酿酒酵母的单拷贝基因位点染色质纯化
Summary
该方案提出了一种基于位点特异性重组的位点特异性染色质分离方法，以从出芽酵母酿酒酵母中纯化其天然染色质环境中感兴趣的单拷贝基因位点 。
Abstract
真核染色质的基本组织单位是核小体核心颗粒 （NCP），它包括包裹在组蛋白八聚体周围 ~1.7 倍的 DNA。染色质被定义为 NCP 和许多其他蛋白质复合物的实体，包括转录因子、染色质重塑和修饰酶。目前尚不清楚在细胞周期的不同阶段，这些蛋白质-DNA相互作用是如何在特定基因组位点的水平上协调的。这主要是由于当前的技术限制，这使得获得这种动态相互作用的精确测量具有挑战性。在这里，我们描述了一种改进的方法，将位点特异性重组与高效的单步亲和纯化方案相结合，以分离处于天然染色质状态的单拷贝目标基因位点。该方法允许在基因组染色质上稳健地富集靶位点，使该技术成为以无偏倚和系统的方式（例如通过质谱法）识别和量化蛋白质相互作用的有效策略。除了这种成分分析之外，通过这种方法纯化的天然染色质可能反映了有关核小体定位和组蛋白修饰的 体内 情况，因此，可以对来自酵母中几乎任何基因组位点的染色质进行进一步的结构和生化分析。
Introduction
真核生物基因组的动态组织成染色质，使DNA紧凑，使其适应细胞核的范围，同时确保基因表达的足够动态和调节因子的可及性。在某种程度上，这种多功能性是由核小体介导的，核小体是染色质的基本单位，它包括一个核心颗粒，其 147 bp 的 DNA 包裹在组蛋白八聚体1 周围 ~1.7 倍。核小体的组成是一种高度动态的结构，在 N 端和 C 端组蛋白尾部有许多组蛋白变体和翻译后修饰 （PTM）。此外，真核染色质与许多其他基本成分相互作用，例如转录因子、DNA 和 RNA 加工机制、结构蛋白、参与染色质重塑和修饰的酶以及与染色质相关的 RNA 分子。这些参与转录、复制和修复的关键机制都需要获得染色质，染色质是这些过程的天然底物。因此，理解这些DNA交易背后的分子机制需要精确定义这些机制汇聚并促进生物反应的特定基因组区域染色质结构的集体改变。
尽管通过遗传学和蛋白质-蛋白质相互作用研究鉴定了许多染色质因子，但对特定基因组位点的染色质相互作用进行直接、无偏倚和全面的分析仍然是一个重大障碍 2,3。最初，只能分离出足够数量和纯度的基因组高丰度区域（即重复位点）或多拷贝质粒，以质谱鉴定相关蛋白质4,5,6,7。一系列基于捕获探针与染色质化 DNA 的直接杂交、使用 CRISPR-dCas9 系统进行邻近生物素化或序列特异性衔接蛋白与目标位点结合的新方法已开始揭示酵母和哺乳动物基因组中单拷贝位点的蛋白质组 8,9,10.然而，所有这些方法都需要甲醛交联来稳定蛋白质-DNA的相互作用，并需要超声处理来溶解染色质以进行后续纯化。总之，这两种操作都排除了对纯化的染色质进行后续结构和功能研究的可能性。
为了克服这些局限性，我们之前设计了一种采用位点特异性重组从酵母中提取靶向染色体结构域的方法11,12。从本质上讲，感兴趣的基因组区域被来自接合酵母的位点特异性 R-重组酶的识别位点 （RS） 包围，同时在同一区域内掺入一组原核转录阻遏蛋白 LexA 蛋白 （LexA） 的三个 DNA 结合位点。酵母细胞包含一个用于同时表达 R-重组酶的表达盒和一个融合到串联亲和纯化 （TAP） 标签的 LexA 蛋白。在诱导R-重组酶后，该酶以环状染色质结构域的形式有效地从染色体上切除目标区域。该结构域可通过 LexA-TAP 接头蛋白纯化，该接头蛋白与 LexA DNA 结合位点以及亲和力支持结合。该方法最近被用于分离包含酵母 III13 号染色体选定复制来源的不同染色质结构域。
这种 离体 方法的一个主要优点是它允许对分离的材料进行功能分析。例如，可以对用该方法纯化的复制原点结构域进行 体外 复制测定，以评估在试管中从天然 体内 组装的染色质模板进行原点烧制的效率。最终，分离材料的生化和功能表征可能允许使用纯化的蛋白质和天然染色质模板重建核过程。总之，这种方法为染色质研究开辟了一条令人兴奋的途径，因为可以跟踪经历特定染色体交易的特定基因组区域的集体组成和结构染色质变化。
Protocol
有关本协议中使用的所有材料和仪器的详细信息，请参阅 材料表 。有关所用溶液、缓冲液和培养基的列表，请参见 表 1 。 1. 重组酵母菌株构建 为了构建具有重组能力的酵母菌株，将 SbfI 消化的质粒 K238 转化为在目标位点13,14 处集成了 LexA 结合位点和 RS 重组位点的酵母菌株。注意?…
Representative Results
~1.4 kb ARS316 染色质结构域的纯化由组成型表达的 LexA-TAP 接头蛋白介导。作为阴性对照，我们使用表达LexA-TAP但不含整合RS和LexA结合位点的同基因菌株进行纯化。 图 3 显示了对对照和靶向 ARS316 位点的重组能力菌株进行的标准纯化实验的 DNA 分析结果。在DNA分离和限制性内切酶消化以线性化环状DNA分子后，进行qPCR分析以评估ARS316靶位点对不相关的基因组对照位点PDC1的纯化效率…
Discussion
鉴定特定靶基因组区域的因子和染色质景观仍然是染色质研究的主要挑战18。该协议描述了一种有效的系统，用于从酵母染色体中特异性切除和纯化不同的染色质结构域。据我们所知，这种单步纯化的纯度和产量克服了位点特异性染色质纯化方法的许多局限性，因此与其他不相关的基因组区域相比，可以实现目标位点的非常高富集。
由于使用位点特异性R-重?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
S.H.实验室的工作得到了DFG通过SFB1064（项目ID 213249687）、欧洲研究委员会（ERC启动资助852798冲突解决）和亥姆霍兹协会的支持。
Materials
