Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Journal Article · Thu Mar 04 00:00:00 EST 2021 · Journal of Visualized Experiments

DOI:https://doi.org/10.3791/61707· OSTI ID:1814619

Zhou, Mowei  ^[1]; Abdali, Shadan H. ^[1]; Dilworth, David ^[2]; Liu, Lifeng ^[2]; Cole, Benjamin ^[2]; Malhan, Neha ^[1]; Ahkami, Amir H. ^[1]; Winkler, Tanya E. ^[1]; Hollingsworth, Joy ^[3]; Sievert, Julie ^[3]; Dahlberg, Jeff ^[3]; Hutmacher, Robert ^[4]; Madera, Mary ^[5]; Owiti, Judith A. ^[5]; Hixson, Kim K. ^[1]; Lemaux, Peggy G. ^[5]; Jansson, Christer ^[1]; Paša-Tolić, Ljiljana ^[1]

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1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
3. University of California Agriculture and Natural Resources, Parlier, CA (United States)
4. University of California, Five Points, CA (United States); Univ. of California, Davis, CA (United States)
5. Univ. of California, Berkeley, CA (United States)

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors including histone PTMs are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for engineering solutions to increase the resilience of crops to climate change. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed as their intact forms by top-down mass spectrometry (MS) coupled to online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow thus yielding the global PTM profile of core histones (H4, H2A, H2B, H3). By comparing the PTM profile among samples corresponding to different conditions (e.g. drought vs. control), potential epigenetic marks can be discovered as targets for further characterization using approaches such as chromatin immunoprecipitation – sequencing (ChIP-seq).

• Research Organization: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• Grant/Contract Number: AC02-05CH11231; AC05-76RL01830
• OSTI ID: 1814619
• Alternate ID(s): OSTI ID: 1788205
• Report Number(s): PNNL-SA-153694; ark:/13030/qt9h02d61k
• Journal Information: Journal of Visualized Experiments, Journal Issue: 169; ISSN 1940-087X
• Publisher: MyJoVE Corp.
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES