Biochemical mechanism of transcriptional activation by GAL4-VP16

Tantin, Dean; Chi, Tianhuai; Hori, Roderick T.; Pyo, Sung-Woon; Carey, Michael

doi:10.1016/s0076-6879(96)74013-2

Cited by 22 publications

(25 citation statements)

References 37 publications

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“…Bound protein was eluted from the immobilized templates in 10 l of 2ϫ Laemmli buffer, fractionated by SDS-PAGE, and immunoblotted. Antibodies used in immunoblotting included MED23 (BD Pharmingen), Pol II C-terminal domain 8WG16 (QED Bioscience), TFIIB (27), and CHD1 (Bethyl Laboratories). All other antibodies were purchased from Santa Cruz Biotechnology.…”

Section: Methodsmentioning

confidence: 99%

Polycomb Repressive Complex 1 (PRC1) Disassembles RNA Polymerase II Preinitiation Complexes

Lehmann

Ferrari

Vashisht

et al. 2012

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Polycomb Repressive Complex 1 (PRC1) Disassembles RNA Polymerase II Preinitiation Complexes

Lehmann

Ferrari

Vashisht

et al. 2012

Journal of Biological Chemistry

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“…The cell lysate was precipitated by polyethyleneimine (PEI) and then by ammonium sulfate (45). The protein was loaded onto Q Sepharose and eluted with a linear gradient of 250 to 450 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

High-Resolution Mapping of Changes in Histone-DNA Contacts of Nucleosomes Remodeled by ISW2

Kassabov

Henry

Zofall

et al. 2002

Molecular and Cellular Biology

102

118

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The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA. The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach. The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed. Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template. In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting. ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA.The process of making DNA more or less accessible in eukaryotes plays a crucial role in the regulation of transcription, replication, recombination, and DNA repair. Several large multisubunit complexes reorganize chromatin in an ATPdependent manner to either activate or repress these cellular processes (26,44,51,53,56,57). ATP-dependent chromatin remodeling complexes contain an ATPase that has a wellconserved seven-domain region, and the ATPase activity is stimulated by DNA and nucleosomes (26). These chromatin remodeling complexes can be divided into four classes: the SWI/SNF and RSC, ISWI (for imitation SWI family), CHD/ Mi-2, and INO80 families (12,26,28,39,43,55). The ISWI protein in Drosophila is assembled into three distinct complexes (NURF, for nucleosome remodeling factor; CHRAC, for chromatin accessibility complex; and ACF, for ATP-utilizing chromatin assembly and remodeling factor), each with different subunit compositions and biochemical activities (9,22,23,47,49,52). In yeast, there are two ISWI proteins, referred to as Isw1p and Isw2p, which are closely related by sequence to the Drosophila ISWI protein and are associated with one to three other polypeptides (48).A general feature of the ISWI family of chromatin remodeling complexes is that they change the translational position of nucleosomes or slide nucleosomes (17,20,28,29). In addition, all of the ISWI complexes except for NURF have been shown to help regularly space nucleosomes. The spacing activities of ISW1 and ISW2 differ in that ISW1 produces more regularly spaced arrays than ISW2, and the spacing varied from ϳ175 to ϳ200 bp for ISW1 and ISW2, respectively (48). ISWI, SWI/SNF, RSC, and Mi-2 have all recently been shown to induce torsional strain on DNA during remodeling (21). A distinguishing characteristic of these complexes is that ISWI requires hist...

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“…Recombinant TFIIB was purified as described previously (22). Gal4-VP16 (amino acids 1 to 147 of Gal4 fused to amino acids 413 to 490 of VP16) was expressed in Escherichia coli and purified as described previously (33). GAL4-E1A (amino acids 1 to 147 of Gal4 fused to amino acids 121 to 223 of the adenovirus 2 large E1A protein) was expressed in E. coli and purified as described previously (42) except that SP-Sepharose (Pharmacia) was used and washed with 0.2 M KCl D buffer and eluted with 0.5 M KCl D buffer.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

Wang

Cantin

Stevens

et al. 2001

Molecular and Cellular Biology

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A number of mammalian multiprotein complexes containing homologs of Saccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (ϳ500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (ϳ2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at ϳ500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecularmass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 ؋ 10 5 to 6 ؋ 10 5 molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the ϳ2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The ϳ2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.The Saccharomyces cerevisiae Mediator complex was originally identified because of its ability to stimulate activated transcription in vitro. Many of the subunits of the purified Mediator complex (24) are encoded by SRB genes, first characterized as suppressors of a deletion in the C-terminal heptapeptide repeat (CTD) of the large subunit of RNA polymerase II (Pol II) (23). Additional Mediator subunits are encoded by genes initially identified in other genetic screens for mutations affecting gene control and are named accordingly (e.g., RGR-1). Mediator subunits not characterized previously were called Med1, Med2, etc. (24). Mediator subunits in yeast have also been purified as part of a still larger holoenzyme complex including Pol II and several general transcription factors (GTFs) (23). The Pol II holoenzyme analyzed by Young and colleagues (see reference 23 for a review) includes Srb8, Srb9, Srb10, and Srb11, whereas the Mediator complex studied by Myers et al. (24) lacks these subunits. The Srb8 to Srb11 subunits form a functional subcomplex of the holoenzyme required for repression by several yeast repressors (3,11,23). These subunits are regulated differently from other yeast Mediator and holoenzyme subunits. The intracellular Srb10 concentration falls dramatically as yeast cells depl...

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Biochemical mechanism of transcriptional activation by GAL4-VP16

Cited by 22 publications

References 37 publications

Polycomb Repressive Complex 1 (PRC1) Disassembles RNA Polymerase II Preinitiation Complexes

Polycomb Repressive Complex 1 (PRC1) Disassembles RNA Polymerase II Preinitiation Complexes

High-Resolution Mapping of Changes in Histone-DNA Contacts of Nucleosomes Remodeled by ISW2

Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

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