使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法
Summary
MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.
Abstract
骨骼肌终末分化开始于多能中胚层前体细胞向成肌细胞的承诺。这些细胞具有仍然增殖的能力，或者它们可以分化和融合成多核肌管，其中，还maturate形成肌纤维。骨骼肌终末分化是由多种转录因子的协调动作协调，特别是肌肉调节因子或回收设施的成员（MyoD的，肌细胞生成素，Myf5的，和MRF4），也称为成肌的bHLH转录因子家族。这些因素精心转录调控网络中的染色质重塑复合物合作，以实现骨骼肌肉发生。在这方面，MyoD基因被认为是引发肌肉终末分化的主生肌转录因子。这个概念是由MyoD的与非肌肉细胞转化成骨骼肌细胞的能力增强。在这里，我们描述了用于识别MyoD的一种方法为了阐明参与骨骼肌终端分化的不同因素以详尽的方式蛋白伙伴。长期目的是了解参与骨骼肌基因的调控， 即 ，MyoD的目标的表观遗传机制。 MyoD的伙伴通过从耦合到质谱（MS）表征的异源系统中，随后有关伙伴的骨骼肌终末分化过程中的作用的验证使用串联亲和纯化（TAP-标记）标识。先天性肌无力，肌强直性营养不良，横纹肌肉瘤和缺陷在肌肉再生：生肌因子，或它们的异常调节异常的形式，与许多肌肉障碍相关联。因此，生肌因素提供潜在治疗靶点池肌肉疾病，既涉及导致疾病本身和再生机制，可以提高疾病的治疗机制。因此，详细understan由生肌因素控制分子间相互作用和基因计划的鼎是有效治疗的合理设计是必不可少的。
Introduction
真核多细胞生物是由不同的器官和组织。各功能性组织具有特定的基因图案表达，这是在各个分化步骤中确定。细胞分化涉及特定基因的活化，维持它们的表达，并且通常沉默一组基因，例如那些参与细胞增殖的。骨骼肌分化，或成肌，因而是一个多步骤的过程，与中胚层干细胞的确定为成肌细胞开始，然后导致这些成肌成第一单核，然后多核，肌管的终端分化。因此，成肌细胞被“确定”的细胞，其仍然能够增殖，但他们致力于骨骼肌谱系，因此可以单独使用分化成期间胚胎发育或成年肌肉再生的骨骼肌细胞。骨骼肌终端的过程不同entiation由特定遗传程序，与来自成肌细胞前体细胞的细胞周期，导致的增殖相关的基因，如E2F靶基因1明确沉默永久退出开始协调。实际上，终端分化的过程中，成肌细胞增殖阻滞是之前的骨骼肌特异性基因表达和成肌细胞的融合成肌管2的关键步骤。这样的程序允许成年肌肉干细胞，也称为卫星细胞，在以下骨骼肌损伤再生过程来区分。
哺乳动物肌形成是严重依赖于一个家庭生肌碱性螺旋-环-螺旋（bHLH结构）转录因子的MyoD，Myf5的，MRF4和肌细胞生成素，经常被称为家庭的骨骼肌确定因素或回收设施（肌肉调节因子3）3。他们每个人都在扮演着规范和DIF至关重要的作用骨骼肌细胞的ferentiation和具有特定的表达图案4 5-7 Myf5的和MyoD的活化构成了承诺细胞生肌谱系的决定性步骤，和肌细胞生成素的后续表达触发肌形成与骨骼肌特异基因，如MCK（肌肉肌酸激酶）的活化。生肌的bHLH转录因子与从先前无声轨迹8在肌肉的基因的激活MEF2家族的成员进行合作。它们也刺激骨骼肌基因的转录与普遍存在的bHLH蛋白，E12和E47，已知为E蛋白，其在各个基因调控区8结合所谓的E-盒异二聚体。捻，ID（分化抑制剂）等因素负调节这一过程中，通过与MyoD的对于E蛋白结合8竞争。
MyoD的是引发肌肉终末分化9视为主要参与者</s向上>因为它具有诱导在许多完全分化非肌肉系统10-13一个生肌判定/分化（反分化）方案的能力。事实上，MyoD的强制表达诱导不同的细胞类型，甚至没有从另一个胚胎起源12衍生的转分化。例如，MyoD的可肝细胞，成纤维细胞，黑素细胞，神经母细胞，以及脂肪细胞转化成肌样细胞。的MyoD的反式分化行动涉及的生肌遗传方案的在非肌肉环境中的异常活化（特别是它的靶基因），伴随的原始遗传程序（尤其是增殖基因）的沉默。
