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Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody
使用Mintbody半定量分析活细胞中H4K20me1的水平   

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Abstract

Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones plays an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. Histone H4 Lys20 monomethylation (H4K20me1) has been shown to be involved in chromosome condensation, segregation, replication and repair. H4K20 methylation is controlled through a few methyltransferases, PR-Set7/Set8, SUV420H1, and SUV420H2, and a demethylase, PHF8. In cycling cells, the level of H4K20me1 increases during G2 and M phases and decreases during G1 phase. To monitor the local concentration and global fluctuation of histone modifications in living cells, we have developed a genetically encoded probe termed mintbody (modification-specific intracellular antibody; Sato et al., 2013 and 2016). By measuring the nuclear to cytoplasmic intensity ratio, the relative level of H4K20me1 in individual cells can be monitored. This detailed protocol allows the semi-quantitative analysis of the effects of methyltransferases on H4K20me1 levels in living cells based on H4K20me1-mintbody described by Sato et al. (2016).

Keywords: Post translational modification(翻译后修饰), Chromatin dynamics(染色质动力学), Live-cell imaging(活细胞成像), Mintbody(Mintbody), Quantitative imaging(定量成像)

Background

Posttranslational modifications of histone proteins play important roles in transcriptional regulation and genome integrity. While the one-dimensional epigenomic landscape has been revealed in many cell types by chromatin immunoprecipitation and sequencing, less is known about the dynamics of histone modifications due to technical limitations (Kimura et al., 2015). Recently, a few techniques for detecting protein modifications in living cells have been developed. One strategy uses sensors based on fluorescence/Förster resonance energy transfer (FRET) to monitor the balance between the modifying and demodifying enzymes. However, the dynamics of endogenous modifications cannot be monitored using FRET sensors. Another strategy that we have developed uses probes based on modification-specific antibodies. Fab-based live endogenous modification labeling (FabLEM) is a live-imaging system using fluorescently labeled antigen-binding fragments (Fabs). Fabs loaded into cells bind to the target modification without disturbing cell function as the binding time is very small (a second to tens of seconds). A genetically encoded system to express a modification-specific intracellular antibody (mintbody) can be applied for observation with a longer period of time or in living animals (Figure 1). Both Fabs and mintbodies are just small enough to pass through the nuclear pore by diffusion. When the level of the target modification increases, more probes become enriched in the nucleus. Therefore, by measuring the nuclear/cytoplasmic intensity ratio, changes of modification level in living cells can be monitored (Hayashi-Takanaka et al., 2011; Sato et al., 2013 and 2016). The live cell modification monitoring system using mintbodies will be particularly useful to evaluate the effects of small chemicals and protein depletion and overexpression.

Histone H4 Lys20 monomethylation (H4K20me1) is an essential modification in mammals, involved in chromosome condensation, segregation, replication and repair, as well as gene regulation (Beck et al., 2012; Jørgensen et al., 2013). The level of H4K20me1 increases during G2 to M phases and the inhibition of PR-Set7/Set8, a methyltransferase responsible for H4K20 monomethylation, causes mitotic defects. In female cells, the enrichment of H4K20me1 in inactive X chromosomes is microscopically observed. H4K20me1-specific mintbody has proven useful for monitoring the dynamic behavior of H4K20me1 in living cells (Sato et al., 2016). In addition, alteration of H4K20me1 level by ectopic expression of a methyltransferase has been evaluated. Among methyltransferases (PR-Set7/Set8, SUV420H1, and SUV420H2) and a demethylase (PHF8), involved in H4K20me1 metabolism, the expression of SUV420H1, which add methyl-groups to monomethylated H4K20 towards to trimethylation, caused a drastic effect. As an example of measuring relative H4K20me1 levels, we here describe the method to evaluate the effect of SUV420H1 on H4K20me1 in living cells.


Figure 1. Schematic diagram of mintbody expression and function. A genetically encoded mintbody, which reversibly binds to specific modification, can be expressed in cells and animals that harbors the expression vector.

