In Vitro Transcription Enhancement by Purified Derivatives of the Glucocorticoid Receptor

Freedman, Leonard P.; Yoshinaga, Steven K.; Vanderbilt, Jeff N.

doi:10.1126/science.2473529

Cited by 108 publications

(40 citation statements)

References 33 publications

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“…netically map an enhancement region [enhl] within the finger domain, extending the findings that this portion of the receptor alone is sufficient for enhancement, al beit with substantially reduced activity, both in vivo ) and in vitro (Freedman et al 1989). In this regard, it is particularly intriguing that deletion mutagenesis (Giguere et al 1986), as well as direct ac tivity assays , identified a second enhancement region, enh2, near the amino terminus of the receptor, and other work has inferred yet another distinct enhancement region within the receptor (Hollenberg and Evans 1988; see also Godowski et al 1988).…”

Section: Discussionmentioning

confidence: 70%

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Schena¹,

Freedman²,

Yamamoto³

1989

Genes Dev.

230

125

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Mammalian glucocorticoid receptors bind specifically to glucocorticoid response element (GRE) DNA sequences and enhance transcription from GRE-linked promoters in mammalian cells and in yeast. We randomly mutagenized a segment of the receptor encompassing sequences responsible for DNA-binding and transcriptional regulation and screened in yeast for receptor defects. The mutations all mapped to a 66-aminoacid subregion that includes two zinc fingers; in general parallel phenotypes were observed in yeast and animal cells. Mutants defective for DNA binding also failed either to enhance or to repress transcription. However, several mutations in the second finger selectively impaired enhancement; we suggest that such 'positive control' mutants may alter protein-protein contacts required for transcriptional activation.

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Section: Discussionmentioning

confidence: 70%

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Schena¹,

Freedman²,

Yamamoto³

1989

Genes Dev.

230

125

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“…Beads were washed three times in pull-down buffer and then incubated with 4 g of GR fragment EX556 (Freedman et al, 1989) containing NL1 for 3 h at 4°C. Bound proteins were resolved and immunoblotted with the BuGR2 antibody.…”

Section: Binding Assaysmentioning

confidence: 99%

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

Freedman

2004

MBoC

Self Cite

192

151

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The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin ␣ selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin ␣-importin ␤ heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin ␣ bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding. INTRODUCTIONTo survive in a complex environment, cells sense external cues and commonly respond by altering gene expression, in part by regulating the intracellular localization and activity of transcriptional regulatory factors. For example, the intracellular localization of the yeast Pho4 and mammalian SREBP factors is altered in response to changes in external phosphate and circulating sterol concentrations, respectively (Kaffman et al., 1998b;Nagoshi et al., 1999). Similarly, changes in blood glucose levels and a wide range of physiological stress signals modulate the circulating level of glucocorticoids, changing the activity and localization of the glucocorticoid receptor (GR). Within minutes of glucocorticoid binding, GR enters the nucleus and activates or represses target gene transcription (Picard and Yamamoto, 1987). However, hormone binding is reversible; after hormone withdrawal, GR locates back to the cytoplasm (t 1/2 ϭ 10 h; Hache et al., 1999).Proteins and RNAs enter and exit the nucleus through the nuclear pore, a 125-MDa complex in the nuclear envelope (Stoffler et al., 1999). Small molecules readily diffuse through the nuclear pore, whereas molecules greater than ϳ40 kDa require import and export machinery for transport (Davis, 1995;Pante and Aebi, 1995). The first nuclear localization sequence (NLS) was identified in the SV40 large T-antigen (Kalderon et al., 1984). Development of an in vitro nuclear import assay facilitated the identification of two proteins that import substrates containing SV40 NLS-like domains. The adapter molecule importin ␣ binds to the NLS of the substrate and the importin ␤ transport receptor, creating a trimeric complex that imports the substrate into the nucleus through the nuclear pore (Strom and Weis, 2001).Based on similarity to importin ␤, a family of nuclear import and export receptors was identified and shown to transport a wide variety of proteins and RNAs into and out of the nucleus. After reaching the nucleoplasm, import receptors bind the...

