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。

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
<ol><li>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）。</li>
	<li>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。</li>
	<li>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。</li>
</ol>2. 将IgG抗体偶联到环氧活化的磁珠上
注：根据以下已发表的方案11，将 IgG 抗体偶联到环氧树脂活化的磁珠上。
<ol><li>将 300 mg 环氧活化珠悬浮在 50 mL 锥形管中的 10 mL 50% 丙酮（300 mg 对应于 ~5.1 ×10 10 个珠子）中。在涡旋混合器上用力摇晃。</li>
	<li>将含有珠子的试管在820× g 和4°C下离心2分钟。除去上清液。</li>
	<li>用 20 mL 0.1 M 磷酸钠缓冲液 （pH 7.4） 洗涤珠子 3 次。按照步骤2.2所述，在每个洗涤步骤后通过离心除去上清液。</li>
	<li>将珠子悬浮在 16 mL 0.1 M 磷酸钠缓冲液 （pH 7.4） 中，并在室温下在杂交炉中轻轻旋转 5 分钟。</li>
	<li>将兔IgGs（100mg）溶解在7mL dH2O（终浓度：14mg / mL）中。</li>
	<li>将IgG悬浮液在13,000× g 和4°C下离心10分钟以澄清悬浮液。</li>
	<li>将 3.5 mL 上清液（相当于 50 mg 兔 IgG）转移到新的 50 mL 锥形管中。</li>
	<li>用 9.85 mL 0.1 M 磷酸钠缓冲液 （pH 7.4） 稀释 IgG 溶液，然后在温和混合下滴加 6.65 mL 3 M 硫酸铵在 0.1 M 磷酸钠 （pH 7.4） 中。 注意：避免在溶液中快速添加硫酸铵，因为局部浓度高的盐会导致兔 IgG 沉淀。</li>
	<li>将IgG溶液在820× g 和4°C下离心3分钟，并将所得上清液加入磁珠悬浮液中。</li>
	<li>将试管孵育过夜或在30°C下至少孵育18小时，并在杂交烘箱中轻轻旋转。</li>
	<li>如步骤2.2所述除去上清液。</li>
	<li>用 20 mL 100 mM 甘氨酸-HCl （pH 2.5） 洗涤珠子。按照步骤2.2中的说明快速除去溶液，以避免IgG多肽变性。</li>
	<li>用 20 mL 10 mM Tris-HCl （pH 8.8） 洗涤珠子一次。如步骤2.2所述吸出上清液。</li>
	<li>加入 20 mL 0.1M 三乙胺溶液 5-10 分钟，轻轻旋转以灭活残留的反应性环氧基团。如步骤2.2所述除去上清液。</li>
	<li>用 20 mL PBS（pH 7.4）洗涤珠子 4 次，轻轻旋转 5 分钟。在每个洗涤步骤后，按照步骤2.2中的说明除去上清液。</li>
	<li>用 20 mL PBS（pH 7.4）和 0.5% Triton X-100 （w/v） 在杂交烘箱中轻轻旋转洗涤珠子 2 次，每次 5 分钟和 15 分钟。如步骤2.2所述除去上清液。</li>
	<li>将珠子悬浮在含有 0.02% 叠氮化钠 （w/v） 的最终体积为 16 mL 的 PBS （pH 7.4） 中。在4°C下以1mL等分试样储存直至使用。</li>
</ol>3. 酵母细胞培养和收获
<ol><li>将YPD板中的对照和重组感受态酵母菌株接种在5mL的YPR培养基中，并在30°C和200rpm下孵育过夜。</li>
	<li>将2mL该培养物接种到100mLYPR培养基中，并在30°C和200rpm下孵育过夜。</li>
	<li>对于每种菌株，将 1,800 mL 高压灭菌的 YP 培养基和 200 mL 高压灭菌的棉子糖溶液 （20% w/v） 分配到两个 5 L 锥形瓶中（每个菌株总共 4 L 培养基，分成两个烧瓶）。</li>
	<li>将生长的酵母细胞以OD600 0.2接种在其各自的培养基中，并按照步骤3.1中的描述孵育约6小时或直到细胞达到所需的OD600 1.0。为确保细胞的正常生长，不受任何污染，每隔2小时检查OD。</li>
	<li>加入 200 mL 半乳糖 （20% w/v） 以诱导位点特异性重组。</li>
	<li>加入 110 μL （50 ng/mL） 的 YMFA（与半乳糖同时，步骤 3.5）以阻止 G1 期的细胞，并孵育 2 小时。 注意：将YMFA添加到细胞中取决于要解决的实验条件和生物学问题。确切地说，对于该实验，细胞被阻滞在 G1 期以获得具有许可复制来源的同质细胞群;在此过程中，复制前复合物在 S 期开始 DNA 复制之前与 G1 期的起点结合。</li>
	<li>将细胞悬液转移到1L离心桶中，并在6,000× g 和4°C下离心10分钟。弃去上清液，并将细胞沉淀重悬于总体积为10-15mLdH 2O的溶液中。</li>
	<li>用鲁尔塞密封 25 mL 注射器，并将其放入装满水的 50 mL 锥形管中。将细胞悬浮液转移到注射器组件上，并在室温下以2,397× g 离心10分钟。弃去上清液。</li>
	<li>从注射器上取下鲁尔塞，将细胞挤出到液氮中以形成细胞“意大利面”。 注意：使用 4 L 培养基可提供 7-10 g 的最终细胞沉淀的湿重。</li>
	<li>将冷冻细胞意大利面转移到50mL锥形管中，并储存在-80°C直至进一步使用。</li>
</ol>4. 染色质位点纯化