Materials and Reagents

  1. Pipette tips (10, 20, 200, 1,000 μl)
  2. 6 well plate (Corning, catalog number: 3516 )
  3. 10 cm dish (Greiner Bio One International, catalog number: 664160-013 )
  4. 24 well glass-bottom plate (IWAKI, catalog number: 5826-024 )
  5. HeLa cells (ATCC, catalog number: CRM-CCL-2 )
  6. Purified plasmid DNA (~1 μg/μl) encoding H4K20me1-mintbody based on pEGFP (Clontech) or a piggybac system (Sato et al., 2016) and Halo-SUV420H1 (Kazusa DNA Research Institute; FlexiHaloTag clone FHC01413)
  7. Dulbecco’s modified Eagle’s medium (DMEM), high glucose (4 g/L), containing L-Gln and sodium pyruvate (Nacalai Tesque, catalog number: 08458-16 )
  8. L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich, catalog number: G1146-100ML )
  9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  10. FuGENE HD (Promega, catalog number: E2312 )
  11. Opti-MEM media (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
  12. G-418 disulfate aqueous solution (Nacalai Tesque, catalog number: 16513-26 )
  13. FluoroBrite DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: A1896701 )
  14. HaloTag TMR ligand (Promega, catalog number: G8251 )

Notes:

  1. Nucleotide sequence of H4K20me1-scFv is available in public databases (DDBJ/EMBL/GenBank) under the accession number LC129890. The plasmid DNA is available upon request to the authors.
  2. The original H4K20me1-specific antibody that was used to generate H4K20me1-mintbody (Hayashi-Takanaka et al., 2015) is available commercially (MBL International, catalog number: MABI0421).

Equipment

  1. CO2 incubator
  2. Pipette (10, 20, 200, 1,000 μl)
  3. 35 mm glass-bottom dishes, No. 1.5 coverslip (MATTEK, catalog number: P35G-1.5-14-C )
  4. Fluorescence microscope (Nikon Instruments, model: ECLIPSE Ti-E ) operated by NIS-elements and equipped with:
    A spinning disk confocal unit (Yokogawa Electric, model: CSU-W1 )
    An EM-CCD camera (Andor, model: iXon3 DU888 X-8465 )
    An objective lens (Plan Apo 40x DIC M N2 [NA 0.95])
    A laser unit (Nikon Instruments, model: LU-N4 )
    A heated stage (Tokai Hit)
    A CO2-control system (Tokken)

Software

  1. NIS-elements ver. 4.30 (Nikon Instruments)
  2. Excel (Microsoft)

Procedure

  1. Establishing stable cell lines expressing the H4K20me1-mintbody (Figure 2)
    Note: When using primary cells, cell sorting can be used instead of establishing antibiotic resistant clones. 


    Figure 2. Scheme for preparing cells expressing H4K20me1-mintbody and Halo-SUV420H1

    1. Maintain HeLa cells in DMEM supplemented with 10% FBS and L-glutamine-penicillin-streptomycin at 37 °C under 5% CO2 atmosphere, according to a standard protocol (Freshney, 2015).
    2. Transfect the plasmid DNA encoding the H4K20me1-mintbody into cells using FuGENE HD, as per standard protocol. Typically, cells were plated onto 6 well plate (2 x 105 cells in 2 ml medium/well) on the day before transfection. Mix 2 μg plasmid and 6 μl FuGENE HD in 100 μl Opti-MEM, leave the mixture for 5 min at room temperature, and add the mixture to one well of a 6-well plate containing 2 ml medium. Put back the plate into a CO2 incubator.
    3. The next day, to obtain the stable cell line expressing the mintbody, harvest the cells, dilute at around 103 cells/ml, and plate cells on to a 6 well plate (2 ml per well) or a 10 cm dish (10 ml per dish) in DMEM with FBS and L-glutamine-penicillin-streptomycin, containing 1 mg/ml G-418.
    4. Approximately two weeks after addition of G-418, isolate and expand GFP-positive colonies. Isolation of cells expressing H4K20me1-mintbody can be done in several ways (Freshney, 2015).
    5. If fluorescent colonies are observed at a high frequency (> 10%), randomly pick up 24 colonies using a micropipette with a 200 μl tip, or using a cloning ring, and transfer into a 24-well glass-bottom plate containing 0.5 ml DMEM containing FBS and L-glutamine-penicillin-streptomycin (without G-418).
    6. If fluorescent colonies are rarely observed, pick up them after marking their position under an inverted fluorescence microscope. A few days later, investigate the level and distribution of fluorescence in each well under an inverted fluorescence microscope using a dry lens with a high numerical aperture and a short working distance (e.g., Plan Apo 40x DIC M N2 [NA 0.95]). Fluorescent cells can also be collected using a cell sorter, if available.

