In Vivo Requirement of Activator-Specific Binding Targets of Mediator

Park, Jin Mo; Kim, Hye-Suk; Han, Sang Jun; Hwang, Moonsun; Lee, Young Chul; Kim, Young-Joon

doi:10.1128/mcb.20.23.8709-8719.2000

Cited by 113 publications

(114 citation statements)

References 50 publications

(54 reference statements)

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“…For example, in some assays direct interactions between Gal4 and Mediator have been detected (Koh et al 1998;Park et al 2000;Jeong et al 2001). Consistent with this possibility, Bryant and Ptashne (2003) reported that Mediator was recruited to the GAL1 promoter in an spt20⌬ mutant, prompting the suggestion that in the initial stages of activation, SAGA and Mediator are independently contacted and recruited by Gal4.…”

Section: An Ordered Protein-protein Interaction Pathway For Transcripsupporting

confidence: 52%

“…The well-characterized acidic activator Gal4 is responsible for the transcriptional stimulation of GAL genes, such as GAL1, which contain Gal4-binding sites in their promoters (Johnston 1987;Johnston and Carlson 1992;Dudley et al 1999). A variety of transcriptional components have been proposed to be the target of Gal4 including TBP (Melcher and Johnston 1995;Wu et al 1996), TFIIB (Wu et al 1996), Srb4 (Koh et al 1998;Park et al 2000), Gal11 (Jeong et al 2001), the Swi/Snf complex (Neely et al 2002), and the SAGA (Spt/Ada/ Gcn5/acetyltransferase) complex (Bhaumik and Green 2001;Brown et al 2001;Larschan and Winston 2001). These proposals are based on either in vitro protein interaction studies or inferences from various indirect in vivo experiments, and to date there is no definitive evidence that Gal4 directly interacts with any of these putative targets in vivo.…”

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confidence: 99%

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In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

Bhaumik¹,

Raha²,

Aiello³

et al. 2004

Genes Dev.

174

217

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Our understanding of eukaryotic transcriptional activation mechanisms has been hampered by an inability to identify the direct in vivo targets of activator proteins, primarily because of lack of appropriate experimental methods. To circumvent this problem, we have developed a fluorescence resonance energy transfer (FRET) assay to monitor interactions with transcriptional activation domains in living cells. We use this method to show that the Tra1 subunit of the SAGA (Spt/Ada/Gcn5/acetyltransferase) complex is the direct in vivo target of the yeast activator Gal4. Chromatin-immunoprecipitation experiments demonstrate that the Gal4-Tra1 interaction is required for recruitment of SAGA to the upstream activating sequence (UAS), and SAGA, in turn, recruits the Mediator complex to the UAS. The UAS-bound Mediator is required for recruitment of the general transcription factors to the core promoter. Thus, our results identify the in vivo target of an activator and show how the activator-target interaction leads to transcriptional stimulation. The FRET assay we describe is a general method that can be used to identify the in vivo targets of other activators. Transcription initiation by RNA polymerase II involves the assembly of general transcription factors (GTFs) on the core promoter to form a preinitiation complex (PIC). A variety of studies indicate that promoter-specific activator proteins (activators) work, at least in part, by increasing PIC formation (Orphanides et al. 1996;Roeder 1996;Ptashne and Gann 1997;. Activator-mediated stimulation of PIC assembly is believed to result from a direct interaction between the activation domain (AD) and one or more components of the transcription machinery, termed the "target." The unambiguous identification of the direct in vivo targets of activators has been a major challenge in the field.Transcriptional induction of genes involved in galactose utilization (GAL genes) has been a model experimental system for studying transcriptional activation mechanisms. The well-characterized acidic activator Gal4 is responsible for the transcriptional stimulation of GAL genes, such as GAL1, which contain Gal4-binding sites in their promoters (Johnston 1987;Johnston and Carlson 1992;Dudley et al. 1999). A variety of transcriptional components have been proposed to be the target of Gal4 including TBP (Melcher and Johnston 1995;Wu et al. 1996), TFIIB (Wu et al. 1996), Srb4 (Koh et al. 1998Park et al. 2000), Gal11 (Jeong et al. 2001), the Swi/Snf complex (Neely et al. 2002), and the SAGA (Spt/Ada/ Gcn5/acetyltransferase) complex (Bhaumik and Green 2001;Brown et al. 2001;Larschan and Winston 2001). These proposals are based on either in vitro protein interaction studies or inferences from various indirect in vivo experiments, and to date there is no definitive evidence that Gal4 directly interacts with any of these putative targets in vivo. The major obstacle has been the lack of an experimental strategy to measure direct interactions with transcriptional ADs in vivo.The development of spectra...