在增殖成肌细胞，MyoD的表达，但不能激活其靶基因甚至当它结合到他们的启动子14-16。因此，对于MyoD的在未分化的成肌细胞不断表达的需求仍然相当埃卢西伯。 MyoD的抑制可能因镇压染色质修饰酶的招聘其靶基因在激活染色质重塑酶14,17加载之前增殖成肌细胞。例如，在增殖成肌细胞，MyoD的与转录共阻遏KAP-1，组蛋白脱乙酰酶（HDACs）和压制性赖氨酸甲基转移（KMTs），包括组蛋白H3赖氨酸9或H3K9和H3K27 KMTs相关联，并且积极地抑制其靶基因的表达通过建立本地镇压染色质结构14,17。重要的是，最近的一份报告指出，MyoD的本身由H3K9 KMT G9A从而抑制其反式激活活性16的直接甲基化。
参与非肌肉细胞的MyoD的这种转分化的表观遗传机制在很大程度上是未知的。值得注意的是，一些细胞系是对MyoD的诱导转分化抗性。因此，在HeLa细胞中，MyoD的或者是不活动的，或甚至还不如阻遏，而不是转录激活功能，由于缺乏染色质重塑复杂SWI / SNF 18 BAF60C亚基的表达。因此，这种模式可以选择，以更好地表征的MyoD诱导基因抑制的机制。它也适合于测定的MyoD在其靶位点以诱导压制性染色质环境及其关联伙伴，因此揭开MyoD的依赖性压制性机制在增殖成肌细胞微调终末分化的能力。
在这里，我们通过使用耦合到质谱（MS）表征串联亲和纯化（TAP-标签）描述的MyoD伙伴的标识的协议。使用的HeLa-S3稳定表达的Flag-HA-的MyoD的许可来获得足够的材料来净化从分馏核提取物MyoD的。在异源系统的MyoD伙伴的识别随后validati在一个相关的系统。
Protocol
Representative Results
Discussion
该方法允许详尽的鉴定转录因子的MyoD的合作伙伴。它揭示了MyoD的来诱导分化，即接种HeLa-S3细胞性异源系统MyoD的合作伙伴。因此，根据定义，确定MyoD的合作伙伴普遍表达。这些包括一般和序列特异性转录因子，染色质修饰酶，RNA加工蛋白质激酶（ 表1和2）。自的HeLa-S3细胞对MyoD的诱导转分化抗性，MyoD的活动主要是压制性以及所识别的伙伴可能是MyoD的辅阻遏。这是通过Id?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Work in the Ait-Si-Ali lab was supported by the Association Française contre les Myopathies Téléthon (AFM-Téléthon); Institut National du Cancer (INCa); Agence Nationale de la Recherche (ANR), Fondation Association pour la Recherche sur le Cancer (Fondation ARC); Groupement des Entreprises Françaises pour la Lutte contre le Cancer (GEFLUC); CNRS; Université Paris Diderot and the ”Who Am I?” Laboratory of Excellence #ANR-11- LABX-0071 funded by the French Government through its ”Investments for the Future” program operated by the ANR under grant #ANR-11-IDEX-0005-01. EB was supported by an INCa grant.