  2. Cell preparation for imaging
    1. Two days before imaging, plate cells expressing mintbody in a 35 mm glass-bottom dish (2 ml medium; ~2 x 105 cells/dish).
    2. The next day, transfect the plasmid DNA encoding Halo-SUV420H1 into cells using FuGENE HD to convert monomethylated H4K20 to trimethylation. Transfection using 2 μg plasmid and 6 μl FuGENE HD is carried out as indicated above. The transaction efficiency typically yields ~30%.
    3. A day after transfection, replace the culture medium to FluoroBrite DMEM with 10% FBS and L-glutamine-penicillin-streptomycin, containing 0.1 μM Halo TMR ligand. Incubate the cells at 37 °C under 5% CO2 atmosphere.

  3. Image acquisition
    1. One hour after the addition of Halo TMR ligand, set the glass-bottom dish onto a heated stage. It is not essential to remove free TMR. However, if the expression level of HaloTag-tagged protein is low and background fluorescence hampers the detection, cells should be washed three times with FluoroBrite DMEM with 10% FBS and L-glutamine-penicillin-streptomycin, before setting the dish onto a heated stage.
    2. Acquire fluorescence images for the H4K20me1-mintbody (green channel) and TMR-labeled Halo-SUV420H1 (red channel) using a confocal microscope with laser lines at 488 and 561 nm, combined with a dichroic mirror DM405/488/561/640 and emission filters 520/30 and 617/73, respectively.

Notes:

  1.  One hour staining with 0.1 μM Halo TMR ligand is usually sufficient to label HaloTag-tagged proteins. However, if the signal intensity is not high enough, samples can be incubated overnight with the TMR ligand. Washing with FluoroBrite DMEM with 10% FBS and L-glutamine-penicillin-streptomycin also improves the signal-to-noise ratio.
  2. To avoid the saturation of fluorescence intensity, we briefly scan multiple areas and set up the laser power and exposure time to cover all cells (except dead ones, which sometimes exhibit brightest fluorescence) are within the dynamic range of the camera for both green and red channels.
  3. Non-transfected cells in the captured images will be used for negative control cells.
  4. Empirically, at least 30 Halo-positive cells should be imaged for a good statistical analysis. Total 200 cells including non-transfected cells are typically imaged.

Data analysis

  1. The relative level of H4K20me1 measured by the nuclear enrichment of mintbody
    Note: To compare the nuclear enrichment of H4K20me1-mintbody in different cells, measure the nucleus/cytoplasm intensity ratio in each cell.
    1. Open the images using NIS-elements.
    2. Select the background ROI by choosing an area without cells.
    3. Obtain the net intensity images by background subtraction.
    4. Draw ROIs for the cell and nucleus manually using ‘Draw Polygonal ROI’ tool (Figure 3). Nuclear region can be selected automatically by thresholding and binarization, but the selecting cell region is often difficult due to the low signal intensity in the cytoplasm. For analysis of tens of cells, manual drawing is often quicker and accurate; as the selected areas are quite large, a difference in a few pixels do not affect much.
    5. Measure the mean intensities [I], areas [A], and total intensity [T = I x A] of the ROIs in green channel.
    6. Export the data to Excel.
    7. Open the exported data in Excel and calculate the intensity of the cytoplasm by the following equation: [I]cytoplasm = ([T]cell - [T]nucleus)/([A]cell - [A]nucleus).
    8. Calculate the nucleus/cytoplasm intensity ratio by dividing the mean intensity of the nucleus with that of cytoplasm (i.e., the ratio = [I]nucleus/[I]cytoplasm).