注：有关此位点特异性染色质纯化方案中涉及的步骤的示意图概述，请参见 图 2 。
<ol><li>通过每次剧烈摇晃咖啡研磨机 2 次，每次 30 秒，研磨 30-50 克干冰来冷却商用咖啡研磨机。冷却后，丢弃干冰粉。</li>
	<li>在预冷的咖啡研磨机中将 3 克冷冻酵母细胞意大利面与 ~30-50 克干冰混合。</li>
	<li>用封口膜密封盖子和研磨机之间的连接处，以防止研磨过程中酵母细胞粉末的损失。</li>
	<li>将酵母细胞 - 干冰混合物研磨10次，每次30秒，每轮之间间隔30秒（总时间估计：~10分钟）。为避免干冰和细胞粉粘在咖啡研磨机壁上，请在研磨过程中不断敲击咖啡研磨机的侧面。</li>
	<li>使用干净干燥的刮刀将得到的酵母细胞干冰粉转移到塑料烧杯中。</li>
	<li>将烧杯保持在室温下以蒸发干冰。</li>
	<li>干冰蒸发后，向酵母细胞粉末中加入 2.25 mL 含有 1x 蛋白酶和磷酸酶抑制剂（750 μL/g 酵母细胞）的 MB 缓冲液。</li>
	<li>用力移液细胞-缓冲液混合物，以确保细胞与缓冲液完全悬浮，然后转移到 15 mL 锥形管中。</li>
	<li>从所得细胞悬液（粗细胞提取物 [CCE]）中取样品进行 DNA （0.1%） 和蛋白质 （0.05%） 分析。储存在-20°C直至进一步处理（如第7节所述）。</li>
	<li>将细胞悬液转移到低结合的2mL反应管中，并在4°C下以21,130× g 离心30分钟，以将细胞碎片与粗细胞裂解物分离。</li>
	<li>将所有试管中的上清液汇集到一个 15 mL 锥形管中。</li>
	<li>从上清液（起始量[IN]）中取样进行DNA（0.1%）和蛋白质分析（0.05%）。储存在-20°C直至进一步处理（如第7节所述）。</li>
	<li>通过用含有1x蛋白酶和磷酸酶抑制剂的500μL冷MB缓冲液在4°C下以20rpm旋转洗涤2次，以平衡500μL偶联磁珠浆液（对于每个酵母菌株）。在每个洗涤步骤后，使用磁性架从珠子中丢弃上清液。</li>
	<li>将IgG偶联的磁珠在500μL冷MB缓冲液中与1x蛋白酶和磷酸酶抑制剂在4°C下以20rpm旋转孵育1小时。使用磁架从珠子中丢弃上清液。</li>
	<li>将平衡的磁珠浆液与细胞裂解物混合，并在4°C下以20rpm旋转孵育2小时。</li>
	<li>将完全的磁珠-细胞裂解物悬浮液转移到低结合反应管中。</li>
	<li>使用磁架将现在携带目标染色质环的磁珠与细胞裂解物分离。</li>
	<li>将剩余的流通 （FT） 从每个试管转移到新鲜的 15 mL 锥形管中，并采集样品进行 DNA （0.1%） 和蛋白质 （0.05%） 分析。储存在-20°C直至进一步处理（如第7节所述）。</li>
	<li>将每个试管中的磁珠悬浮在 300 μL 冷 MB 缓冲液中，并将磁珠汇集到一个反应管中。用含有1x蛋白酶和磷酸酶抑制剂的750μL冷MB缓冲液在4°C下以20rpm旋转洗涤5次，每次10分钟。用不含蛋白酶和磷酸酶抑制剂的 750 μL 冷 MB 缓冲液进行最终洗涤。</li>
	<li>将微珠悬浮于不含蛋白酶和磷酸酶抑制剂的 40 μL 冷 MB 缓冲液中。 注：洗脱液的最终体积可根据其预期的下游应用进行调整。TEV 洗脱通常在 100-300 μL 范围内有效工作。</li>
</ol>5. TEV蛋白酶介导的洗脱
<ol><li>为了从珠子中释放LexA-CBP染色质环复合物，将珠子与2μL6x His标记的重组TEV蛋白酶在热混合器中4°C和450rpm孵育过夜。</li>
	<li>使用磁架将珠子与最终洗脱液分离。将含有裂解染色质环的洗脱液转移到新的 1.5 mL 反应管中。</li>
	<li>再次将试管放在磁架上，以将任何残留的珠子与最终洗脱液分离。</li>
	<li>将珠子 （TB） 重悬于 750 μL 冷 AC 缓冲液中，并取样品进行 DNA （0.1%） 和蛋白质 （0.05%） 分析。储存在-20°C直至进一步处理（如第7节所述）。</li>
	<li>从最终洗脱液 （TE） 中取样进行 DNA （0.5%） 和蛋白质 （0.25%） 分析。储存在-20°C直至进一步处理（如第7节所述）。 注：由于最终洗脱液的体积较小，因此增加了用于 DNA 和蛋白质分析的样品的百分比，以便在分析过程中更好地表示其蛋白质和 DNA 含量。</li>
</ol>6. 变性洗脱
<ol><li>在750μL冷AC缓冲液中洗涤珠子2x，每次在4°C下以20rpm旋转20分钟。</li>
	<li>为了提取剩余的结合的LexA染色质复合物，通过向珠子中加入500μL的0.5M NH4OH进行变性洗脱，并在室温下孵育30分钟。</li>
	<li>使用磁架将珠子从悬浮液中分离出来，并将洗脱液转移到低结合反应管中。</li>
	<li>如步骤6.2所示再次孵育珠子，以恢复洗脱液中的最大染色质位点。</li>
	<li>使用磁架将珠子从悬浮液中分离出来，并将所得洗脱液汇集到包含步骤6.3中的洗脱液的同一管中。</li>
	<li>将珠子 （DB） 重悬于 750 μL dH2O 中，并取样进行 DNA （0.1%） 和蛋白质 （0.05%） 分析。</li>
	<li>从最终洗脱液 （DE） 中取样进行 DNA （0.5%） 和蛋白质 （0.25%） 分析。</li>
</ol>7. DNA和蛋白质分析
<ol><li>DNA分析<ol><li>样品制备<ol><li>向 DNA 样品中加入 100 μL IRN 缓冲液，并用 dH2O 增加体积至最终体积为 200 μL。</li>
			<li>加入 1 ng K071 加标质粒 DNA。</li>
			<li>加入1μLRNAse A（10mg / mL），并在37°C下孵育1小时。</li>
