Dynamics of global histone acetylation and deacetylation in vivo: rapid restoration of normal histone acetylation status upon removal of activators and repressors

Katan-Khaykovich, Yael; Struhl, Kevin

doi:10.1101/gad.967302

Cited by 195 publications

(153 citation statements)

References 52 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: 60%

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

311

261

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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: 60%

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Hoffman

Frey

Smith

et al. 2015

Journal of Biological Chemistry

311

261

View full text Add to dashboard Cite

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“…Moreover, SIRT1-deficient cells exhibit p53 hyperacetylation after DNA damage and ionizing radiation-induced thymocyte apoptosis, further supporting a role for SIRT1-mediated p53 deacetylation in p53-dependent stress responses (57). Recent studies indicate that targeted deacetylation and acetylation occur very quickly amidst a global equilibrium of genomic acetylation and deacetylation (58,59). Although the steady-state level of acetylated p53 is low even in stressed cells, we have found that p53 is fully acetylated (at the p300͞CBP sites) in response to DNA damage when p53 deacetylation is inhibited (Fig.…”

Section: Discussionmentioning

confidence: 99%

Acetylation of p53 augments its site-specific DNA binding both in vitro and in vivo

Luo

Tang

et al. 2004

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

373

318

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p53 promotes tumor suppression through its ability to function as a transcriptional factor and is activated by posttranslational modifications that include acetylation. Our earlier study demonstrated that p53 acetylation can enhance its sequence-specific DNA binding in vitro, and this notion was later confirmed in several other studies. However, a recent study has reported that in vitro acetylation of p53 fails to stimulate its DNA binding to large DNA fragments, raising an important issue that requires further investigation. Here, we show that unacetylated p53 is able to bind weakly to its consensus site within the context of large DNA fragments, although it completely fails to bind the same site within short oligonucleotide probes. Strikingly, by using highly purified and fully acetylated p53 proteins obtained from cells, we show that acetylation of the C-terminal domain can dramatically enhance site-specific DNA binding on both short oligonucleotide probes and long DNA fragments. Moreover, endogenous p53 apparently can be fully acetylated in response to DNA damage when both histone deacetylase complex 1 (HDAC1)-and Sir2-mediated deacetylation are inhibited, indicating dynamic p53 acetylation and deacetylation events during the DNA damage response. Finally, we also show that acetylation of endogenous p53 indeed significantly augments its ability to bind an endogenous target gene and that p53 acetylation levels correlate well with p53-mediated transcriptional activation in vivo. Thus, our results clarify some of the confusion surrounding acetylation-mediated effects on p53 binding to DNA and suggest that acetylation of p53 in vivo may contribute, at least in part, to its transcriptional activation functions.ChIP ͉ transcription ͉ CBP͞p300

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“…In one case, TBP binding to chromatin-assembled SV40 minichromosomes was facilitated by histone acetylation at an underlying nucleosome (Sewack et al, 2001). However, in yeast gene systems, binding of TBP to promoters does not appear to strictly correlate with the acetylation status of promoter histones (Sekinger and Gross 2001;Katan-Khaykovich and Struhl 2002;Kristjuhan et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of MMTV transcription by HDAC inhibitors occurs independent of changes in chromatin remodeling and increased histone acetylation

2003

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Increased histone acetylation has been associated with activated gene transcription and decreased acetylation with repression. However, there is a growing number of genes known, which are downregulated by histone deacetylase (HDAC) inhibitors through unknown mechanisms. This study examines the mechanism by which the mouse mammary tumor virus (MMTV) promoter is repressed by the HDAC inhibitor, trichostatin A (TSA). We find that this repression is transcriptional in nature and that it occurs in the presence and absence of glucocorticoids. TSA decreases MMTV transcription at a rapid rate, reaching maximum in 30-60 min. In contrast with previous reports, the repression does not correlate with an inhibition of glucocorticoid-induced nuclease hypersensitivity or NF1-binding at the MMTV promoter. Surprisingly, TSA does not induce sizable increases in histone acetylation at the MMTV promoter nor does it inhibit histone deacetylation, which accompanies deactivation of the glucocorticoid-activated MMTV promoter. Repression of MMTV transcription by TSA does not depend on the chromatin organization of the promoter because a transiently transfected MMTV promoter construct with a disorganized nucleoprotein structure was also repressed by TSA treatment. Mutational analysis of the MMTV promoter indicates that repression by TSA is mediated through the TATA box region. These results suggest a novel mechanism that involves acetylation of nonhistone proteins necessary for basal transcription.

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Dynamics of global histone acetylation and deacetylation in vivo: rapid restoration of normal histone acetylation status upon removal of activators and repressors

Cited by 195 publications

References 52 publications

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

Acetylation of p53 augments its site-specific DNA binding both in vitro and in vivo

Inhibition of MMTV transcription by HDAC inhibitors occurs independent of changes in chromatin remodeling and increased histone acetylation

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