      Figure 3. Drawing ROIs to define cell and nuclear regions. A representative image file analyzed using NIS-elements. Blue, Hoechst; Green, H4K20me1-mintbody; Red, Halo-SUV420H1. Note that live Hoechst staining was employed here, but this is not essential because the nuclear regions are clearly distinguished from the cytoplasm. Scale bar = 10 μm.

  2. The expression level of Halo-SUV420H1
    1. The ROI area of the nucleus defined above can also be used for the relative quantification of Halo-SUV420H1 expression level.
    2. Measure the mean intensity of the nucleus in red channel.

  3. Relationship between SUV420H1 expression and H4K20me1
    1. Draw a scatter plot to visualize the relationship between the levels of SUV420H1 expression and H4K20me1 (Figure 4).


      Figure 4. H4K20me1 levels in living cells monitored with H4K20me1-mintbody. This figure is adapted from Sato et al., 2016. Halo-SUV420H1 was transiently expressed in HeLa cells that stably express H4K20me1-mintbody. The mintbody was more diffuse in the cytoplasm in the cell that expressed SUV420H1 (arrowhead). Nuclear/Cytoplasm intensity ratios in single cells were plotted (right graph). Nuclear/Cytoplasm ratios decreased depending on Halo-SUV420H1 expression. Bar = 10 µm.

Notes

This protocol can be applicable to many other cell lines and modification enzymes. To confirm the live cell observations, immunofluorescence analysis based on fixed cells can be employed using the original H4K20me1-specific antibody that is used to generate H4K20me1-mintbody (Hayashi-Takanaka et al., 2015; Sato et al., 2016).

Acknowledgments

This work was supported by JSPS KAKENHI Grants JP25118714, JP262910711, JP25116005. The protocol has been adapted from Sato et al. (2016).

References

  1. Beck, D. B., Oda, H., Shen, S. S. and Reinberg, D. (2012). PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription. Genes Dev 26(4): 325-337.
  2. Freshney, R. I. (2015). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition. John Wiley & Sons.
  3. Hayashi-Takanaka, Y., Maehara, K., Harada, A., Umehara, T., Yokoyama, S., Obuse, C., Ohkawa, Y., Nozaki, N., Kimura, H. (2015). Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies. Chromosome Res 23(4):753-766.
  4. Hayashi-Takanaka, Y., Yamagata, K., Wakayama, T., Stasevich, T. J., Kainuma, T., Tsurimoto, T., Tachibana, M., Shinkai, Y., Kurumizaka, H., Nozaki, N., Kimura, H. (2011). Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling. Nucleic Acids Res 39(15): 6475-6488.
  5. Jørgensen, S., Schotta, G. and Sorensen, C. S. (2013). Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity. Nucleic Acids Res 41(5): 2797-2806.
  6. Kimura, H., Hayashi-Takanaka, Y., Stasevich, T. J., Sato, Y. (2015). Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo. Histochem Cell Biol 144(2): 101-109.
  7. Sato, Y., Kujirai, T., Arai, R., Asakawa, H., Ohtsuki, C., Horikoshi, N., Yamagata, K., Ueda, J., Nagase, T., Haraguchi, T., Hiraoka, Y., Kimura, A., Kurumizaka, H. and Kimura, H. (2016). A genetically encoded probe for live-cell imaging of H4K20 monomethylation. J Mol Biol 428(20): 3885-3902.
  8. Sato, Y., Mukai, M., Ueda, J., Muraki, M., Stasevich, T. J., Horikoshi, N., Kujirai, T., Kita, H., Kimura, T., Hira, S., Okada, Y., Hayashi-Takanaka, Y., Obuse, C., Kurumizaka, H., Kawahara, A., Yamagata, K., Nozaki, N. and Kimura, H. (2013). Genetically encoded system to track histone modification in vivo. Sci Rep 3: 2436.

