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In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease   

Edited by
Yanjie Li
Reviewed by
Anonymous reviewer
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Original research article

A brief version of this protocol appeared in:
Yeast
Jun 2016

Abstract

Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic clipping of histone tails is continuously attracting interest of researchers in the field of chromatin biology. We can recapitulate H3-clipping by performing in vitro H3 cleavage assay. Here, we are presenting the detailed protocol to perform in vitro H3 cleavage assay.

Keywords: Histone H3, Chicken liver H3 protease, Yeast H3 protease, Histone clipping, Chromatin

Background

Histone H3 clipping is the least understood mechanism of chromatin modification and regulation. It is expected that H3 clipping will permanently erase PTMs from the nucleosomes that might affect chromatin related events. Moreover, the fate of cleaved histones is still under investigation and it has been suggested that the cleaved histones might be recycled at specific regions of chromatin or they are targeted for degradation. There are various reports that describe in vivo clipping of histone H3 in different organisms, while in vitro assays for histone H3-specific clipping are limited. We need an efficient and robust in vitro assay for characterizing histone specific proteases. To this end, we present a protocol that can be used to examine the in vitro histone H3 clipping activity of yeast and chicken liver histone H3 proteases. We have optimized temperature and pH conditions for the assay. Under our optimized conditions, proteases were found to specifically cleave histone H3 out of all core histones. We have extensively used this protocol in our recent publications (Chauhan et al., 2016; Chauhan and Tomar, 2016; Azad and Tomar, 2016; Mandal et al., 2014; Mandal et al., 2013; Mandal et al., 2012). This protocol can be used to identify and characterize histone H3 specific proteases from different organisms ranging from yeast to mammals.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chauhan, S., Azad, G. K. and Tomar, R. S. (2017). In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease. Bio-protocol 7(1): e2085. DOI: 10.21769/BioProtoc.2085.
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