Chemical and Genetic Approaches to Study Histone Modifications

Dhall, Abhinav; Chatterjee, Champak

doi:10.1016/b978-0-12-801080-8.00008-9

Cited by 3 publications

(2 citation statements)

References 107 publications

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“…The discovery of multiple functional analogs of modified histones provide convenient alternatives that are accessible by one or two-step transformations on recombinant proteins with commercially available reagents. 16 The two methods presented to generate analogs of acetylated and sumoylated histones are readily accomplished in a two-week period, starting from heterologous expression, and provide access to milligram quantities of the modified histones. Although disulfide-linked sumoylated histones cannot be used in the presence of reducing agents, these can easily be excluded from in vitro assays, and may even be tolerated based on the solvent accessibility of the modified residue.…”

Section: Discussionmentioning

confidence: 99%

“…Several thiol-directed strategies provide rapid access to close functional analogs of wild-type PTMs of lysines and arginines. 16 In this chapter, we provide detailed methods for chemical strategies that yield analogs of acetylated and sumoylated histones. 17 A high-yielding disulfide forming reaction 18 is used to generate H4 site-specifically sumoylated at Lys 12 (suH4 ss ) and radical-mediated thiol-ene coupling chemistry is used to introduce acetylated thialysine in histone H3 at positions 14 and 56.…”

Section: Introductionmentioning

confidence: 99%

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Rapid Semisynthesis of Acetylated and Sumoylated Histone Analogs

Dhall

Weller

Chatterjee

2016

Methods in Enzymology

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The density and diversity of post-translational modifications (PTMs) observed in histone proteins typically limits their purification to homogeneity from biological sources. Access to quantities of uniformly modified histones is, however, critical for investigating the downstream effects of histone PTMs on chromatin-templated processes. Therefore, a number of semisynthetic methodologies have been developed to generate histones bearing precisely defined PTMs or close analogs thereof. In this chapter, we present two optimized and rapid strategies for generating functional analogs of site-specifically acetylated and sumoylated histones. First, we describe a convergent strategy to site-specifically attach the small ubiquitin-like modifier-3 (SUMO-3) protein to the site of Lys12 in histone H4 by means of a disulfide linkage. We then describe the generation of thialysine analogs of histone H3 acetylated at Lys 14 or Lys 56, using thiol-ene coupling chemistry. Both strategies afford multi-milligram quantities of uniformly modified histones that are easily incorporated into mononucleosomes and nucleosome arrays for biophysical and biochemical investigations. These methods are readily extendable to any desired sites in the four core nucleosomal histones and their variant forms.

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Section: Discussionmentioning

confidence: 99%