Materials
| Cell lines | |||
| HeLa-S3 | ATCC | CCL-2.2 | |
| C2C12 | ATCC | CRL-1772 | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Spinner | Corning | 778531 | |
| Homogenizer : Dounce homogenizer | Wheaton | 432-1273 and 432-1271 | |
| Agitating device for spinners | Bellco | 778531 | |
| Sonicator | Diagenode | UCD 200 | |
| Hemocytometer | Marienfeld Superior | 640610 | |
| Low-binding tubes | Sorenson | 27210 | |
| Empty spin column | Bio-Rad | 7326204 | |
| Name | Company | Catalog Number | Comments |
| Reagents | |||
| SDS-polyacrylamide 4-12 % gel | Life technologies | NP0336BOX | |
| 4X loading buffer | Life technologies | NP0007 | |
| 10X reducing agent | Life technologies | NP0004 | |
| Silver staining kit | Life technologies | A8592 | |
| Centrifugal filter units, 10K | Millipore | UFC501024 | |
| Protein G agarose beads | Sigma-Aldrich | P4691 | |
| Protein A/G Resin | Thermo Scientific | 53132 | |
| Flag resin (anti-Flag M2-agarose affinity gel) | Sigma-Aldrich | A2220 | |
| HA resin (monoclonal Anti-HA agarose) | Sigma-Aldrich | A2095 | |
| MNase | Sigma-Aldrich | N3755 | |
| Flag free peptide (DYKDDDDK) | Ansynth Service BV | Custom synthesis | Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8 |
| HA free peptide (YPYDVPDYA) | Ansynth Service BV | Custom synthesis | Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8 |
| Bicinchoninic acid based protein assay kit : BCA kit | Thermo Scientific | 23225 | |
| Protease inhibitors | Sigma-Aldrich | S8830 | |
| Luminol-based enhanced chemiluminescence (ECL) HRP substrate | Life technologies | 34075 | |
| BSA | Sigma-Aldrich | A9647 | |
| Sheared salmon sperm DNA (Deoxyribonucleic acid sodium salt from salmon testes) | Sigma-Aldrich | D1626 | |
| Spermine tetrahydrochloride | Sigma-Aldrich | S1141 | |
| Spermidine | Sigma-Aldrich | S0266 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| PBS | Sigma-Aldrich | D8537 | |
| MOPS running buffer | Life technologies | NP0001 | |
| DMEM (high glucose) | Sigma-Aldrich | D0822 | Pre-warm at 37 °C before use |
| Trypsin-EDTA (0.05% phenol red | Life technologies | 25300-054 | |
| Serum | GE healthcare life sciences | PAA A15-102 | Each lot of serum has to be first tested for your cells. |
| Penicillin and Streptomycin | Life technologies | 15140-122 | |
| Trypan Blue Solution, 0.4% | Life technologies | 15250-061 | |
| Water (sterile-filtered) | Sigma-Aldrich | W3500 | |
| Name | Company | Catalog Number | Comments |
| Antibodies | |||
| HA from rat (12CA5) | Roche | 11583816001 | |
| Flag | Sigma-Aldrich | A8592 | |
| MyoD | Santa Cruz | sc-32758 | To use for western blotting |
| MyoD | Santa Cruz | SC-760 | To use for immunoprecipitation and western blotting |
| CBFbeta | Santa Cruz | FL-182 | |
| HP1alpha | Euromedex | 2HP2G9 | |
| HP1beta | Euromedex | 1MOD1A9AS | |
| HP1gamma | Euromedex | 2MOD1GC | |
| Suv39h1 | Cell Signaling Technology | 8729 | |
| BAF47 | BD Biosciences | 612111 | |
| Myf5 | Santa Cruz | SC-302 | |
| Tubulin | Sigma-Aldrich | T9026 | |
| Actin | Sigma-Aldrich | A5441 | |
| IgG Mouse | Santa Cruz | SC-2025 | |
| IgG Rabbit | Santa Cruz | SC-2027 | |
| Goat anti-rat -HRP | Sigma-Aldrich | A9037 | |
| Goat anti-rabbit -HRP | Sigma-Aldrich | A6154 | |
| Goat anti-mouse -HRP | Sigma-Aldrich | A4416 |
References
- Ait-Si-Ali, S., et al. A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells. Embo J. 23 (3), 605-615 (2004).
- Buckingham, M. Skeletal muscle development and the role of the myogenic regulatory factors. Biochem Soc Trans. 24 (2), 506-509 (1996).
- Arnold, H. H., Winter, B. Muscle differentiation: more complexity to the network of myogenic regulators. Curr Opin Genet Dev. 8 (5), 539-544 (1998).
- Buckingham, M. Skeletal muscle formation in vertebrates. Curr Opin Genet Dev. 11 (4), 440-448 (2001).
- Yun, K., Wold, B. Skeletal muscle determination and differentiation: story of a core regulatory network and its context. Curr Opin Cell Biol. 8 (6), 877-889 (1996).
- Molkentin, J. D., Olson, E. N. Defining the regulatory networks for muscle development. Curr Opin Genet Dev. 6 (4), 445-453 (1996).
- Arnold, H. H., Braun, T. Targeted inactivation of myogenic factor genes reveals their role during mouse myogenesis: a review. Int J Dev Biol. 40 (1), 345-353 (1996).
- Puri, P. L., Sartorelli, V. Regulation of muscle regulatory factors by DNA-binding, interacting proteins, and post-transcriptional modifications. J Cell Physiol. 185 (2), 155-173 (2000).
