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, Immunocytochemistry, H2Aub, in vivo
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
Equipment
Procedure
Data analysis
Check several fields containing together at least 50 nuclei for statistical analysis. Choose fields with similar background signal because some regions on the slide may have high background from inadequate rinsing. Figure 2 shows a strategy for quantitative analysis of assay results. Look for nuclei by DAPI signal (Figures 1A and 1B, left panel) and use the CFP signal to select nuclei that were successfully transfected with pUBC-H3.3-CFP (control construct) or pUBC-UBP12-CFP (test construct) (Figures 1A and 1B, center panel). Compare transfection rates (Figure 2A); ideally, transfection rates should be similar for control and test constructs. Count the number of nuclei with fluorescent signals for H2Aub or H4 (Figures 1A and 1B, right panel). Detection efficiency of immunocytochemistry can be expressed as number of H4-positive nuclei as percentage of all transfected nuclei (Figure 2B). Similar values indicate that the detection process (fixing, staining, washing etc.) was comparable for control and test constructs. The effects of control and test constructs on target PTM can be seen in a comparison of the percentages of PTM-positive nuclei after transfection (Figure 2C). Smaller values for the test than for the control construct indicate that the test construct specifically reduced the target PTM. An alternative display is to compare the relative frequencies of PTM-positive nuclei (Figure 2D). For this, the frequency of PTM-positive nuclei after transfection with the control construct is set as 1 and the frequency of PTM-positive nuclei after transfection with the test construct is expressed relative to it. A well-working test construct should affect the target PTM and thus show a relative frequency of PTM-positive nuclei < 1. This display directly shows the effect size of the transfected test construct and is recommended when transfection or detection rates differ; results should be compared across several experiments or when multiple test constructs are included. Figure 2. Strategy for quantitative analysis of assay results for a histone posttranslational modification (PTM; here: H2Aub). Control and test constructs (here: pUBC-H3.3-CFP and pUBC-UBP12-CFP) are represented by light grey and dark grey bars, respectively. A. Transfection rate, i.e., number of CFP-positive nuclei as percentage of all detected nuclei. Similar bar heights indicate similar transfection rates for control and test constructs. B. Detection efficiency of immunocytochemistry, i.e., number of H4-positive nuclei as percentage of all transfected nuclei. Similar bar heights indicate that the detection process (fixing, staining, washing etc.) was similar for control and test constructs. C. Effect of control and test construct on target PTM, i.e., number of PTM-positive nuclei as percentage of all transfected nuclei. Smaller values for the test than for the control construct indicate that the test construct specifically reduced the target PTM. D. Alternative display of the effect of control and test constructs on target PTM using relative frequencies. The frequency of PTM-positive nuclei transfected with the pUBC-H3.3-CFP control construct was set to 1 and the frequency of PTM-positive nuclei transfected with the pUBC-UBP12-CFP test construct expressed relative to this. A well-working test construct (here: pUBC-UBP12-CFP) should affect the target PTM and thus result in a relative intensity < 1.
Notes
Recipes
Acknowledgments
Seeds of N. benthamiana and the construct harboring viral RNA silencing suppressor p19 were kindly provided by E. Savenkov (SLU, Uppsala). This protocol was developed from the following published paper: Derkacheva et al. (2016). This work was supported by a grant from the Knut-and-Alice-Wallenberg Foundation. The authors declare no conflicts of interest or competing interests.
References
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