Alfred C. Johnson

Molecular Cloning and Characterization of a Transcription Regulator with Homology to GC-binding Factor

GC-binding factor (GCF) represses transcription of certain genes and is encoded by a 3.0-kilobase mRNA (Kageyama, R., and Pastan, I. (1989) Cell 59, 815–825). The GCF cDNA hybridizes to two additional mRNA species, 4.2 and 1.2 kilobases. We have used differential hybridization to identify a cDNA clone (termed GCF2) for the 4.2-kilobase mRNA and find that it is highly expressed in HUT-102 cells. The open reading frame consists of 2256 nucleotides and encodes a protein of 752 amino acids with a calculated molecular mass of 83 kilodaltons. GCF2 expressedin vitro using reticulocyte lysates and Escherichia coli migrates as a 160-kilodalton protein in SDS-polyacrylamide gel electrophoresis but has a molecular mass of 83 kilodaltons as determined by mass spectrum analysis. GCF2 binds to epidermal growth factor receptor promoter fragments, and the major binding site is located between nucleotides −249 and −233. Cotransfection assays show that GCF2 acts to repress transcription from the epidermal growth factor receptor promoter in constructs containing the major GCF2 binding site and not when the site had been mutated. Thus, GCF2 is a newly identified transcriptional repressor with aberrant electrophoretic mobility.

Expression of epidermal growth factor receptor proto-oncogene mRNA in regenerating rat liver

The expression of EGF receptor mRNAs in regenerating rat liver was measured using two nonoverlapping cDNA probes for the human gene from a highly conserved region. These probes (pE7 and pE62) both hybridized to RNA species of 10 and 6 kb. The 10 and 6 kb RNA species were shown to decrease in the first 12 hours after partial hepatectomy. However, significant increases above control levels were noted at 24h and 72h. The level of alpha-actin mRNA increased as has been previously reported. These results suggest that a transcriptional and/or a posttranscriptional regulatory mechanism exists in regenerating rat liver with respect to EGF receptor gene expression.

Interferon regulatory factor-1 is a major regulator of epidermal growth factor receptor gene expression

Overexpression of the epidermal growth factor receptor (EGFR) occurs in many tumors and in breast cancer correlates with poor prognosis for treatment. Here, we report that interferon regulatory factor‐1 (IRF‐1) induces EGFR promoter activity up to 200‐fold compared to 3–10‐fold induction by other regulators. The region of the promoter that is required for this induction was defined using deletion mutants. In addition, we found that IRF‐1 and tricostatin A, a deacetylase inhibitor, have a synergistic effect on EGFR promoter activity. This indicates that the increase in EGFR promoter activity by IRF‐1 may also involve changes in chromatin structure. These results identify IRF‐1 as a major regulator of EGFR gene expression.

Cloning and Characterization of the Promoter Region of the Rat Epidermal Growth Factor Receptor Gene and Its Transcriptional Regulation by Nerve Growth Factor in PC12 Cells

Our previous studies have shown that treatment of PC12 cells with nerve growth factor (NGF) causes a profound down-regulation of the epidermal growth factor receptor (EGFR) mRNA and protein. Further, the NGF-induced down-regulation of the EGFR is under transcriptional control. To elucidate the molecular mechanism of this down-regulation we have cloned a 2.7-kilobase sequence from the promoter region of the rat EGFR from a rat P1 library. Six transcriptional start sites were identified by 5′-rapid amplification of cDNA ends and primer extension. Sequence analysis showed a 62% overall homology with the human EGFR promoter region. To investigate its transcription, 1.1 kilobases of the 5′-flanking sequence were fused to a luciferase reporter gene. This sequence exhibited functional promoter activity in transient transfection experiments with PC12, C6, and CV-1 cells. Treatment of PC12 cells with NGF inhibited promoter activity. By transfection of promoter deletion constructs, a silencer element was found between nucleotides −260 and −181, and TCC repeat sequences appeared to be at least partially responsible for the down-regulation of the EGFR by NGF. Supportive evidence for the relevance of this sequence was obtained from gel mobility shift assays and by transfection of TCC mutation constructs. Our results demonstrate that TCC repeat sequences are required for the down-regulation of rat EGFR by NGF in PC12 cells and may lead to the identification of the NGF-responsive transcription factors.

GC-binding factor 2 interacts with dishevelled and regulates Wnt signaling pathways in human carcinoma cell lines.

GC‐binding factor 2 (GCF2), a transcriptional repressor that decreases the activity of several genes is capable of binding directly to the GC‐rich sequence of the EGFR promoter and repressing the transcriptional activity of EGFR. In addition to its function as a transcriptional repressor, GCF2 can directly interact with other proteins such as flightless‐1 (Fli‐1). Many previous findings pertaining to the function of Fli‐1 have suggested a role for fli‐1 in providing a direct link between molecules involved in signal transduction pathways and the actin cytoskeleton. We hypothesized that GCF2, together with Fli‐1, plays a role in regulating cytoskeleton function, cell migration, and/or morphology. In our study, we observed that GCF2 is crucial for the activation of RhoA, a small GTPase that plays a key role in the regulation of the actin cytoskeleton. RhoA was markedly inactivated as a result of the decreased expression of GCF2. Co‐immunoprecipitations were subsequently performed to further investigate the mechanism for the repressive function. We identified dishevelled (Dvl), which is the key mediator for the Wnt pathway, as a binding partner with GCF2. These results strongly suggest that GCF2 plays a role in the Wnt‐noncanonical planar cell polarity (PCP) signaling pathway. Consequently, GCF2 may regulate the cytoskeleton or migration via Dvls and RhoA.

Insulin increases transcription of rat gene 33 through cis-acting elements in 5'-flanking DNA

Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, we have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequences in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme. This expression was increased by insulin treatment in a fashion resembling the effect of this hormone on transcription of the native gene. In vitro transcription assays in nuclear extracts also revealed increased transcription of the chimeric plasmids when the extracts were prepared from insulin-treated rat hepatoma cells. The results demonstrate that induction by insulin is mediated by cis-acting nucleotide sequences located between bp -480 to +27 relative to the tsp.