- Tapscott, S. J. The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription. Development. 132 (12), 2685-2695 (2005).
- Lassar, A. B., Paterson, B. M., Weintraub, H. Transfection of a DNA locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts. Cell. 47 (5), 649-656 (1986).
- Davis, R. L., Weintraub, H., Lassar, A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 51, 987-1000 (1987).
- Tapscott, S. J., et al. MyoD1: a nuclear phosphoprotein requiring a myc homology region to convert fibroblasts to myoblasts. Science. 242, 405-411 (1988).
- Weintraub, H., et al. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci U S A. 86 (14), 5434-5438 (1989).
- Mal, A., Harter, M. L. MyoD is functionally linked to the silencing of a muscle-specific regulatory gene prior to skeletal myogenesis. Proc Natl Acad Sci U S A. 100 (4), 1735-1739 (2003).
- Ohkawa, Y., Marfella, C. G., Imbalzano, A. N. Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1. Embo J. 25 (3), 490-501 (2006).
- Ling, B. M., et al. Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation. Proc Natl Acad Sci U S A. 109 (3), 841-846 (2012).
- Zhang, C. L., McKinsey, T. A., Olson, E. N. Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation. Mol Cell Biol. 22 (20), 7302-7312 (2002).
- Forcales, S. V., et al. Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex. The EMBO journal. 31 (2), 301-316 (2011).
- Nakatani, Y., Ogryzko, V. Immunoaffinity purification of mammalian protein complexes. Methods Enzymol. 370, 430-444 (2003).
- Ouararhni, K., et al. The histone variant mH2A1.1 interferes with transcription by down-regulating PARP-1 enzymatic activity. Genes Dev. 20 (23), 3324-3336 (2006).
- Fritsch, L., et al. A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex. Mol Cell. 37 (1), 46-56 (2010).
- Robin, P., Fritsch, L., Philipot, O., Svinarchuk, F., Ait-Si-Ali, S. Post-translational modifications of histones H3 and H4 associated with the histone methyltransferases Suv39h1 and G9a. Genome Biol. 8 (12), R270 (2007).
- Huang da, W., Sherman, B. T., Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 4 (1), 44-57 (2009).
- Huang da, W., Sherman, B. T., Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37 (1), 1-13 (2009).
- Ohta, S., et al. The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics. Cell. 142 (5), 810-821 (2010).
- Yahi, H., et al. Differential cooperation between heterochromatin protein HP1 isoforms and MyoD in myoblasts. J Biol Chem. 283 (35), 23692-23700 (2008).
- Philipot, O., et al. The core binding factor CBF negatively regulates skeletal muscle terminal differentiation. PloS one. 5 (2), (2010).
- Joliot, V., et al. The SWI/SNF subunit/tumor suppressor BAF47/INI1 is essential in cell cycle arrest upon skeletal muscle terminal differentiation. PloS one. 9 (10), e108858 (2014).
- Tanese, N. Small-scale density gradient sedimentation to separate and analyze multiprotein complexes. Methods. 12 (3), 224-234 (1997).
- Singh, K., et al. A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation. Genes Dev. 29 (5), 513-525 (2015).
- Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339 (6121), 819-823 (2013).
- Yahi, H., Philipot, O., Guasconi, V., Fritsch, L., Ait-Si-Ali, S. Chromatin modification and muscle differentiation. Expert Opin Ther Targets. 10 (6), 923-934 (2006).
- Sun, L., Liu, L., Yang, X. J., Wu, Z. Akt binds prohibitin 2 and relieves its repression of MyoD and muscle differentiation. J Cell Sci. 117. 117 (Pt 14), 3021-3029 (2004).
- Caretti, G., et al. The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation. Dev Cell. 11 (4), 547-560 (2006).
- Knoepfler, P. S., et al. A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1. Nucleic Acids Res. 27 (18), 3752-3761 (1999).
- Albini, S., Puri, P. L. SWI/SNF complexes, chromatin remodeling and skeletal myogenesis: it’s time to exchange!. Exp Cell Res. 316 (18), 3073-3080 (2010).
- Yahi, H., Fritsch, L., Philipot, O., Guasconi, V., Souidi, M., Robin, P., Polesskaya, A., Losson, R., Harel-Bellan, A., Ait-Si-Ali, S. Differential Cooperation between Heterochromatin Protein HP1 Isoforms and MyoD in Myoblasts. J Biol Chem. 283 (35), 23692-23700 (2008).