			<li>加入5μL蛋白酶K（10mg / mL）和10μLSDS（20%），并在56°C下孵育1小时。</li>
			<li>加入 200 μL 苯酚/氯仿/异丙醇 （25：24：1），每次彻底涡旋 2 次，持续 10 秒。</li>
			<li>将样品以 21,130 × g 离心 7 分钟以分离有机相和水相。</li>
			<li>将上清液转移到含有 1.5 μL 糖原 （5 mg/mL） 和 2.5x 100% 乙醇的新 1.5 mL 管中。</li>
			<li>将试管在-20°C孵育至少2小时，最多过夜以沉淀DNA。</li>
			<li>以21,130× g，4°C离心45分钟。弃去上清液。</li>
			<li>加入150μL的70%乙醇，不重悬DNA沉淀，并在4°C下以21,130× g 再次离心10分钟。</li>
			<li>将DNA沉淀在室温下干燥10-15分钟，并重悬于40μL dH2O中。</li>
			<li>进行限制性内切酶消化，用HpaI限制性核酸内切酶线性化步骤7.1.1.11中分离的DNA。对于50μL反应混合物，将40μL分离的DNA + 2μLHpaI酶+ 5μL限制性内切酶缓冲液+ 3μL dH2O在37°C下孵育过夜。</li>
		</ol></li>
		<li>qPCR分析<ol><li>以 1：5 的比例稀释限制性酶切的 DNA（10 μL 从步骤 7.1.1.12 获得的限制性酶切 DNA 在 40 μL dH2O 中）。</li>
			<li>使用专为 ARS316 区域、PDC1 和刺突质粒 DNA 设计的引物对。</li>
			<li>使用 表2中给出的程序运行qPCR。</li>
			<li>使用 ARS316 区域和加标质粒 DNA 作为靶标，以 PDC1（或任何其他基因或基因组区域）为参考位点，通过相对定量分析 qPCR 结果。</li>
			<li>通过将 ARS316 和 PDC1 引物相对定量值归一化为每个样品所取馏分体积的百分比来评估 PDC1 上的 ARS316 位点回收百分比。例如，将流通液的 0.1% 乘以 1,000 倍，将 TEV 洗脱液的 0.5% 乘以 200 倍。</li>
			<li>使用加标质粒 DNA 相对定量值对 ARS316 和 PDC1 回收率百分比进行归一化。</li>
			<li>通过使用公式（1）评估获得的相对定量靶标/参考值，评估ARS316位点在总染色质上的富集。 <img alt="Equation 1" src="/files/ftp_upload/65460/65460eq01v2.jpg" style="margin: auto;">（1）</li>
		</ol></li>
		<li>Southern印迹分析<ol><li>向 20 μL 限制性内切酶消化的 DNA 样品中加入 5 μL 凝胶上样染料。</li>
			<li>在110V的1%琼脂糖凝胶中运行样品~3小时。</li>
			<li>将凝胶转移到托盘中，并将其浸泡在脱嘌呤溶液中，轻轻摇晃20分钟。</li>
			<li>用dH2O冲洗凝胶，并在变性溶液中摇动浸泡15分钟。丢弃变性溶液。</li>
			<li>将凝胶浸泡在变性溶液中15分钟，轻轻摇晃。</li>
			<li>用dH2O冲洗凝胶，并用转印缓冲液轻轻摇动，每次洗涤2次，每次15分钟。</li>
			<li>用 1-1.5 L 转印缓冲液填充托盘，并将其放在稳定的平台上。</li>
			<li>剪下一张长 Whatman 纸，并将其放在平台上，使纸的两端浸泡在转印缓冲液中。</li>
			<li>切下一条正性尼龙膜和四条与凝胶大小相等的 Whatman 纸条。</li>
			<li>将两张浸泡在平台上转印缓冲液中的 Whatman 纸。</li>
			<li>将凝胶面朝下放在 Whatman 纸片的顶部。</li>
			<li>在凝胶的顶部，放置正性尼龙膜，然后将剩余的两片 Whatman 纸浸泡在转印缓冲液中。确保使用转印缓冲液保持烟囱湿润。</li>
			<li>使用玻璃棒将任何滞留的气泡滚落，以去除它们。</li>
			<li>将一叠纸巾放在组装好的纸巾上，然后放一个 0.5 公斤的物体（例如玻璃瓶）。</li>
			<li>将组件在室温下放置过夜转移。</li>
			<li>转移后，紫外线交联膜，总能量输出为 1,200 J/cm2。</li>
			<li>为了检测 ARS316 位点，使用使用参考 DNA 标记系统合成的放射性探针进行 Southern 印迹杂交。 注意：在冰上执行以下所有步骤，直到另有说明。</li>
			<li>在低结合反应管中，在19μL dH2O中稀释25-40ngPCR片段（探针），并在95°C下孵育5分钟。立即在冰上冷却。</li>
			<li>向试管中加入 500 μM dCTP、500 μM dGTP 和 500 μM dTTP 各 1 μL。</li>
			<li>加入 20 μL 含有试剂盒随附的随机核苷酸六聚体的缓冲液。</li>
			<li>向试管中加入 5 μL 放射性标记的核苷酸 [α-32P] dATP。在放射性批准的保护环境中执行涉及放射性标记材料的所有步骤。</li>
			<li>加入1μL的Klenow片段，并在37°C下在热混合器上孵育15分钟，用于单链DNA合成。</li>
			<li>加入 5 μL 终止缓冲液以停止反应。</li>
			<li>将尺寸排阻柱以 1,500 × g 离心 2 分钟以除去储存缓冲液。弃去流过的液体，并将色谱柱放入新管中。</li>
			<li>将探针混合物通过色谱柱以去除未掺入的放射性核苷酸。以 1,500 × g 离心 30 秒以收集探针。</li>