简介

真核核DNA包裹组蛋白,形成核小体,是染色质的基本单位。组蛋白的翻译后修饰在基因调控和染色体重复中起重要作用。一些修饰是相当稳定的,作为表观遗传记忆,其他修饰在细胞周期中表现出快速更替或波动。组蛋白H4 Lys20单甲基化(H4K20me1)已显示参与染色体凝聚,分离,复制和修复。通过几种甲基转移酶PR-Set7 / Set8,SUV420H1和SUV420H2以及脱甲基酶PHF8控制H4K20甲基化。在循环细胞中,H4K20me1的水平在G2期和M期增加,G1期下降。为了监测活细胞中组蛋白修饰的局部浓度和全局波动,我们开发了一种基因编码的探针,称为薄荷素(修饰特异性细胞内抗体; Sato等人,2013和2016)。通过测量核细胞与细胞质的强度比,可以监测单个细胞中H4K20me1的相对水平。该详细方案允许甲基转移酶对基于HatoK等人的质粒H4K20me1-mintbody活性细胞中H4K20me1水平的影响进行半定量分析(2016)。

背景 组蛋白蛋白的翻译后修饰在转录调控和基因组完整性中起重要作用。虽然通过染色质免疫沉淀和测序在许多细胞类型中已经揭示了一维表观基因组景观,但由于技术限制,对组蛋白修饰的动力学知之甚少(Kimura等人,2015)。近来,已经开发了几种用于检测活细胞中蛋白质修饰的技术。一种策略使用基于荧光/Förster共振能量转移(FRET)的传感器来监测修饰和解调酶之间的平衡。然而,使用FRET传感器不能监测内生修饰的动力学。我们开发的另一个策略是使用基于修饰特异性抗体的探针。基于Fab的活内源性修饰标记(FabLEM)是使用荧光标记的抗原结合片段(Fab)的活体成像系统。加载到细胞中的Fab结合目标修饰而不干扰细胞功能,因为结合时间非常小(秒至数十秒)。用于表达修饰特异性细胞内抗体(薄荷体)的遗传编码系统可以用于较长时间的观察或在活的动物中观察(图1)。 Fab和薄荷都足够小以通过扩散穿过核孔。当目标修饰水平增加时,核中会增加更多的探针。因此,通过测量核/细胞质强度比,可以监测活细胞中修饰水平的变化(Hayashi-Takanaka等人,2011; Sato等人, 2013和2016)。使用薄荷体的活细胞修饰监测系统对于评估小化学品和蛋白质耗尽和过表达的影响将特别有用。
 组蛋白H4 Lys20单甲基化(H4K20me1)是哺乳动物的重要修饰,涉及染色体凝聚,分离,复制和修复以及基因调控(Beck等人,2012;Jørgensen et al,2013)。 H4K20me1的水平在G2至M期增加,PR-Set7 / Set8(负责H4K20单甲基化的甲基转移酶)的抑制引起有丝分裂缺陷。在雌性细胞中,显微镜观察到H4K20me1在非活性X染色体中的富集。 H4K20me1特异性薄荷醇已被证明可用于监测活细胞中H4K20me1的动态行为(Sato et al。,2016)。另外,通过甲基转移酶的异位表达改变了H4K20me1的水平。参与H4K20me1代谢的甲基转移酶(PR-Set7 / Set8,SUV420H1和SUV420H2)和脱甲基酶(PHF8)中,将甲基化成甲基化H4K20的SUV420H1表达为三甲基化引起了显着的效果。作为测量相对H4K20me1水平的一个例子,我们在这里描述了评估SUV420H1对活细胞中H4K20me1的影响的方法。


