The VP16 Activation Domain Interacts with Multiple Transcriptional Components as Determined by Protein-Protein Cross-linking in Vivo

Hall, Daniel B.; Struhl, Kevin

doi:10.1074/jbc.m208911200

Cited by 126 publications

(97 citation statements)

References 69 publications

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“…The facile detection of protein-DNA complexes following incubation times of 30 min or less suggests that macromolecular crosslinking occurs relatively rapidly, as suggested by the earliest ChIP experiments (14,40,81). Relatively rapid formaldehyde reactivity in cells is also consistent with the ability to distinguish ChIP signals over short time intervals (seconds to minutes) (63,82,83). Formaldehyde crosslinks are quite stable in vivo when compared with the durations of most crosslinking experiments, with crosslink half-lives of ϳ10 -20 h depending on the cell type and conditions (15).…”

Section: Crosslinking Kinetics Stability and Reversalmentioning

confidence: 59%

“…Because DNA site-specific transcription factors can also bind to nonspecific sites (59 -62), crosslinking of non-specifically bound proteins to DNA would be expected to occur and may account in part for binding events detected in genome-wide studies that cannot be readily explained physiologically. Non-DNA-binding proteins are not crosslinked to chromatin (14,63), and non-specifically bound factors are presumably bound to a multitude of disparate sites at low levels consistent with their relative occupancies (64,65). Analyses of chromatin binding by a series of mutants in the methyl CpGbinding protein 2 gene led to the conclusion that there is a threshold interaction lifetime of about 5 s required for crosslinking (26).…”

Section: Capture Of Protein-dna Complexesmentioning

confidence: 99%

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Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

310

256

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Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.Prior to its use in the chromatin field, formaldehyde use had a long history in a number of fields, including vaccine production (1, 2) and histology (3). In this review, we focus on its use in chromatin immunoprecipitation approaches and protein-protein interaction studies applied to understand the location and abundance of transcription factor binding along DNA. A recent complementary perspective highlights gaps in knowledge with a particular focus on how formaldehyde crosslinking data have been used to interpret aspects of chromatin three-dimensional organization (4). Here, we briefly review prior work describing formaldehyde reactivity toward proteins, DNA, and their constituent monomers. This information provides a basis for understanding how formaldehyde functions in widely used assays in the chromatin field, and conversely, highlights less well understood aspects of formaldehyde behavior in cells. These issues are of significance for designing crosslinkingbased studies as well as for properly interpreting the resulting data. The analysis of formaldehyde-fixed chromatin has provided fundamental insights into where and when regulatory factors associate with the DNA template in vivo, but it in general does not provide unambiguous information about chromatin binding kinetics. A major goal of ongoing work is to understand kinetic and thermodynamic aspects of chromatin complex assembly at single copy loci in vivo. Development of experimental strategies to achieve these goals will require a deeper and more comprehensive understanding of the effects mediated by formaldehyde in cells.The following discussion provides a framework for understanding aspects of formaldehyde function when used to trap macromolecular complexes in cells, with the main features shown in Fig. 1. Beginning with basic chemical reactivity, this review will explore the ability of formaldehyde to crosslink with proteins and DNA to form protein-protein or protein-DNA complexes, common molecular quenchers, and the potential for crosslink reversal. Progress in capturing crosslinked complexes will also be discussed with an emphasis on the impact of a better understanding of formaldehyde chemistry in vivo. Basic ChemistryFormaldehyde is the smallest aldehyde, an electrophilic molecule susceptible to chemical attack by a wide range of nucleophilic species of ...

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Section: Crosslinking Kinetics Stability and Reversalmentioning

confidence: 59%

Section: Capture Of Protein-dna Complexesmentioning

confidence: 99%

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

310

256

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“…This indicates that basic mechanisms of transcription activation are conserved amongst eukaryotes. The VP16 TAD targets basal Pol II transcription factors, including TFIIA, TFIIB, TFIID, the TFIIH subunit Tfb1/p62 (yeast/human) and the Mediator co-activator [18][19][20][21][22][23] . The VP16 TAD binds the Mediator subunit Med25 (also called Arc92) [24][25][26][27] , which is specific to higher eukaryotes.…”

