Conversion of Cysteine into Dehydroalanine Enables Access to Synthetic Histones Bearing Diverse Post‐Translational Modifications

Chalker, Justin M.; Lercher, Lukas; Rose, Nathan R.; Schofield, Christopher J.; Davis, Benjamin G.

doi:10.1002/anie.201106432

Cited by 183 publications

(155 citation statements)

References 45 publications

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“…Additionally, the disulfide attachment chemistry used has the attractive feature that the modification can be "erased" through simple reduction, thereby offering the potential to study, in a time-resolved way, the functional and structural consequences of PTM removal. Moreover, it may be possible to adapt the strategy to give nonreducible thioether linkages through use of alternate thiol-directed chemistries (36,37). Indeed, analogs of other PTMs can, in principle, be introduced chemically into preassembled chromatin using known cysteine derivatization routes-for example, lysine methylation (2) and acetylation (38).…”

Section: Discussionmentioning

confidence: 99%

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Holt

David

Pollock

et al. 2015

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

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Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure-activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub-induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.H istone posttranslational modifications (PTMs) modulate chromatin structure and function either by directly altering the intrinsic physical properties of the chromatin fiber or by nucleating the recruitment and activity of a host of transacting nuclear factors (1-3). The chemical diversity, differential dynamics, and sheer number (currently over 100) (4, 5) of these PTMs, along with their combinatorial occurrence at the level of the nucleosome, create a complex and nonstatic molecular architecture in which all chromatin-related processes function. A central challenge in the field of epigenetics is to disentangle how distinct chromatin states control biochemical outputs, which requires the elucidation of the critical determinants governing histone PTM readout.A particularly fascinating histone PTM is the ubiquitylation of H2B at lysine 120 (H2B-Ub). H2B-Ub is enriched near the 5′ end of highly expressed genes and has been implicated in transcriptional elongation, as well as chromatin structure definition (6-9). Moreover, H2B-Ub directly regulates the H3K4 and H3K79 methyltransferases Set1 and Dot1, respectively (10-13). The mechanistic principles underlying these various phenomena remain poorly understood. At 8.5 kDa, ubiquitin is nearly as large as the histone to which it is linked (13.8 kDa in the case of H2B), increasing the nucleosome surface by as much as 4,800 A 2 (14). Thus, compared with smaller PTMs such as acetylation and methylation, ubiquitin is "information rich" in that it alters the steric and electrostatic properties around its attachment site, as well as presenting a large surface area for the recruitment of binding factors. Structural and biochemical studies of ubiquitin-ligand complexes, including the ubiquitylation of histone H2A at lysine 15, have revealed a canonical binding hotspot on ubiquitin involving a hydrophobic patch centered on Leu8/I...

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Section: Discussionmentioning

confidence: 99%

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Holt

David

Pollock

et al. 2015

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

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“…This reaction has been used to install a number of thioether mimics of natural protein modifications such as lipidation, glycosylation, phosphorylation and lysine methylation/acetylation 164,165,167,168 , as well as installing reactive handles for further modification such as S-allyl cysteine for olefin metathesis. 131 These reactions typically (but not always) proceed with low substrate control in their diastereoselection and so a mixture of D/L-epimers is produced at the site of modification.…”

Section: Review Nature Communications | Doi: 101038/ncomms5740mentioning

confidence: 99%

Selective chemical protein modification

2014

Self Cite

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“…6). Maleimides react rapidly, selectively, and irreversibly with thiolate-bearing residues such as the amino acid cysteine even in the presence of other nucleophiles such as lysine (39). We previously demonstrated that MLAmaleimide reacts covalently to ␣7 nAChR cysteine mutants in a time-dependent manner (32).…”

Section: Resultsmentioning

confidence: 99%

Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor

Absalom

Quek

Lewis

et al. 2013

Journal of Biological Chemistry

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Background:Methyllycaconitine is an antagonist at subtypes of the nicotinic acetylcholine receptor. Results: A reactive methyllycaconitine probe was covalently trapped by a cysteine introduced on the complementary face of the ␣4 subunit and only in the (␣4) 3 (␤2) 2 nAChR stoichiometry. Conclusion:The ␣4-␣4 interface on the ␣4␤2 nAChR contains a methyllycaconitine binding site. Significance: Defining the molecular interactions of nAChR ligands at the ␣4-␣4 interface may lead to superior therapeutics.

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Conversion of Cysteine into Dehydroalanine Enables Access to Synthetic Histones Bearing Diverse Post‐Translational Modifications

Cited by 183 publications

References 45 publications

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Selective chemical protein modification

Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor

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