			<li>加入 150 μL 鲑鱼精子 DNA （1：100） 以阻断探针与膜的非特异性结合。</li>
			<li>将探针混合物在95°C孵育5分钟，使双链DNA变性。</li>
			<li>在冰上快速冷冻几秒钟，然后以 500 × g 离心几秒钟，以降低凝结在盖子上的水滴。</li>
			<li>用杂交缓冲液短暂洗涤南部印迹膜5-10分钟，旋转。</li>
			<li>将膜与杂交缓冲液在55°C下预杂交1小时，并旋转。丢弃杂交缓冲液。</li>
			<li>将15mL新鲜杂交缓冲液（在55°C预热）与制备的探针一起加入膜中。将膜在55°C下旋转孵育过夜。</li>
			<li>在55°C下用0.3x SSC，0.1%SDS进行杂交后洗涤2次，每次15分钟，并旋转。</li>
			<li>在55°C下用0.1x SSC，0.1%SDS进行杂交后洗涤2次，每次15分钟，旋转。</li>
			<li>在55°C下用0.1x SSC，1.5%SDS进行杂交后洗涤2次，每次15分钟，旋转。</li>
			<li>从杂交管中取出膜，并将膜暴露在磷化成像仪屏幕中长达 3 天，以获得薄膜上的放射性印记。</li>
			<li>使用磷光成像激光扫描系统获取胶片图像。</li>
		</ol></li>
	</ol></li>
	<li>蛋白质分析<ol><li>样品制备<ol><li>通过加入 1 μL β-巯基乙醇、PAGE LDS 样品缓冲液 （1x） 和 dH2O，将所有蛋白质样品调整至其最终体积（粗细胞提取物、输入、流通、珠子和洗脱液样品分别为 200 μL、100 μL、100 μL、20 μL 和 20 μL）。</li>
			<li>样品在95°C下变性5分钟。</li>
		</ol></li>
		<li>蛋白质印迹分析 注：使用标准方案15,16 进行 SDS-PAGE 和蛋白质印迹。<ol><li>将每个样品的 15 μL 上样到 1.5 mm SDS-PAGE 凝胶的每个孔中。</li>
			<li>使用PAP（1：2,000）和LexA（1：1,000）抗体对印迹进行免疫染色，并在4°C下孵育过夜。 用5%奶粉/PBST溶液稀释。 注：在 PAP 分析后，在使用第二种 LexA 抗体进行免疫染色之前，可以使用温和的剥离方案17 剥离印迹。</li>
		</ol></li>
	</ol></li>
</ol>

从酵母细胞中分离基因座特异性染色质

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D., Lorch, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-five+years+of+the+nucleosome,+fundamental+particle+of+the+eukaryote+chromosome.">Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome.</a> Cell. 98 (3), 285-294 (1999).</li><li>Gauchier, M., van Mierlo, G., Vermeulen, M., D&#233;jardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+enrichment+of+specific+chromatin+loci.">Purification and enrichment of specific chromatin loci.</a> Nature Methods. 17 (4), 380-389 (2020).</li><li>Korthout, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+chromatin+proteome+of+a+single+genomic+locus+by+DNA+sequencing.">Decoding the chromatin proteome of a single genomic locus by DNA sequencing.</a> PLoS Biology. 16 (7), e2005542 (2018).</li><li>Ant&#227;o, J. M., Mason, J. M., D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+landscape+at+Drosophila+melanogaster+telomere-associated+sequence+repeats.">Protein landscape at Drosophila melanogaster telomere-associated sequence repeats.</a> Molecular and Cellular Biology. 32 (12), 2170-2182 (2012).</li><li>D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins+associated+with+specific+genomic+loci.">Purification of proteins associated with specific genomic loci.</a> Cell. 136 (1), 175-186 (2009).</li><li>Ide, S., Dejardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-targeting+proteomics+of+isolated+chromatin+segments+of+a+mammalian+ribosomal+RNA+gene+promoter.">End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.</a> Nature Communications. 6, 6674 (2015).</li><li>Unnikrishnan, A., Gafken, P. R., Tsukiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+histone+acetylation+regulate+origins+of+DNA+replication.">Dynamic changes in histone acetylation regulate origins of DNA replication.</a> Nature Structural &amp;amp; Molecular Biology. 17 (4), 430 (2010).</li><li>Buxton, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+protein-DNA+interactions+in+human+alphoid+chromatin+via+hybridization+capture+and+mass+spectrometry.">Elucidating protein-DNA interactions in human alphoid chromatin via hybridization capture and mass spectrometry.</a> Journal of Proteome Research. 16 (9), 3433-3442 (2017).</li><li>Kennedy-Darling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+chromatin-associated+proteins+via+sequence-specific+capture+and+mass+spectrometric+protein+identification+in+Saccharomyces+cerevisiae.">Discovery of chromatin-associated proteins via sequence-specific capture and mass spectrometric protein identification in Saccharomyces cerevisiae.</a> Journal of Proteome Research. 13 (8), 3810-3825 (2014).</li><li>Liu, S. 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D., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+yeast+transformation+using+the+LiAc/SS+carrier+DNA/PEG+method.">High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.</a> Nature Protocols. 2 (1), 31-34 (2007).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (9), 4350-4354 (1979).</li><li>Western blot membrane stripping for restaining protocol. Abcam Available from: <a target="_blank" href="https://www.abcam.com/protocols/western-blot-membrane-stripping-for-restaining-protocol">https://www.abcam.com/protocols/western-blot-membrane-stripping-for-restaining-protocol</a> (2023)</li><li>Vermeulen, M., D&#233;jardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locus-specific+chromatin+isolation.">Locus-specific chromatin isolation.</a> Nature Reviews. Molecular Cell Biology. 21 (5), 249-250 (2020).</li></ol>

作者没有利益冲突需要披露。

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control Strain: MATa; ura3&#916;0; leu2&#916;0; his3&#916;1; met15&#916;0; bar1::kanMX4; Chr I 212kb::LEU2 pTEF2-LEXA-TAP pGAL1-10 RecR</td>
			<td></td>
			<td></td>
			<td>Section 1, see references 13 and 14</td>
		</tr>
		<tr>
			<td>Recombination Strain: MATa; ura3&#916;0; leu2&#916;0; his3&#916;1; met15&#916;0; bar1::kanMX4; RS_LEXA_NS-3_ARS316_NS+3_RS; Chr I 212kb::LEU2 pTEF2-LEXA-TAP pGAL1-10 RecR</td>
			<td></td>
			<td></td>
			<td>Section 1, see reference 13 and 14</td>
		</tr>
		<tr>
			<td>Plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>K238 plasmid</td>
			<td></td>
			<td></td>
			<td>Section 1, see reference 13 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>K071 Spike-in plasmid DNA</td>
			<td></td>
			<td></td>
			<td>Section 7.1, see reference 13 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Carl Roth</td>
			<td>5025.1</td>
			<td>Section 2 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Ammonium acetate (NH4Ac)</td>
			<td>Sigma Aldrich</td>
			<td>A7262</td>
			<td>Section 6 and 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Ammonium solution (NH4OH) 25%</td>
			<td>Merck Millipore</td>
			<td>533003</td>
			<td>Section 6 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Santa Cruz</td>
			<td>Sc-29085</td>
			<td>Section 2 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD (VWR)</td>
			<td>90000-760</td>
			<td>Section 3 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD (VWR)</td>
			<td>211820</td>
			<td>Section 3 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>07604</td>
			<td>Section 7.2 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Chemiluminescent substrate kit</td>
			<td>ThermoFisher</td>
			<td>34580</td>
			<td>Section 7.2 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Di-Sodium Hydrogen phosphate dodecahydrate</td>
			<td>Merck</td>
			<td>1.06579.1000</td>
			<td>Section 2 and 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>ThermoFisher</td>
			<td>15508013</td>
			<td>Section 4 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>ED</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Galactose (20% (w/v) stock)</td>
			<td>Sigma Aldrich</td>
			<td>G0625-1KG&#160; /&#160; 5KG</td>
			<td>Section 3 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Gel loading dye (6x)</td>
			<td>BioLabs</td>
			<td>B7024A</td>
			<td>Section 7.1 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Glusose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Section 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth</td>
			<td>.0079.4</td>
			<td>Section 2 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Glycogen (5 mg/mL)</td>
			<td>Invitrogen</td>
			<td>AM9510</td>