| Yeast strains | |||
| Control Strain: MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; bar1::kanMX4; Chr I 212kb::LEU2 pTEF2-LEXA-TAP pGAL1-10 RecR | Section 1, see references 13 and 14 | ||
| Recombination Strain: MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; bar1::kanMX4; RS_LEXA_NS-3_ARS316_NS+3_RS; Chr I 212kb::LEU2 pTEF2-LEXA-TAP pGAL1-10 RecR | Section 1, see reference 13 and 14 | ||
| Plasmid | |||
| K238 plasmid | Section 1, see reference 13 Storage: Store at -20 °C |
||
| K071 Spike-in plasmid DNA | Section 7.1, see reference 13 Storage: Store at -20 °C |
||
| Reagents | |||
| Acetone | Carl Roth | 5025.1 | Section 2 Storage: Store at room temperature |
| Ammonium acetate (NH4Ac) | Sigma Aldrich | A7262 | Section 6 and 7.1 Storage: Store at room temperature |
| Ammonium solution (NH4OH) 25% | Merck Millipore | 533003 | Section 6 Storage: Store at room temperature |
| Ammonium sulfate | Santa Cruz | Sc-29085 | Section 2 Storage: Store at room temperature |
| Bacto agar | BD (VWR) | 90000-760 | Section 3 Storage: Store at room temperature |
| Bacto peptone | BD (VWR) | 211820 | Section 3 Storage: Store at room temperature |
| β-Mercaptoethanol | Sigma Aldrich | 07604 | Section 7.2 Storage: Store at 4 °C |
| Chemiluminescent substrate kit | ThermoFisher | 34580 | Section 7.2 Storage: Store at 4 °C |
| Di-Sodium Hydrogen phosphate dodecahydrate | Merck | 1.06579.1000 | Section 2 and 7.1 Storage: Store at room temperature |
| Dithiothreitol (DTT) | ThermoFisher | 15508013 | Section 4 Storage: Store at 4 °C |
| Ethanol | Merck | 100983 | Section 7.1 Storage: Store at room temperature |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma Aldrich | ED | Section 7.1 Storage: Store at room temperature |
| Galactose (20% (w/v) stock) | Sigma Aldrich | G0625-1KG / 5KG | Section 3 Storage: Store at room temperature |
| Gel loading dye (6x) | BioLabs | B7024A | Section 7.1 Storage: Store at -20 °C |
| Glusose | Sigma-Aldrich | G8270 | Section Storage: Store at room temperature |
| Glycine | Carl Roth | .0079.4 | Section 2 Storage: Store at room temperature |
| Glycogen (5 mg/mL) | Invitrogen | AM9510 | Section 7.1 Storage: Store at -20 °C |
| Hydrochloric acid (HCl) | PanReac AppliChem | 182109.1211 | Section 2, 4 and 7.1 Storage: Store at room temperature |
| Magnesium Acetate (MgAc) | Bernd Kraft | 15274.2600/C035 | Section 4 Storage: Store at room temperature |
| Magnesium chloride (MgCl2) | Sigma Aldrich | M8266 | Section 6 Storage: Store at room temperature |
