本氏烟草中的组蛋白去泛素化分析

Shujing  Liu; Lars  Hennig

doi:10.21769/BioProtoc.2746

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Peer-reviewed

Histone Deubiquitination Assay in Nicotiana benthamiana

本氏烟草中的组蛋白去泛素化分析

Shujing Liu email

Lars Hennig

发布: 2018年03月05日第8卷第5期 DOI: 10.21769/BioProtoc.2746 浏览次数: 5882

评审: Tohir BozorovAnonymous reviewer(s)

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Cover of Nature Plants, featuring study using the protocol.

Aug 2016

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Abstract

Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo assay for histone deubiquitination activity. After infiltrating UBP12 into Nicotiana benthamiana leaves, H2Aub was visualized by immunocytochemistry. Nicotiana benthamiana leaves, which show high agro infiltration efficiency, were used for transient UBP12 expression for a labor- and time-saving protocol. Reduced H2Aub levels indicated histone deubiquitination activity of UBP12. The clear visualization of nuclei of N. benthamiana leaves makes this method able to easily measure the level of histone modification in vivo by using specific antibodies, providing robust clues of protein function. Thus, this protocol is a powerful complementation to in vitro assays of histone deubiquitination activity.

Keywords: Histone deubiquitination (组蛋白去泛素化)

Immunocytochemistry (免疫细胞组化)

H2Aub (H2Aub)

in vivo (体内试验)

Background

Histone modifications play important roles in regulating chromatin structure and gene expression. Best studied histone modifications include methylation, acetylation, phosphorylation, ubiquitination and sumoylation. However, enzymes introducing or removing specific histone modifications are not always known. Powerful in vitro assays can establish the catalytic potential of histone modifying enzymes but in vivo methods are desirable to confirm that in vitro specificity reflects in vivo activity. Here, we describe a flexible protocol to test activity of histone modifying enzymes in the plant N. benthamiana. Although we used the protocol to test activity of ubiquitin specific protease (UBP) on ubiquitylated H2A, it can also easily be adopted to other histone modifications for which specific antibodies are available.

Materials and Reagents

50 ml and 15 ml screw cap tubes (SARSTEDT, catalog numbers: 62.547.254 and 62.554.002 )
2 ml and 1.5 ml microtubes (SARSTEDT, catalog numbers: 72.695.500 and 72.690.001 )
5 ml syringe (BD plastipak, BD, catalog number: 302187 )
Razor blade (Feather Safety Razor, catalog number: 02.015.00.024 )
Petri dish, 9.2 cm diameter (SARSTEDT, catalog number: 82.1473.001 )
CellTrics^®(pore size 30 μm) (Sysmex, catalog number: 04-0042-2316 )
Absorbent paper (Kimberly-Clark, catalog number: 3020 )
Microscopic slides and cover slips (VWR, catalog number: 631-1551 and 631-9430 )
Nicotiana benthamiana wild-type plants (seeds kindly provided by Dr. Savenkov, Uppsala), 4-6 weeks old (cultivated at 24 °C，16 h light, 8 h dark, 60% humidity); mature leaves (flat surface and edge) are used
Agrobacterium tumefaciens strain GV3101 harboring pUBC-UBP12-CFP plasmid (Derkacheva et al., 2016)
Agrobacterium tumefaciens strain GV3101 harboring pUBC-H3.3-CFP plasmid (Derkacheva et al., 2016)
Agrobacterium tumefaciens strain GV3101 harboring viral RNA silencing suppressor p19 (kindly provided by E. Savenkov, Uppsala)
LB broth high salt (Duchefa Biochemie, catalog number: L1704.2500 )
Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
Antibody anti-H2Aub (Cell Signaling Technology, catalog number: 8240 )
Antibody anti-H4 (Merck, EMD Millipore, catalog number: 05-858 )
Antibody Alexa Fluor 555 conjugated goat anti-rabbit Kit (Thermo Fisher Scientific, Invitrogen, catalog number: A31629 )
VECTASHIELD^® mounting medium with 1 μg/ml DAPI (Vector Laboratories, catalog number: H-1200 )
Bovine serum albumin (BSA) (Carl Roth, catalog number: 3737.2 )
MES (Duchefa Biochemie, catalog number: M1503 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M9272 )
Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
Tris base (VWR, catalog number: 28811.364 )
EDTA disodium salt (Na₂-EDTA) (Sigma-Aldrich, catalog number: ED2SS )
Sodium chloride (NaCl) (Merck, catalog number: 567441 )
37% HCl (Merck, catalog number: 1.00317.2500 )
Spermine (Sigma-Aldrich, catalog number: 85590 )
Potassium chloride (KCl) (Merck, catalog number: 529552 )
Triton X-100 (Sigma-Aldrich, catalog number: X100 )
Sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O) (Merck, EMD Millipore, catalog number: 106342 )
Sodium hydrogen phosphate (Na₂HPO₄) (Sigma-Aldrich, catalog number: S3264 )
Infiltration medium (see Recipes)
Tris buffer (see Recipes)
LB01 lysis buffer (see Recipes)
PBS buffer (see Recipes)

Equipment

Centrifuges (Eppendorf, model: 5804 R and Thermo Fisher Scientific, Thermo Scientific^TM, model: Heraeus^TM Pico^TM 17 )
Surgical scissors (DIMEDA Instrumente, catalog number: 08.340.11 )
Laminar hood
Pipettes (Gilson, model: PIPETMAN^® Classic, P2 , P20 , P200 , P1000 )
Humid incubator
Confocal microscope (ZEISS, model: LSM 780 )