			<td>Section 7.1 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>PanReac AppliChem</td>
			<td>182109.1211</td>
			<td>Section 2, 4 and 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Magnesium Acetate (MgAc)</td>
			<td>Bernd Kraft</td>
			<td>15274.2600/C035</td>
			<td>Section 4 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td>Section 6 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Nu PAGE LDS sample buffer (4x)</td>
			<td>Invitrogen</td>
			<td>2399549</td>
			<td>Section 7.2 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamyl alcohol (25:24:1 v/v)</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
			<td>Section 7.1 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>Section 4 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Radioactively labeled &#945;-32P dATP (3,000 Ci/mmol, 10 mCi/mL)</td>
			<td>Hartmann Analytic</td>
			<td>SRP-203</td>
			<td>Section 7.1 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>RadPrime labeling system</td>
			<td>ThermoFisher</td>
			<td>18428-011</td>
			<td>Section 7.1 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Raffinose (20% (w/v) stock)</td>
			<td>SERVA</td>
			<td>34140.03</td>
			<td>Section 3 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Merck</td>
			<td>K53710504142</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Sodium citrate (Na3C6H5O7)</td>
			<td>Sigma-Aldrich</td>
			<td>71402</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Sodium n-dodecyl sulfate (SDS) (5% stock (w/v) )</td>
			<td>Alfa Aesar</td>
			<td>A11183</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>71496</td>
			<td>Section 2 and 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Sodium&#160;azide</td>
			<td>Santa Cruz&#160;Biotechnology</td>
			<td>sc-208393</td>
			<td>Section 2 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>&#160;Triethylamine</td>
			<td>Sigma Aldrich</td>
			<td>90340</td>
			<td>Section 2 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Chem Cruz</td>
			<td>SC-3715B</td>
			<td>Section 2 and 4 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>Section 2 and 4 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bernd Kraft</td>
			<td>18014332</td>
			<td>Section 4 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD (VWR)</td>
			<td>212720</td>
			<td>Section 3 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Yeast mating factor alpha (1 &#181;g/mL stock )</td>
			<td>&#160;Biomol</td>
			<td>Y2016.5</td>