图1.薄荷体表达和功能的示意图可逆转地结合特异性修饰的遗传编码薄荷体可以在含有表达载体的细胞和动物中表达。

关键字:翻译后修饰, 染色质动力学, 活细胞成像, Mintbody, 定量成像

材料和试剂

  1. 移液器吸头(10,20,200,1,000μl)
  2. 6孔板(Corning,目录号:3516)
  3. 10厘米盘(Greiner Bio One International,目录号:664160-013)
  4. 24孔玻璃底板(IWAKI,目录号:5826-024)
  5. HeLa细胞(ATCC,目录号:CRM-CCL-2)
  6. 基于pEGFP(Clontech)或piggybac系统(Sato等人,2016)编码H4K20me1薄荷体的纯化质粒DNA(〜1μg/μl)和Halo-SUV420H1(Kazusa DNA Research Institute; FlexiHaloTag克隆FHC01413)
  7. 含有L-Gln和丙酮酸钠(Nacalai Tesque,目录号:08458-16)的Dulbecco改良的Eagle培养基(DMEM),高葡萄糖(4g/L)
  8. L-谷氨酰胺 - 青霉素 - 链霉素溶液(Sigma-Aldrich,目录号:G1146-100ML)
  9. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  10. FuGENE HD(Promega,目录号:E2312)
  11. Opti-MEM介质(Thermo Fisher Scientific,Gibco TM ,目录号:31985070)
  12. G-418二硫酸盐水溶液(Nacalai Tesque,目录号:16513-26)
  13. FluoroBrite DMEM(Thermo Fisher Scientific,Gibco TM,目录号:A1896701)
  14. HaloTag TMR配体(Promega,目录号:G8251)

注意:

  1. H4K20me1-scFv的核苷酸序列可在公开数据库(DDBJ/EMBL/GenBank)中以登录号LC129890获得。质粒DNA可根据要求提供给作者。
  2. 用于产生H4K20me1薄荷体的原始H4K20me1特异性抗体(Hayashi-Takanaka等,2015)可商购(MBL International,目录号:MABI0421)。

设备

  1. CO 2 孵化器
  2. 移液器(10,20,200,1,000μl)
  3. 35毫米玻璃底盘,1.5号盖玻片(MATTEK,目录号:P35G-1.5-14-C)
  4. 荧光显微镜(Nikon Instruments,型号:ECLIPSE Ti-E)由NIS元件操作并配备:
    旋转盘共焦单元(横河电机,型号:CSU-W1)
    EM-CCD相机(Andor,型号:iXon3 DU888 X-8465)
    物镜(Plan Apo 40x DIC M N2 [NA 0.95])
    激光单元(Nikon Instruments,型号:LU-N4)
    加热阶段(东海命中)
    CO 2子控制系统(Tokken)

软件

  1. NIS元素ver。 4.30(尼康乐器)
  2. Excel(Microsoft)

程序

  1. 建立表达H4K20me1薄荷体的稳定细胞系(图2)
    注意:当使用原代细胞时,可以使用细胞分选,而不是建立抗生素抗性克隆。


    图2.制备表达H4K20me1薄荷体和Halo-SUV420H1的细胞的方案

    1. 根据标准方案(Freshney,2015),在37℃,5%CO 2气氛下,在补充有10%FBS和L-谷氨酰胺 - 青霉素 - 链霉素的DMEM中维持HeLa细胞。
    2. 根据标准方案,使用FuGENE HD将编码H4K20me1-薄荷体的质粒DNA转染入细胞。通常,在转染前一天将细胞接种到6孔板(2×10 5个细胞在2ml培养基/孔中)。将2μg质粒和6μlFuGENE HD在100μlOpti-MEM中混合,将混合物在室温下放置5分钟,并将混合物加入含有2ml培养基的6孔板的一个孔中。将平板放入CO 2 孵化器。
    3. 第二天,为了获得表达薄荷体的稳定细胞系,收获细胞,稀释至约10 3个细胞/ml,将平板细胞置于6孔板上(每孔2ml)或在含有1mg/ml G-418的FBS和L-谷氨酰胺 - 青霉素 - 链霉素的DMEM中的10cm皿(10ml /皿)。
    4. 加入G-418后约两周,分离并扩增GFP阳性菌落。表达H4K20me1薄荷体的细胞的分离可以通过几种方式进行(Freshney,2015)。
    5. 如果以高频(> 10%)观察到荧光菌落,则使用具有200μl尖端的微量移液管或使用克隆环随机挑取24个菌落,并转移到含有0.5ml的24孔玻璃底板中含有FBS和L-谷氨酰胺 - 青霉素 - 链霉素(不含G-418)的DMEM。
    6. 如果荧光菌落很少被观察到,则在倒置的荧光显微镜下标记它们的位置之后拿起它们。几天后,使用具有高数值孔径和较短工作距离的干透镜(例如,Plan Apo 40x DIC M N2),在倒置荧光显微镜下研究每个孔中荧光的水平和分布[NA 0.95])。如果可用,也可以使用细胞分选仪收集荧光细胞。

