Regulation of Progesterone Receptor Activity in Cell Culture Systems and Cell-Free Transcription

Vaßen, Lothar; Klotzbücher, Michael; Ulber, Verena; Ryffel, Gerhart U.; Klein-Hitpaß, Ludger

doi:10.1007/978-3-662-03011-0_13

Cited by 3 publications

(5 citation statements)

References 43 publications

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“…As shown in Fig. 1A, 35 S-labeled dTAF II 110 was retained on beads containing DNA-bound MH 6 -hPR0, whereas no binding was observed on DNA beads lacking MH 6 -hPR0 (compare lanes 6 and 5), demonstrating that dTAF II 110 does not stably bind to DNA on its own. In contrast, control beads lacking DNA fragments, which did not contain bound PR after washing (data not shown), did not bind dTAF II 110 (lanes 4 and 3).…”

Section: Resultsmentioning

confidence: 92%

“…In contrast, control beads lacking DNA fragments, which did not contain bound PR after washing (data not shown), did not bind dTAF II 110 (lanes 4 and 3). To explore the specificity of the interaction, we tested whether DNA-bound MH 6 -hPR0 would interact under the same conditions with 35 S-labeled luciferase (Fig. 1B) or human TFIIB (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For analysis of the transcriptional activation potential of the DBD of the hPR in vitro, we used a cell-free transcription system, which uses rat liver nuclear extract as a source for general transcription factors (29,34,35). Two different test genes containing a TATA box or two PREs and a TATA box in front of a G-free cassette (33) were transcribed simultaneously in all reactions.…”

Section: Sequences Comprising the Dtaf II 110 Interaction Domain Of Hmentioning

confidence: 99%

“…E. coli mock extract, recombinant MH 6 -hPR(DBD), and recombinant MH 6 -hPR0 added to some of the reactions were expressed in E. coli or Sf21 cells infected with recombinant baculovirus, respectively, and purified as described above. The supercoiled transcription templates PRE 2 TATA-G300 and TATA-G400 containing G-free cassettes (33), reaction conditions, and analysis of transcripts were as previously described (29,34,35). Quantitation of transcripts was performed by autoradiography and subsequent densitometry.…”

Section: Hpr-dtaf II 110 Interactionmentioning

confidence: 99%

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Identification of a Transactivation Function in the Progesterone Receptor That Interacts with the TAFII110 Subunit of the TFIID Complex

Schwerk

Klotzbücher

Sachs

et al. 1995

Journal of Biological Chemistry

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Transcriptional activation of target genes by the human progesterone receptor is thought to involve direct or indirect protein-protein interactions between the progesterone receptor and general transcription factors. A key role in transcription plays the general transcription factor TFIID, a multiprotein complex consisting of the TATA-binding protein and several tightly associated factors (TAFs). TAFs have been shown to be required for activated transcription and are, thus, potential targets of activator proteins. Using in vitro interaction assays, we could identify specific interactions between the progesterone receptor and the TATA-binding protein-associated factor dTAF II 110. The dTAF II 110 domain responsible for the interaction is distinct from that reported to suffice for binding to Sp1. Somewhat surprisingly, deletion analysis indicated that the previously identified activation functions 1 and 2 of the progesterone receptor are not required for this interaction but pointed to an important role of the DNA binding domain. In cotransfection experiments and an in vitro transcription assay, the DNA binding domain of the progesterone receptor displayed significant activation potential. These findings, taken together, suggest that an interaction between the progesterone receptor and TAF II 110 may represent an important step in the mechanism of activation.In eukaryotes, at least seven basal transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIJ) are required for basal levels of transcription at promoters by RNA polymerase II in vitro (reviewed in Ref. 1). In mammalian systems, these factors assemble into functional initiation complexes in a highly ordered, stepwise fashion beginning with the binding of TFIID to the TATA box (1-3), while in yeast a number of factors seem to be brought in as a preassembled RNA polymerase II holoenzyme complex (4). Sequence-specific activators that bind to specific regulatory elements within distal promoter regions or enhancers are capable of stimulating the rate of transcription initiation. Although the precise mechanism is presently unknown, there is increasing support for models proposing that protein-protein interactions between activators and basal transcription factors play a decisive role in this process (5). Such interactions might help to recruit these basal transcription factors or the preassembled RNA polymerase II holoenzyme complex to the TATA box, stabilize intermediates in the assembly of the initiation complex, or activate certain components by inducing conformational changes.Biochemical and molecular characterization of TFIID has shown that it is a multi-subunit complex consisting of the TATA-binding protein (TBP) 1 and a number of TBP-associated factors (TAFs) (6 -8), ranging from 20 to 250 kDa in size. In cell-free transcription systems reconstituted from partially purified factors, recombinant TBP is able to support basal transcription of TATA-only promoters (9, 10). However, unlike TFIID, TBP fails to mediate transcriptional stimulation b...

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Sequences Comprising the Dtaf II 110 Interaction Domain Of Hmentioning

confidence: 99%

Section: Hpr-dtaf II 110 Interactionmentioning

confidence: 99%

See 2 more Smart Citations

Identification of a Transactivation Function in the Progesterone Receptor That Interacts with the TAFII110 Subunit of the TFIID Complex

Schwerk

Klotzbücher

Sachs

et al. 1995

Journal of Biological Chemistry

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“…Type I antiprogestins (13α-methyl substituted 19-nor-steroids) represented by onapristone (46), have been shown to prevent the association of the hPR to DNA. Type II antagonists (13β-methyl 19-nor-steroids) represented by RU-486 or ZK-98734 (lilopristone), appeared to promote stable binding of the receptor to DNA (29, 47,48) in in vitro binding studies. Based on these findings, it was proposed that the type I compounds may hamper hPR-mediated transactivation by preventing binding of hPR to progesterone response elements (PRE), whereas the type II compounds may act at steps downstream of DNA binding by preventing interaction with coactivators and/or enhancing interaction with corepressors (29, 44,49).…”

Section: Different Types Of Antagonistsmentioning

confidence: 99%

Progesterone receptor antagonists (antiprogestins)

Hess‐Stumpp¹,

Hoffmann²,

Fuhrmann³

2002

Drugs of the Future

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Recent developments in molecular action of antihormones

Fuhrmann¹,

Parczyk²,

Klotzbücher³

et al. 1998

Journal of Molecular Medicine

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Antihormones are by definition antagonists of steroid hormone action. They interact with the ligand binding domains of steroid hormone receptors and competitively inhibit the action of the receptors by mechanisms that are not quite understood. In certain cases antihormones also exhibit agonistic activity especially in connection with certain naturally occurring receptor mutants. These observations together with findings of indiscriminate interaction of antihormones with several classes of steroid receptors have necessitated a search of more effective and reliable antihormones. Recent advances in the resolution of the crystal structure of the ligand binding domains of certain members of the steroid receptor family and identification of non-liganded activation of steroid receptors have produced considerable information that can be harnessed into a fruitful search for a new generation of antihormones.

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Regulation of Progesterone Receptor Activity in Cell Culture Systems and Cell-Free Transcription

Cited by 3 publications

References 43 publications

Identification of a Transactivation Function in the Progesterone Receptor That Interacts with the TAFII110 Subunit of the TFIID Complex

Identification of a Transactivation Function in the Progesterone Receptor That Interacts with the TAFII110 Subunit of the TFIID Complex

Progesterone receptor antagonists (antiprogestins)

Recent developments in molecular action of antihormones

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