithelioid cell lines. Between in 2 X SSC with 10 yg/ml RNase A, twice with 0.2 X SSC, 1%SDS tissues or cell lines expressing the EGFreceptor, large variations in receptor number (Adamson and Rees, 1981) and steady-state EGFR RNA levels are observed. We have measured the distribution of RNA polymerase I1 actively engaged in transcription at defined exons of the EGF receptor gene in the human carcinoma cell lines, and single-stranded templates. Single-strandedtemplateDNA was preparedas script elongation in exons 2-26 between 120 and 240 min and described (Messing et al, 1981), quantitated by agarose gel electrothe magnitude of increased readthrough transcription suggest the block to RNA elongation may be a point at which EGF receptor RNA synthesis is controlled.

RESULTS

PTremrmaitnuaretion of EGFR Transcription

Premature Termination of EGFR Transcription

DISCUSSION