  2. 成像细胞准备
    1. 在成像前两天,将平板细胞在35mm玻璃底盘(2ml培养基;〜2×10 5细胞/皿)中表达薄荷体。
    2. 第二天,使用FuGENE HD将编码Halo-SUV420H1的质粒DNA转染到细胞中,将单甲基化的H4K20转化为三甲基化。使用2μg质粒和6μlFuGENE HD进行转染如上所述进行。横切效率通常会达到〜30%
    3. 转染后一天,用含有0.1μMHalo TMR配体的10%FBS和L-谷氨酰胺 - 青霉素 - 链霉素代替培养基至FluoroBrite DMEM。在37℃,5%CO 2气氛下孵育细胞。

  3. 图像采集
    1. 加入Halo TMR配体一小时后,将玻璃底盘放入加热台上。删除免费的TMR并不重要。然而,如果HaloTag标记的蛋白质的表达水平低,背景荧光妨碍检测,则应将细胞用含10%FBS和L-谷氨酰胺 - 青霉素 - 链霉素的FluoroBrite DMEM洗涤三次,然后将培养皿置于加热阶段。
    2. 采用共焦焦显微镜,采用488和561 nm的激光线采集H4K20me1薄荷体(绿色通道)和TMR标记的Halo-SUV420H1(红色通道)的荧光图像,结合二向色镜DM405/488/561/640和发射过滤器分别为520/30和617/73

注意:

  1. 用0.1μMHalo TMR配体染色一小时通常足以标记HaloTag标记的蛋白质。然而,如果信号强度不够高,则样品可以与TMR配体孵育过夜。用10%FBS和L-谷氨酰胺 - 青霉素 - 链霉素洗涤氟乙酸DMEM也可提高信噪比。
  2. 为了避免荧光强度的饱和,我们简要扫描多个区域,并设置激光功率和曝光时间,以覆盖所有细胞(除了有时表现出最亮荧光的死细胞)在相机的动态范围内绿色和红色频道。
  3. 捕获的图像中的非转染细胞将用于阴性对照细胞。
  4. 经验上,至少需要30个光晕阳性细胞进行成像才能进行良好的统计分析。总共200个细胞,包括非转染细胞通常成像。

数据分析

  1. H4K20me1的相对含量通过薄荷核的浓缩测定 注意:为了比较不同细胞中H4K20me1-mintbody的核富集,测量每个细胞中的细胞核/细胞质强度比。
    1. 使用NIS元素打开图像。
    2. 选择没有单元格的区域来选择背景ROI。
    3. 通过背景减法获取净强度图像。
    4. 使用"绘制多边形ROI"工具手动绘制单元格和核心的ROI(图3)。核区域可以通过三维化和二值化自动选择,但由于细胞质中的信号强度低,通常难以选择细胞区域。对于数十个细胞的分析,手动绘图通常更快速准确;由于所选区域相当大,所以几个像素的差异不会太大。
    5. 测量绿色通道中ROI的平均强度[I],面积[A]和总强度[T = I x A]。
    6. 将数据导出到Excel。
    7. 在Excel中打开导出的数据,通过以下方程计算细胞质的强度:[I]细胞质=([T]细胞 - [T]核)/([A]细胞 - [A]核)。
    8. 通过将细胞核的平均强度与细胞质的平均强度(即,比例= [I]细胞核/[I]细胞质)的比例计算核/细胞质强度比例。


      图3.绘制ROI以定义单元格和核区域。使用NIS元素分析的代表性图像文件。蓝色,Hoechst;绿色,H4K20me1薄荷;红色,光环SUV420H1。注意,在这里使用活的Hoechst染色,但这并不是必需的,因为核区域与细胞质明显区分开来。比例尺= 10μm