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Section: An Ordered Protein-protein Interaction Pathway For Transcripsupporting

confidence: 52%

mentioning

confidence: 99%

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

Bhaumik¹,

Raha²,

Aiello³

et al. 2004

Genes Dev.

174

217

View full text Add to dashboard Cite

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“…Mutations in the tail module components sin4 and pgd1 showed both growth defects and suppression when combined with swi6, but a third tail component, gal11, showed neither. It has been suggested that GAL11 mutants prevent association of other tail module subunits (Lee et al 1999;Park et al 2000). If that were the case, we would expect the gal11 phenotypes to include all the phenotypes associated with loss of Sin4 and Pgd1.…”

Section: Discussionmentioning

confidence: 99%

Genetic Interactions Between Mediator and the Late G1-Specific Transcription Factor Swi6 in Saccharomyces cerevisiae

Quinton²,

Miles

et al. 2005

Genetics

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Swi6 associates with Swi4 to activate HO and many other late G 1 -specific transcripts in budding yeast. Genetic screens for suppressors of SWI6 mutants have been carried out. A total of 112 of these mutants have been identified and most fall into seven complementation groups. Six of these genes have been cloned and identified and they all encode subunits of the mediator complex. These mutants restore transcription to the HO-lacZ reporter in the absence of Swi6 and have variable effects on other Swi6 target genes. Deletions of other nonessential mediator components have been tested directly for suppression of, or genetic interaction with, swi6. Mutations in half of the known subunits of mediator show suppression and/or growth defects in combination with swi6. These phenotypes are highly variable and do not correlate with a specific module of the mediator. Mutations in tail module components sin4 and pgd1 showed both growth defects and suppression when combined with swi6, but a third tail component, gal11, showed neither. A truncated form of the essential Srb7 mediator subunit also suppresses swi6 mutations and shows a defect in recruitment of the tail module components Sin4, Pgd1, and Gal11 to the mediator complex.

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“…This is one of the few examples of the complete elimination of AD function by mutation of a Mediator subunit. Other studies have shown partial loss of AD function for specific activators in yeast (Suzuki et al, 1988;Park et al, 2000), mammalian (Ito et al, 2000) and Drosophila cells as the result of elimination or knockdown of specific Mediator subunits. Significant AD function is retained in these examples, suggesting that alternative activation mechanisms still function in the absence of these Mediator subunits.…”

Section: E1a Conserved Region 3 Activation Of Early Viral Gene Expresmentioning

confidence: 99%

Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus

2005

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Adenovirus continues to be an important model system for investigating basic aspects of cell biology. Interactions of several cellular proteins with E1A conserved regions (CR) 1 and 2, and inhibition of apoptosis by E1B proteins are required for oncogenic transformation. CR2 binds RB family members, de-repressing E2F transcription factors, thus activating genes required for cell cycling. E1B-19K is a BCL2 homolog that binds and inactivates proapoptotic BAK and BAX. E1B-55K binds p53, inhibiting its transcriptional activation function. In productively infected cells, E1B-55K and E4orf6 assemble a ubiquitin ligase with cellular proteins Elongins B and C, Cullin 5 and RBX1 that polyubiquitinates p53 and one or more subunits of the MRN complex involved in DNA doublestrand break repair, directing them to proteosomal degradation. E1A CR3 activates viral transcription by interacting with the MED23 Mediator subunit, stimulating preinitiation complex assembly on early viral promoters and probably also the rate at which they initiate transcription. The viral E1B-55K/E4orf6 ubiquitin ligase is also required for efficient viral late protein synthesis in many cell types, but the mechanism is not understood. E1A CR1 binds several chromatin-modifying complexes, but how this contributes to stimulation of cellular DNA synthesis and transformation is not clear. E1A CR4 binds the CtBP corepressor, but the mechanism by which this modulates the frequency of transformation remains to be determined. Clearly, adenovirus has much left to teach us about fundamental cellular processes.

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In Vivo Requirement of Activator-Specific Binding Targets of Mediator

Cited by 113 publications

References 50 publications

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

Genetic Interactions Between Mediator and the Late G1-Specific Transcription Factor Swi6 in Saccharomyces cerevisiae

Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus

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