mentioning

confidence: 99%

Structure and VP16 binding of the Mediator Med25 activator interaction domain

Vojnic

Mourão

Seizl

et al. 2011

Nat Struct Mol Biol

109

161

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4 0 4 VOLUME 18 NUMBER 4 APRIL 2011 nAture structurAl & moleculAr biologyActivator proteins regulate eukaryotic RNA polymerase II (Pol II) transcription in response to developmental and environmental signals. They bind to the DNA recognition sites of target genes with a sequence-specific DNA-binding domain, and recruit components of the Pol II machinery through a transcriptional activation domain (TAD) 1,2 . Activators have been classified as acidic, glutamine-rich, proline-rich and serine/threonine-rich, depending on the preponderance of amino acids in their TAD 3 . An archetypal acidic activator is the herpes simplex virus protein 16 (VP16), which activates the expression of immediate early viral genes during infection 4-8 . VP16 exerts its activating function through a C-terminal TAD that includes residues 413-490 (refs. 9-13). The VP16 TAD has been intensely used for studying transcriptional activation. Usually, the VP16 TAD is fused with its N terminus to the DNA-binding domain of the yeast transcription factor Gal4. The resulting Gal4-VP16 activator fusion protein stimulates transcription from promoters that contain Gal4-binding sites in the yeast and mammalian transcription systems in vivo 14,15 and in vitro 16,17 . This indicates that basic mechanisms of transcription activation are conserved amongst eukaryotes. The VP16 TAD targets basal Pol II transcription factors, including TFIIA, TFIIB, TFIID, the TFIIH subunit Tfb1/p62 (yeast/human) and the Mediator co-activator [18][19][20][21][22][23] . The VP16 TAD binds the Mediator subunit Med25 (also called Arc92) [24][25][26][27] , which is specific to higher eukaryotes. Med25 consists of two domains, the activator interaction domain (ACID) 24 that binds the VP16 TAD, and a 'von Willebrand factor type A' domain that anchors Med25 to Mediator 24 . Mediator generally conveys regulatory information by forming a bridge between activators and the basal Pol II machinery 28,29 . Whereas information on the core Mediator structure is emerging, structural information about its more peripheral activator-binding domains is limited to the KIX domain in subunit Med15 (or Arc105) 30,31 .The VP16 TAD is intrinsically flexible, whereas the VP16 core domain (residues 49-402) forms a stable structure 23,[32][33][34] . The TAD contains two functional subdomains, H1 (residues 410-452) and H2 (residues 453-490), which activate transcription independently 35,36 . Both H1 and H2 contain acidic amino acids, but specific bulky hydrophobic and aromatic residues are required for their function [36][37][38][39] . H2 has been proposed to adopt an α-helical conformation when bound to TFIIB 40 . NMR analysis revealed a nine-residue amphipathic α-helix in H2 that docks onto a PH fold in Tfb1 21 . On the basis of these and other results it is assumed that acidic TADs are flexible in their free state, but become transiently structured upon interaction with their target proteins.Here we report the NMR structure of the Mediator Med25 ACID and analyze its structural and functional interaction wi...

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“…Moreover, cross-linking of multisubunit complexes may be very efficient because of the large number of intracomplex contacts. Efficient cross-linking is suggested by studies of transcriptional activators and the proteosome (8,9) and the fact that numerous members of a given complex generally provide comparable ChIP signals.…”

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confidence: 99%

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Tardiff

Abruzzi

Rosbash

2007

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

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To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.mass spectrometry ͉ nascent RNP ͉ RNA processing ͉ transcription ͉ affinity purification P urification and mass spectrometric analysis of protein complexes is a ubiquitous approach in modern molecular biology (1). The identification of interacting proteins and their subsequent characterization provides valuable insight into the complexes that carry out essential cellular functions. Purifications can use multiple biochemical fractionations (e.g., ion exchange, gradients, gel filtration, and affinity purification) or a streamlined tandem affinity purification (TAP) approach (2). Conventional purification methods, however, often preclude the accurate identification of in vivo-relevant complexes. Major complications include dissociation of protein complexes, as a function of off-rates and extract dilution. Moreover, strong affinities can promote in vitro associations that do not exist in vivo. These can occur as a consequence of protein exchange and the absence of subcellular localization. Finally, major complexes may be insoluble, or harsh methods required to effect solubility can perturb complex composition. Ribonucleoprotein particles (RNPs) and especially transcription complexes are particularly subject to these caveats.The synthesis of protein-encoding mRNAs is directed by RNA polymerase II (pol II), an extensively studied macromolecular machine. The composition of pol II is dynamic and changes with the stage of transcription, namely, initiation, elongation, and termination (3, 4). Difficulties with complex purification underlie controversies, for example, to what extent initiation occurs by stepwise assembly of a preinitiation complex on DNA. Moreover, elongating pol II is tightly associated with DNA, which makes its characterization challenging (5).ChIP is a major tool for characterizing DNA-associated proteins and complexes, including transcription and cotranscriptional ...

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The VP16 Activation Domain Interacts with Multiple Transcriptional Components as Determined by Protein-Protein Cross-linking in Vivo

Cited by 126 publications

References 69 publications

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Structure and VP16 binding of the Mediator Med25 activator interaction domain

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

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