Multiple Transcription Start Sites, DNase I-Hypersensitive Sites, and an Opposite-Strand Exon in the 5′ Region of the CHO dhfr Gene

Abstract

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.

Proximal and Long-Range Alterations in Chromatin Structure Surrounding the Chinese Hamster Dihydrofolate Reductase Promoter

“…The gene encoding this enzyme is constitutively expressed in all tissues. The DHFR genes from mouse, human, and Chinese hamster have been cloned, mapped, and partially sequenced (Azizkhan et al, 1986;Carothers et al, 1983;Chen et al, 1984;Crouse et al, 1985; Hamlin & Ma, 1990;McGrogan et al, 1985;Milbrandt et al, 1983;Mitchell et al, 1986; Nunberg et al, 1980;Yang et al, 1984). Each gene has six exons, and the sequences of the coding regions and the 3' untranslated region up to the first polyadenylation site, as well as the positions of intron-exon boundaries, are highly conserved.…”

“…In the promoter region of the murine, Chinese hamster, and human genes, four highly conserved elements are arranged very similarly with respect to one another (Azizkhan et al, 1986;Chen et al, 1984;McGrogan et al, 1985;Mitchell et al, 1986). None of the three promoters has canonical TATA or CAAT boxes.…”

“…None of the three promoters has canonical TATA or CAAT boxes. Instead, each gene displays several potential Spl binding sites (GC boxes) lying both proximal and distal to the major DHFR transcription start sites (Azizkhan et al, 1986;Chen et al, 1984;Famham et al, 1986;McGrogan et al, 1985;Mitchell et al, 1986). In the case of the murine DHFR gene, at least one of the GC boxes has been shown to be required for efficient gene fThis work was supported by NIH Grant ROl GM26108, by an NIH Fogarty International Research Collaboration Award, and by the Russian State Program, "Frontiers in Genetics".…”

“…The DHFR promoter is considered to be bidirectional, since a divergently transcribed gene has been identified in murine, Chinese hamster, and human systems (Crouse et al, 1985;Fujii et al, 1992;Linton et al, 1989;Mitchell et al, 1986;Shimada et al, 1989;Smith et al, 1990). In the murine and Chinese hamster genes, these transcripts initiate at ~-300 and -195 bp, respectively, relative to the start codon of the DHFR gene.…”

“…In the murine and Chinese hamster genes, these transcripts initiate at ~-300 and -195 bp, respectively, relative to the start codon of the DHFR gene. The distal clusters of GC boxes mapping between -700 and -400 relative to the start codons of the DHFR genes are therefore incorporated into the bodies of the divergent genes (Crouse et al, 1985;Famham & Schimke, 1986;Mitchell et al, 1986).…”