Structure of the VP16 transactivator target in the Mediator

Milbradt, Alexander G.; Kulkarni, Madhura; Yi, Tongxun; Takeuchi, Koh; Sun, Zhenyu; Luna, Rafael E.; Selenko, Philipp; Näär, Anders M.; Wagner, Gerhard

doi:10.1038/nsmb.1999

Cited by 79 publications

(151 citation statements)

References 44 publications

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“…We present several lines of evidence consistent with the idea that ATF6␣ recruits Mediator to ER stress response genes via direct interactions with its MED25 subunit. First, consistent with our previous observation that the VP16 AD competes with ATF6␣ for binding to Mediator in nuclear extracts (12), we observe that the purified ATF6␣ AD binds stably and specifically in vitro to the purified MED25 VBD, a MED25 domain previously shown to be the binding site on Mediator for the viral VP16 AD (13)(14)(15)(16)(17)(18). Second, in reconstitution experiments, we obtained evidence that Mediator containing a MED25 mutant that lacks the VBD fails to bind efficiently to ATF6␣ but binds to liganded thyroid receptor, which is known to bind to Mediator through LXXLL motifs in its MED1 subunit (33)(34)(35).…”

Section: Med25 Is Needed For Optimal Binding Of Atf6␣ To Mediator Andsupporting

confidence: 73%

“…Furthermore, we discovered that the VP16 AD potently competes with ATF6␣ for binding to the Mediator, raising the possibility that the ATF6␣ and VP16 ADs might bind to the same or overlapping surfaces on Mediator. In light of previous studies demonstrating that the VP16 AD binds to the Mediator through its MED25 subunit (13)(14)(15)(16)(17)(18), we initiated experiments to test the possibility that the ATF6␣ AD might also target Mediator through MED25. Below we present our findings, which identify MED25 as a docking site for transcription factor ATF6␣ on the multisubunit human Mediator complex.…”

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confidence: 99%

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Role for Human Mediator Subunit MED25 in Recruitment of Mediator to Promoters by Endoplasmic Reticulum Stress-responsive Transcription Factor ATF6α

Sela

Conkright

Chen

et al. 2013

Journal of Biological Chemistry

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Background: ATF6␣ recruits Mediator to activate endoplasmic reticulum stress response genes. Results: Mediator subunit MED25 contains an ATF6␣ binding site. Conclusion:The ATF6␣-MED25 interaction contributes to Mediator recruitment to genes. Significance: Learning how DNA binding transcription activators recruit coactivators to genes is important for understanding gene regulatory mechanisms.

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Section: Med25 Is Needed For Optimal Binding Of Atf6␣ To Mediator Andsupporting

confidence: 73%

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confidence: 99%

Role for Human Mediator Subunit MED25 in Recruitment of Mediator to Promoters by Endoplasmic Reticulum Stress-responsive Transcription Factor ATF6α

Sela

Conkright

Chen

et al. 2013

Journal of Biological Chemistry

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“…The function of most tested ADs is conserved among eukaryotes (15,16), even though some key activator targets are not conserved. For example, the herpes virus protein VP16 strongly activates transcription in both yeast and mammalian cells, although human Med25, a critical target of VP16 in humans, is not found in yeast (17,18). Thus, the ability to adapt to different coactivator targets is seemingly a key property of ADs.…”

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confidence: 99%

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

Warfield

Tuttle

Pacheco

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

100

173

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Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.Mediator complex | protein NMR T ranscription activators are regulators of cell identity, cell growth, and the response to environmental conditions. These highly regulated factors contain one or more activation domains (ADs) that typically bind coactivators-the complexes that contact the transcription machinery and/or have chromatin modifying activity (1-5). AD-coactivator binding initiates a cascade of events leading to productive transcription including targeted chromatin remodeling and stimulation of both RNA polymerase II preinitiation complex formation and transcription elongation (6). Many broadly acting ADs bind several coactivators, allowing them to function at a wide range of promoters with different coactivator requirements (7)(8)(9)(10)(11)(12)(13)(14). The function of most tested ADs is conserved among eukaryotes (15, 16), even though some key activator targets are not conserved. For example, the herpes virus protein VP16 strongly activates transcription in both yeast and mammalian cells, although human Med25, a critical target of VP16 in humans, is not found in yeast (17,18). Thus, the ability to adapt to different coactivator targets is seemingly a key property of ADs.Defining what constitutes a functional AD has been difficult, because there is little apparent sequence similarity among different ADs. All structurally characterized eukaryotic ADs lack a stable 3D structure and are disordered in the absence of a coactivator target (19)(20)(21)(22)(23)(24)(25). Initial studies classified ADs based on enriched residues: acidic proline-, glutamine-, or serine-rich activators (26). However, these residue types later were found to be generally enriched in intrinsically disordered proteins and were termed "disorder-promoting residues" (27, 28).Attempts to define functional sequence motifs within ADs have led to ambiguous results. For many ...

