Modulation of epidermal growth factor receptor proto-oncogene transcription by a promoter site sensitive to S1 nuclease.

Abstract

The epidermal growth factor (EGF) receptor is the functional target of the mitogen EGF and the cellular homolog of the avian erythroblastosis virus erbB oncogene product. Regulation of expression of the proto-oncogene encoding the EGF receptor can be elucidated by studying the structure and function of the gene promoter outside the confines of the cell. Previously, we reported the isolation of the human EGF receptor gene promoter. The promoter is highly GC rich, contains no TATA or CAAT box, and has multiple transcription start sites. An Si nuclease-sensitive site has now been found 80 to 110 base pairs (bp) upstream from the maijor in vivo transcription initiation site. Two sets of direct repeat sequences were found in this area; both conform to the motif TCCTCCTCC. When deletion mutations were made in this region of the promoter by using either Bal 31 exonuclease or S1 nuclease, we found that in vivo activity dropped three-to fivefold, on the basis of transient-transfection analysis. Examination of nuclear protein binding to normal and mutated promoter DNAs by gel retardation analysis and DNase I footprinting revealed that two specific factors bind to the direct repeat region but cannot bind to the S1 nuclease-mutated promoter. One of the specific factors is the transcription factor Spl. The results suggest that these nuclear trans-acting factors interact with the S1 nuclease-sensitive region of the EGF receptor gene promoter and either directly or indirectly stimulate transcription.The epidermal growth factor (EGF) is a potent mitogen capable of stimulating protein and RNA synthesis, DNA replication, and, ultimately, cellular proliferation (5,19,30). It elicits these cellular responses by binding to the Nterminus of a 170-kilodalton cell surface glycoprotein, the EGF receptor. The receptor possesses a very active tyrosine kinase at its C-terminus and is capable of phosphorylating itself and other substrates upon EGF binding (4). By virtue of its extensive homology to the erbB oncogene product of the avian erythroblastosis virus, the EGF receptor is considered the cellular erbB proto-oncogene (7,33,47,53). This proto-oncogene has been found to be overexpressed and occasionally amplified in a variety of malignant cell types (6,27,31,37,38,54 Promoter regions of active genes are frequently hypersensitive to the endonucleases DNase I and S1 (10,29,50). This sensitivity is thought to be the result of changes in the structure of the active chromatin, possibly because of the differential dissociation of nucleosomes and local alteration in DNA superhelicity (29,50). Isolated eucaryotic promoter regions cloned into plasmid vectors maintain sensitivity to S1 nuclease, provided the plasmid is supercoiled (12,14,34,35,40,44,55 (#1) is designated by the bent arrow at position -258. Heavy arrows underscore the location of the four sequences making up two sets of direct repeats (designated A and B). The cross-hatched box marks the general region sensitive to Si nuclease. The underlined sequences are those deleted by S1 nuc...

Conservation of mononucleotide repeats within 3′ and 5′ untranslated regions and their instability in MSI-H colorectal cancer

“…For example, a variety of repeats including poly(T) (Winter and Varshavsky, 1989;Buchman and Kornberg, 1990), poly(C) (Goller et al, 1994) and poly(GA) (Aharoni et al, 1993;Yee et al, 1991) are located within binding sites for transcriptional regulatory proteins. Direct evidence of microsatellites serving as transcriptional activating elements are seen with the (TCC) repeat directly upstream of the EGF receptor coding region (Johnson et al, 1988) and the (T) 9 repeat at the 3' end of the DED1 promoter (Lue et al, 1989). Correlations between the absence of these repeats and reduced levels of transcriptional activity have been shown.…”

Structural Analysis of the Proximal Region of the Microtubule‐Associated Protein 1B Promoter

“…One group contained consensus elements, such as Spl and CRE; transcription factors that interact with them are known to be present in many tissues. A second group included two common motifs previously identified in the promoter region (Liu and Fischer, 1996), the neuronal motif, found in promoter regions of other neuronal genes (Thompson and Ziff, 1989;Maue et al, 1990;Mon et al, 1992;Nedivi et al, 1992), and the TCC repeat motif, found in the promoter region of many growth factor receptor, steroid hormone receptor, and protooncogenes (Johnson et al, 1988). The functional importance of these two motifs is poorly understood.…”

“…SI digestion of plasmid DNA was performed as described (Yu and Manley, 1986;Johnson et al, 1988) with some modifications. DNA samples for the SI assay were prepared using a Qiagen midiprep kit (Qiagen, Studio City, CA, U.S.A.).…”

Characterization of a Polypyrimidine/Polypurine Tract in the Promoter of the Gene for Chicken Malic Enzyme

“…Standard errors of the mean are provided to indicate the degree of variability in the data. (6,17,18). We tested the functional significance of the PPY/PPU sequence in the malic enzyme promoter by comparing transcription from a promoter containing the wild-type sequence (pBH147CAT) with that in a construct lacking all but 4 bp of the PPY/PPU tract (pBH147⌬PPYCAT).…”

“…On a statistical basis, PPY/PPU sequences are overrepresented in eukaryotic genomes (14 -16). Second, triplex-forming PPY/PPU sequences have been discovered in the promoters of several genes that lack TATA-boxes; the protooncogenes hEGFR, c-ets-2, and c-Kiras are examples (6,17,18). Deletion of the PPY/PPU sequences from these promoters decreases their promoter activities in transient transfection assays.…”