| Nu PAGE LDS sample buffer (4x) | Invitrogen | 2399549 | Section 7.2 Storage: Store at room temperature |
| Phenol/Chloroform/Isoamyl alcohol (25:24:1 v/v) | Invitrogen | 15593-031 | Section 7.1 Storage: Store at 4 °C |
| Potassium chloride (KCl) | Sigma | P9541 | Section 4 Storage: Store at room temperature |
| Radioactively labeled α-32P dATP (3,000 Ci/mmol, 10 mCi/mL) | Hartmann Analytic | SRP-203 | Section 7.1 Storage: Store at 4 °C |
| RadPrime labeling system | ThermoFisher | 18428-011 | Section 7.1 Storage: Store at -20 °C |
| Raffinose (20% (w/v) stock) | SERVA | 34140.03 | Section 3 Storage: Store at room temperature |
| Sodium chloride (NaCl) | Merck | K53710504142 | Section 7.1 Storage: Store at room temperature |
| Sodium citrate (Na3C6H5O7) | Sigma-Aldrich | 71402 | Section 7.1 Storage: Store at room temperature |
| Sodium hydroxide (NaOH) | Sigma Aldrich | S5881 | Section 7.1 Storage: Store at room temperature |
| Sodium n-dodecyl sulfate (SDS) (5% stock (w/v) ) | Alfa Aesar | A11183 | Section 7.1 Storage: Store at room temperature |
| Sodium phosphate monobasic | Sigma-Aldrich | 71496 | Section 2 and 7.1 Storage: Store at room temperature |
| Sodium azide | Santa Cruz Biotechnology | sc-208393 | Section 2 Storage: Store at -20 °C |
| Triethylamine | Sigma Aldrich | 90340 | Section 2 Storage: Store at room temperature |
| Tris base | Chem Cruz | SC-3715B | Section 2 and 4 Storage: Store at room temperature |
| Triton X-100 | Sigma Aldrich | X100 | Section 2 and 4 Storage: Store at room temperature |
| Tween-20 | Bernd Kraft | 18014332 | Section 4 Storage: Store at room temperature |
| Yeast extract | BD (VWR) | 212720 | Section 3 Storage: Store at room temperature |
| Yeast mating factor alpha (1 µg/mL stock ) | Biomol | Y2016.5 | Section 3 Storage: Store at -20 °C |
| Yeast Synthetic Drop-out medium Supplements without LEUCINE | Sigma Aldrich | Y1376 | Section 1, see reference 14 |
| Enzymes | |||
| HpaI restriction enzyme (5,000 U/mL) | NEB | R0105S | Section 7.1 Storage: Store at -20 °C |
| Protease and Phosphatase Inhibitor Cocktail (100x) | ThermoFisher Scientific | 78446 | Section 4 Storage: Store at4 °C |
| Proteinase K (10 mg/mL) | SERVA | 33756 | Section 7.1 Storage: Store at -20 °C |
| RNase A (10 mg/mL) | ThermoFisher | EN0531 | Section 7.1 Storage: Store at -20 °C |
| TEV protease (10000 U/µL) | NEB | P8112S | Section 5 Storage: Store at -20 °C |
| Materials | |||