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“…When the IE62 TAD and SRT were moved to the C-terminal end of the IR2P, the IR2P-62TS lost most of its trans-activation activity (Fig. 6C, bar 6 (45). VZV IE62 TAD shows 27% amino acid sequence identity with EHV-1 IEP TAD (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…6). Both the IE62 TAD and VP16 TAD interact with the VP16-binding domain (VBD; Med25 aa 390 to 553) (17,45) of Mediator 25 which is the same domain with an activator-interacting domain (ACID; Med25 aa 391 to 543) (61). However, the interaction between the IE62 TAD and Mediator 25 was considerably weaker than that involving the VP16 (17,25).…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62

Kim

Shakya

Kim

et al. 2016

J Virol

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The immediate early 62 protein (IE62) of varicella-zoster virus (VZV), a major viral trans-activator, initiates the virus life cycle and is a key component of pathogenesis. The IE62 possesses several domains essential for trans-activation, including an acidic trans-activation domain (TAD), a serine-rich tract (SRT), and binding domains for USF, TFIIB, and TATA box binding protein (TBP). Transient-transfection assays showed that the VZV IE62 lacking the SRT trans-activated the early VZV ORF61 promoter at only 16% of the level of the full-length IE62. When the SRT of IE62 was replaced with the SRT of equine herpesvirus 1 (EHV-1) IEP, its trans-activation activity was completely restored. Herpes simplex virus 1 (HSV-1) ICP4 that lacks a TAD very weakly (1.5-fold) trans-activated the ORF61 promoter. An IE62 TAD-ICP4 chimeric protein exhibited trans-activation ability (10.2-fold), indicating that the IE62 TAD functions with the SRT of HSV-1 ICP4 to trans-activate viral promoters. When the serine and acidic residues of the SRT were replaced with Ala, Leu, and Gly, trans-activation activities of the modified IE62 proteins IE62-SRT⌬Se and IE62-SRT⌬Ac were reduced to 46% and 29% of wild-type activity, respectively. Bimolecular complementation assays showed that the TAD of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with Mediator 25 in human melanoma MeWo cells. The SRT of IE62 interacted with the nucleolar-ribosomal protein EAP, which resulted in the formation of globular structures within the nucleus. These results suggest that the SRT plays an important role in VZV viral gene expression and replication. V aricella-zoster virus (VZV), a member of the Alphaherpesvirinae, is a ubiquitous human pathogen that causes varicella (chicken pox) in primary lytic infection and zoster (shingles) during reactivation from latent infection (1). VZV has a linear DNA genome of approximately 125 kbp that encodes at least 70 open reading frames (ORFs) (1). Herpesviruses with genomes of group D, such as equine herpesvirus 1 (EHV-1), bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and varicella-zoster virus (VZV), have a fixed long region (UL) covalently linked to a short (S) genomic region comprised of a pair of inverted repeat sequences that bracket a unique short segment (US) (2-4). Two alphaherpesviruses, VZV and EHV-1, are members of genus Varicellovirus and have very similar genomic structures (2, 4). The VZV immediate early 62 protein (IE62) of 1,310 amino acids (aa) is the major viral trans-activator and is essential for viral growth (1, 5, 6). IE62 is encoded by duplicated ORFs 62 and 71 and, as a tegument protein, is delivered to newly infected cell nuclei, where it initiates VZV replication by transactivating viral immediate early, early, and late genes (7,8). IE62 binds several viral proteins, such as ORF4 (9), ORF9 (10), ORF47 (11), and ORF63 (12), and cellular transcription factors, including upstream stimulatory factor (USF), TATA-binding protein (TBP), TFIIB,. IMPORTANCE The immediate early 62 protein (IE62) o...

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Structure of the VP16 transactivator target in the Mediator

Cited by 79 publications

References 44 publications

Role for Human Mediator Subunit MED25 in Recruitment of Mediator to Promoters by Endoplasmic Reticulum Stress-responsive Transcription Factor ATF6α

Role for Human Mediator Subunit MED25 in Recruitment of Mediator to Promoters by Endoplasmic Reticulum Stress-responsive Transcription Factor ATF6α

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62

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