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“…To create an in-frame fusion, these intermediate plasmids were restricted with XmaI and XhoI, filled in with Klenow, and religated to yield pETI407-5251-HMK and pET(407-525-K461Aj-HMK. Finally, a BstBI-XbaI fragment from T7-EX556 (Freedman et al 1989) was cloned into the BstBI and XbaI sites of pET(407-525t HMK and pET[407-525-K461Al-HMK to create PET-EX525-HMK and PET-EX525(K461AtHMK. The resulting derivatives have a 5 amino-acid heart muscle kinase site (Arg-Arg-AlaSer-Val) fused at their carboxyl termini.…”

Section: Plasmidsmentioning

confidence: 99%

“…Protein purification EX525-HMK and EX525(K461A)-HMK were overexpressed in, and purified from, the BL21/DE3 strain of Escherichia coli as described previously (Freedman et al 1989) except that only one column, CM-Sepharose, was used. Briefly, the cell pellets were processed as described and dialyzed in HZ buffer [lo% glycerol, 50 mM HEPES (pH 8.0), 0.5 mM EDTA] to 50 mM NaC1.…”

Section: Plasmidsmentioning

confidence: 99%

Intracellular receptors use a common mechanism to interpret signaling information at response elements.

1996

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The glucocorticoid receptor (GR) activates transcription in certain glucocorticoid response element (GRE) contexts, and represses or displays no activity in others. We isolated point mutations in one GRE, plfG, at which GR activated transcription under conditions in which the wild-type element was inactive or conferred repression, implying that GREs may carry signals that are interpreted by bound receptors. Consistent with this notion, we identified a mutant rat GR, K461A, which activated transcription in all GRE contexts tested, implying that this residue is important in interpretation of GRE signals. In a yeast screen of 60,000 GR mutants for strong activation from plfG, all 13 mutants isolated contained substitutions at K461. This lysine residue is highly conserved in the zinc-binding region (ZBR) of the intracellular receptor (IR) superfamily; when it was mutated in MR and RARP, the resulting receptors similarly activated transcription at response elements that their wild-type counterparts repressed or were inactive. We suggest that IR response elements serve in part as signaling components, and that a critical lysine residue serves as an allosteric "lock" that restricts IRs to inactive or repressing configurations except in response element contexts that signal their conversion to transcriptional activators. Therefore, mutation of this residue produces altered receptors that activate in many or all response element contexts.[Key Words: Glucocorticoid receptor; transcriptional repression; DNA-binding domain; context-dependent activity; intracellular receptor] Received January 1, 1996; accepted in revised form March 27, 1996.Regulation of transcription typically commences with the specific binding of a regulatory protein to a response element close to the target promoter; the regulator also interacts with other regulatory factors, with the transcription machinery andlor chromatin components, therefore modulating the rate of transcription initiation (for review, see Sheldon and Reinberg 1995;Hori and Carey 1994; Tjian and Maniatis 1994). Numerous regulators have been shown to activate transcription in certain cell types and at certain promoters, and to repress in other settings (Meyer et al. 1980;Keleher et al. 1988;Pedersen et al. 1991). Therefore, it is apparent that the precise activity of a regulator can be determined largely by its context-cell type, promoter, response element, and extracellular signals. The mechanisms by which regulators sense and interpret their contexts and thereby display distinct activities in different settings remain to be determined.The glucocorticoid receptor (GR) is a member of the intracellular receptor (IR) superfamily, which encompasses a large group of transcriptional regulators (Tsai Present addresses: 2Department

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In Vitro Transcription Enhancement by Purified Derivatives of the Glucocorticoid Receptor

Cited by 108 publications

References 33 publications

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities.

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

Intracellular receptors use a common mechanism to interpret signaling information at response elements.

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