  2. Halo-SUV420H1的表达水平
    1. 上述定义的核的ROI区也可用于Halo-SUV420H1表达水平的相对量化。
    2. 测量红色通道中核的平均强度
  3. SUV420H1表达与H4K20me1的关系
    1. 绘制散点图以显示SUV420H1表达水平与H4K20me1之间的关系(图4)。


      图4.H4K20me1-mintbody监测的活细胞中的H4K20me1水平。该图由Sato等人于2016年改编。Halo-SUV420H1在HeLa细胞中瞬时表达,稳定表达H4K20me1-mintbody。在表达SUV420H1(箭头)的细胞中,薄荷体在细胞质中更加扩散。绘制单细胞的核/细胞质强度比(右图)。核/细胞质比率取决于Halo-SUV420H1表达。 Bar = 10μm

笔记

该方案可适用于许多其他细胞系和修饰酶。为了确认活细胞观察,可以使用用于产生H4K20me1薄荷体的原始H4K20me1特异性抗体(Hayashi-Takanaka等人,2015; Sato ,使用基于固定细胞的免疫荧光分析, em> et al。,2016)。

致谢

这项工作得到JSP KAKENHI授予JP25118714,JP262910711,JP25116005的支持。协议已经由Sato等人(2016)进行了改编。

参考

  1. Beck,DB,Oda,H.,Shen,SS and Reinberg,D。(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22345514"target ="_ blank"> PR-Set7和H4K20me1:在基因组完整性,细胞周期,染色体凝聚和转录的十字路口。 Genes Dev 26(4):325 -337。
  2. Freshney,RI(2015)。动物文化细胞:基本技术和专业应用手册,第7版。 John Wiley&儿子。
  3. Hayashi-Takanaka,Y.,Maehara,K.,Harada,A.,Umehara,T.,Yokoyama,S.,Obuse,C.,Ohkawa,Y.,Nozaki,N.,Kimura,H。(2015)。   组蛋白H4修饰的分布由具体的单克隆抗体。染色体抗体23(4):753-766。
  4. Hayashi-Takanaka,Y.,Yamagata,K.,Wakayama,T.,Stasevich,TJ,Kainuma,T.,Tsurimoto,T.,Tachibana,M.,Shinkai,Y.,Kurumizaka,H.,Nozaki,N. ,Kimura,H。(2011)。  跟踪表观遗传组蛋白使用基于Fab的活内源性修饰标记对单细胞进行修饰。 核酸Res 39(15):6475-6488。
  5. Jørgensen,S.,Schotta,G.和Sorensen,CS(2013)。  组蛋白H4赖氨酸20甲基化:基因组完整性的表观遗传调控的关键参与者。核酸Res 41(5):2797-2806。
  6. Kimura,H.,Hayashi-Takanaka,Y.,Stasevich,TJ,Sato,Y。(2015)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih。 gov/pubmed/26138929"target ="_ blank">可视化体内内源性蛋白质的翻译后和表观遗传修饰。 Histochem Cell Biol 144(2):101 -109。
  7. Sato,Y.,Kujirai,T.,Arai,R.,Asakawa,H.,Ohtsuki,C.,Horikoshi,N.,Yamagata,K.,Ueda,J.,Nagase,T.,Haraguchi, Hiraoka,Y.,Kimura,A.,Kurumizaka,H。和Kimura,H。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27534817"target ="_ blank">用于H4K20单甲基化的活细胞成像的遗传编码探针.Mol Biol。428(20):3885-3902。 >
  8. Sato,Y.,Mukai,M.,Ueda,J.,Muraki,M.,Stasevich,TJ,Horikoshi,N.,Kujirai,T.,Kita,H.,Kimura,T.,Hira,S.,Okada ,Y.,Hayashi-Takanaka,Y.,Obuse,C.,Kurumizaka,H.,Kawahara,A.,Yamagata,K.,Nozaki,N.and Kimura,H。(2013)。在体内追踪组蛋白修饰的遗传编码系统/a> 3:2436.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sato, Y. and Kimura, H. (2017). Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody. Bio-protocol 7(10): e2276. DOI: 10.21769/BioProtoc.2276.
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