Nucleotide sequence and nuclease hypersensitivity of the Chinese hamster dihydrofolate reductase gene promoter region

Azizkhan, Jane Clifford; Vaughn, James M.; Christy, Robert J.; Hamlin, Joyce L.

doi:10.1021/bi00368a059

Cited by 44 publications

(34 citation statements)

References 41 publications

(74 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…8B, the 4.6-kb E3a EcoRI band lies just upstream from the transcription start site for the DHFR gene (which lies in fragment E9). Interestingly, we have previously shown that this 4.6-kb fragment contains a prominent DNaseI-hypersensitive site (1). The data in Fig.…”

Section: Resultsmentioning

confidence: 56%

“…An equal volume of either 4 M NaCl or 10 mM LIS buffer was added, and the matrices were collected by centrifugation in the HB-4 rotor at 5,000 rpm for 20 min at 20°C. The pellet from 108 cells was washed at room temperature in matrix wash buffer containing 1 mM EDTA instead of MgCl2 and was then dissolved in 1 The relative amount of DNA remaining attached to the matrix after digestion was estimated by determining the proportion of acid-precipitable [3H]thymidine in this fraction relative to the total amount of label in the matrix preparation prior to digestion. DNA concentrations were additionally determined by a fluorimetric assay based on Hoechst staining (20).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Dijkwel¹,

Hamlin²

1988

Mol. Cell. Biol.

174

102

View full text Add to dashboard Cite

Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping -120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments.In the eucaryotic genome, the chromatin loop represents a basic structural unit. The loops are generated by periodic attachment of the chromatin fiber to a nonhistone chromosomal protein scaffolding in the nucleus, which has been referred to as the nuclear matrix (3,8,9,36). During mitosis, part of this matrix probably rearranges to become the metaphase chromosomal scaffold (2,19). Although neither the interphase nuclear matrix nor the mitotic chromosomal scaffold has been fully characterized as yet, topoisomerase II has been identified as an integral component of both (5,14). This finding has led to the suggestion that the nuclear matrix may somehow poise individual chromatin domains for transcription by allowing torsional stress to be introduced into defined regions (7). Nonrandom organization of the chromatin is further suggested by the presence at the base of the loops of specific DNA sequences that associate with the nuclear matrix. In Drosophila cells, specific matrix attachment regions (MARs) have been found in the 5'-flanking regions of several active genes (12,26,33), and these MARs coincide with enhancerlike elements (12). However, in the murine K light-chain gene, an attachment site is found at an intragenic site...

show abstract

Section: Resultsmentioning

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Dijkwel¹,

Hamlin²

1988

Mol. Cell. Biol.

174

102

View full text Add to dashboard Cite

show abstract

“…Spl binding sites vary in number, relation to the transcription start site, and to each other as well as among heterologous regulatory elements in the different promoters in which they are found. The most proximal Spl binding site is frequently positioned 40-60 bp upstream ofthe transcription start site (5,(32)(33)(34). Activation of transcription by Spl has been demonstrated in vitro and in vivo for several of these genes.…”

Section: Effect Ofmithramycin On Intracellular Dhfr Transcriptionmentioning

confidence: 99%

Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo.

Blume¹,

Snyder²,

Ray³

et al. 1991

J. Clin. Invest.

308

237

View full text Add to dashboard Cite

show abstract

“…The DHFR promoter contains multiple copies of GC boxes, which are capable of interacting with the transcription factor Spl (5). An additional conserved sequence including the major transcription start site has been identified in the DHFR promoter (1).…”

mentioning

confidence: 99%

Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo.

Blake¹,

Azizkhan²

1989

Mol. Cell. Biol.

290

252

View full text Add to dashboard Cite

The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors, Spl and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two-to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.Dihydrofolate reductase (DHFR) is a metabolic enzyme involved in the synthesis of purines, thymidylate, and glycine (23). It is considered a housekeeping enzyme, since it is found in small amounts in nearly all cell types and is required for cell growth (21). Although the gene encoding DHFR is widely expressed, this expression is influenced by several factors, including the growth state of the cells, the cell cycle, and the presence of viral infection (6,21,27). The DHFR promoter lacks TATAA and CCAAT DNA sequence motifs, which are regulatory sequence elements found in most eucaryotic promoters transcribed by RNA polymerase II. The DHFR promoter contains multiple copies of GC boxes, which are capable of interacting with the transcription factor Spl (5). An additional conserved sequence including the major transcription start site has been identified in the DHFR promoter (1).The eucaryotic transcription factor E2F was originally identified as a DNA-binding activity in HeLa nuclear extracts (12). This factor binds to two sites in a region of the adenovirus E2 promoter known to be required for E2 promoter activity. E2F binding activity increases upon infection of HeLa cells with wild-type adenovirus; this increase depends on the presence of a functional EIA gene (12). The adenovirus ElA gene product is known to trans-activate several viral and cellular genes by a variety of mechanisms. E2F is hypothesized to be one of the targets of the adenovirus ElA gene product early in adenovirus infection. E2F binding sites have also been identified in the ElA enhancer (13) and the c-myc promoter (10, 24); E2F binds to the sequence 5'-TTTCGCGC-3'. DHFR expression is increased by adenovirus infection, although the molecular level at which this increase occurs is unclear (7,27).One study of the mouse DHFR promoter reported that a fraction of HeLa nuclear extract enriched in Spl bound to the consensus Spl binding sites in the mouse DHFR promoter (5). It also showed that HeLa nuclear extracts depleted of this fraction were inc...

show abstract

Nucleotide sequence and nuclease hypersensitivity of the Chinese hamster dihydrofolate reductase gene promoter region

Cited by 44 publications

References 41 publications

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo.

Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo.

Contact Info

Product

Resources

About