| BcMag Epoxy-Activated Magnetic Beads | Bioclone Inc. | FC-102 | Section 2 Storage: Store at 4 °C |
| Dry ice | Section 4 | ||
| Low-binding centrifuge tubes 2.0 mL | Eppendorf | 22431102 | Section 4 |
| Microspin G-25 Columns | Cytiva | 27-5325-01 | Section 7.1 Storage: Store at room temperature |
| Parafilm | Merck | P7793 | Section 4 |
| Positive nylon membrane | Biozol | 11MEMP0001 | Section 7.1 Storage: Store at room temperature |
| PVDF transfer membrane | Immobilon-Merck Millipore | IPVH00010 | Section 7.2 Storage: Store at room temperature |
| SDS-PAGE gel 4-12% bis-tris (15 well, 1.5 mm) | Invitrogen | NP0336BOX | Section 7.2 Storage: Store at 4 °C |
| Syringe (25 mL) with luer fitting | Henke Sass Wolf | 4200-000V0 | Section 3 |
| Whatman paper (Grade 3MM CHR Cellulose Western Blotting Paper Sheet) | Cytiva | 3030-917 | Section 7.1 Storage: Store at room temperature |
| Antibodies | |||
| Anti-LexA, rabbit polyclonal IgG, DNA binding region antibody | Merck Millipore | 06-719 | Section 7.2 Storage: Store at -20 °C |
| Goat Anti-Rabbit IgG (H+L), Horseradish peroxidase conjugate | Invitrogen | G21234 | Section 7.2 Storage: Store at -20 °C |
| Peroxidase Anti-Peroxidase (PAP) antibody produced in rabbit for the detection of TAP-tagged proteins | Sigma Aldrich | P1291-500UL | Section 7.2 Storage: Store at -20 °C |
| Rabbit IgG antibodies | Sigma | I5006-100MG | Section 2 Storage: Store at 4 °C |
| Primers (10 µM) | |||
| ARS316: fwd 5'- CGGCATTATCGTACACAACCT, rev 5'- GTTCTTCGTTGCCTACATTTTCT | Section 7.1 | ||
| K071 Spike-in plasmid DNA: fwd: 5'-TTTTCGCTGCTTGTCCTTTT, rev 5'- CATTTTCGTCCTCCCAACAT | Section 7.1 | ||
| PCR fragment from yeast genomic DNA as a template for ARS316 amplification (for southern blot): fwd 5’- AAATTCTGCCCTTGATTCGT rev 5’- TTTGTTTATCTCATCACTAAT | Section 7.1 | ||
| PDC1: fwd 5'- CATGATCAGATGGGGCTTCA, rev 5'-ACCGGTGGTAGCGACTCTGT | Section 7.1 | ||
| Equipment | |||
| Coffee grinder | Gastroback | 42601 | Section 4 |
| Dewar flask | NAL GENE | 4150-2000 | Section 3 |
| DynaMag TM-2 magnetic rack | Invitrogen | 12321D | Section 4, 5 and 6 |
| Hybridization oven | Hybaid Mini10 | Ri418 | Section 2 |
| Microcentrifuge | Eppendorf | 5424R | Section 4 and 7.1 |
| UV-crosslinker | Analytikjena | 95-0174-02 | Section 7.1 |
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Cite This Article
Sajid, A., Hamperl, S. Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae. J. Vis. Exp. (201), e65460, doi:10.3791/65460 (2023).