酿酒酵母的单拷贝基因位点染色质纯化

摘要

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 。

摘要

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。

引言

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。^....

研究方案

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

1. 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：转化后的 SbfI 限制性片段包含用于组成型表达 LexA-TAP 融合蛋白和 R-重组酶的表达盒以及酵母染色体 I 的同源序列，用于通过同源重组对表达盒进行基因组整合（图 1）。
2. 为了构建对照菌株，取缺乏整合 RS 和 LexA 结合位点的同基因酵母菌株，并在与重组能力菌株相同的条件下用 SbfI 消化的 K238 质粒转化它。
3. 根据 SCD-LEU 琼脂平板¹⁴ 上的 LEU2 选择标记物选择感受态酵母细胞。

2. 将IgG抗体偶联到环氧活化的磁珠上

讨论

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?.......

材料

Name	Company	Catalog Number	Comments
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