Functional analysis of GC element binding and transcription in the hamster dihydrofolate reductase gene promoter

Swick, Andrew G.; Blake, M C; Kahn, Jeanne; Azizkhan, Jane Clifford

doi:10.1093/nar/17.22.9291

Cited by 122 publications

(111 citation statements)

References 38 publications

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“…1A. This promoter fragment, which has maximal activity in in vitro transcription and transient expression assays (44), contains four GC boxes that will be referred to as GC boxes I, II, III, and IV. The study described here addresses the functional role of GC boxes in controlling initiation of transcription and start site utilization in the DHFR promoter.…”

Section: Resultsmentioning

confidence: 99%

Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.

Blake¹,

Jambou²,

Swick³

et al. 1990

Mol. Cell. Biol.

260

161

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Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.Transcription of eucaryotic promoters by RNA polymerase II involves multiple sequence elements and protein factors that associate with these sequences. Certain DNAprotein interactions regulate the efficiency of transcriptional initiation, while others have the additional role of specifying the transcriptional initiation site. In many class II gene promoters, a TATAA sequence element is located approximately 25 to 30 bp upstream of the transcription start site (5, 17); interaction of a factor(s) with this sequence specifies the site of initiation in many of these promoters (24, 41). However, in other eucaryotic promoters (7,25,47), interaction with TATAA appears not to specify the start site but rather to control the efficiency of transcription from a downstream initiation site. Another common important control element, CCAAT, is the target of factors that regulate the efficiency of transcription (13,24).A large subclass of polymerase II promoters lacks both TATAA and CCAAT sequence motifs but contains multiple GC boxes. This promoter class includes several housekeeping genes (e.g., the genes encoding dihydrofolate reductase [DHFR] (26), GCF-1 (27), and AP2 (35) have also been shown to interact with GC boxes. Determining the functional role of multiple GC boxes in the absence of TATAA and CCAAT motifs is crucial to the understanding of transcriptional regulation of this important class of promoters.It has been shown that GC boxes are required for efficient promoter activity in the...

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Section: Resultsmentioning

confidence: 99%

Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.

Blake¹,

Jambou²,

Swick³

et al. 1990

Mol. Cell. Biol.

260

161

View full text Add to dashboard Cite

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“…The role of E2F in cell growth regulated transcription of the dhfr promoter has been emphasized by the groups of Azizkhan Wade et al, 1992) and Farnham (Means et al, 1992;Slansky et al, 1993;Li et al, 1994). On the other hand, Sp1 powerfully activates DHFR transcription (Farnham and Shimke, 1986;Swick et al, 1989;Blake et al, 1990;Ciudad et al, 1992), and given this essential role of Sp1 in activating transcription, we have explored the possibility of the interaction of Sp1 with the cell cycle regulator Rb. The main conclusion of this study is that the retinoblastoma gene product Rb forms a complex with Sp1, as indicated by the supershift, co-immunoprecipitation, and immunodepletion experiments.…”

Section: Discussionmentioning

confidence: 99%

Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter

et al. 1998

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The dihydrofolate reductase (dhfr) promoter is powerfully activated by the transcription factor Sp1. It has been suggested that Sp1 is a potential target for transcriptional regulation by the cell cycle regulator retinoblastoma protein (Rb), and so we have explored this possibility using the hamster dhfr gene as a model. By the use of DNA probes from the hamster dhfr gene promoter, containing the most proximal GC box (minimal promoter), and nuclear extracts from cultured hamster cells (CHO K1), we show that polyclonal and monoclonal antibodies against Rb supershift the binding of Sp1. Nuclear extract immunoprecipitation with antiRb followed by Western analysis using anti-Sp1 also shows that Rb is complexed to Sp1. Complementary Immunoprecipitation/WB analysis shows both forms of Rb protein in the anti-Sp1 immunoprecipitates. Moreover, nuclear extract immunodepletion of Rb abolishes Sp1 gel-shift. The interaction between Rb and Sp1 is maintained in all the phases of the cell cycle. Transient overexpression of Rb in dhfr negative cells co-transfected with a dhfr minigene driven by its minimal promoter increases DHFR activity and potentiates transcription when overexpressing Sp1. Both e ects are severely reduced when the co-transfections are performed with a homologous dhfr minigene containing a single point mutation in the GC box. Thus, the activation by Rb of the dhfr gene may be exerted through Sp1. Stable transfectants of pCMVRb in K1 cells show an increase in both mRNA and DHFR activity. It is concluded that Sp1 is physically associated with Rb, and that this association increases Sp1-mediated transcription of the hamster dhfr gene.

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“…Radiolabeled probes for standard and quantitative protein/DNA binding assays were prepared from six double-stranded 60-mers, each carrying a single promoter-derived Spbinding site flanked by common nucleotide sequences. The promoterderived sequences utilized for these experiments are as follows: p21, 5Ј-CCCGCCTCCT-3Ј (25); MDR-1, 5Ј-CGCCGGGGCGTGGGC-3Ј (25); DHFR1, 5Ј-AGGGCGTGGC-3Ј (29); DHFR2, 5Ј-GAGGCGGGGC-3Ј (29); DHFR3, 5Ј-GAGGCGGAGT-3Ј (29); DHFR4, 5Ј-TGGGCGGGG-C-3Ј (29).…”

Section: Methodsmentioning

confidence: 99%

“…Vivo-To begin to analyze Sp2 function, we compared its capacity to stimulate the transcription of DHFR, a well characterized target of Sp-mediated transcription, with that of Sp1 and Sp3 in Drosophila SL2 cells (23,24,29). SL2 cells are a convenient milieu for such studies, since they lack transcription factors closely related to Sp family members although they support Sp-mediated transcription.…”

Section: Sp2 Is a Relatively Weak Trans-activator Of The Hamster Dhfrmentioning

confidence: 99%

Sp2 DNA Binding Activity and trans-Activation Are Negatively Regulated in Mammalian Cells

Moorefield

Fry

Horowitz

2004

Journal of Biological Chemistry

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Functional analysis of GC element binding and transcription in the hamster dihydrofolate reductase gene promoter

Cited by 122 publications

References 38 publications

Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.

Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.

Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter

Sp2 DNA Binding Activity and trans-Activation Are Negatively Regulated in Mammalian Cells

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