			<td>Section 3 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Yeast Synthetic Drop-out medium Supplements without LEUCINE</td>
			<td>Sigma Aldrich</td>
			<td>Y1376</td>
			<td>Section 1, see reference 14</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HpaI restriction enzyme (5,000 U/mL)</td>
			<td>NEB</td>
			<td>R0105S</td>
			<td>Section 7.1 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Protease and Phosphatase Inhibitor Cocktail (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>78446</td>
			<td>Section 4 
			Storage: Store at4 &#176;C</td>
		</tr>
		<tr>
			<td>Proteinase K (10 mg/mL)</td>
			<td>SERVA</td>
			<td>33756</td>
			<td>Section 7.1 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>RNase A (10 mg/mL)</td>
			<td>&#160;ThermoFisher</td>
			<td>EN0531</td>
			<td>Section 7.1 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>TEV protease (10000 U/&#181;L)</td>
			<td>NEB</td>
			<td>P8112S</td>
			<td>Section 5 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BcMag Epoxy-Activated Magnetic Beads</td>
			<td>Bioclone Inc.</td>
			<td>FC-102</td>
			<td>Section 2 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td></td>
			<td></td>
			<td>Section 4</td>
		</tr>
		<tr>
			<td>Low-binding centrifuge tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>22431102</td>
			<td>Section 4</td>
		</tr>
		<tr>
			<td>Microspin&#160;G-25 Columns</td>
			<td>Cytiva</td>
			<td>27-5325-01</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Merck</td>
			<td>P7793</td>
			<td>Section 4</td>
		</tr>
		<tr>
			<td>Positive nylon membrane</td>
			<td>Biozol</td>
			<td>11MEMP0001</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>PVDF transfer membrane</td>
			<td>Immobilon-Merck Millipore</td>
			<td>IPVH00010</td>
			<td>Section 7.2 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>SDS-PAGE gel 4-12% bis-tris (15 well, 1.5 mm)</td>
			<td>Invitrogen</td>
			<td>&#160;NP0336BOX</td>
			<td>Section 7.2 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Syringe (25 mL) with luer fitting</td>
			<td>Henke Sass Wolf</td>
			<td>4200-000V0</td>
			<td>Section 3</td>
		</tr>
		<tr>
			<td>Whatman paper (Grade 3MM CHR Cellulose Western Blotting Paper Sheet)</td>
			<td>Cytiva</td>
			<td>3030-917</td>
			<td>Section 7.1 
			Storage: Store at room temperature</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-LexA, rabbit polyclonal IgG, DNA binding region antibody</td>
			<td>Merck Millipore</td>
			<td>06-719</td>
			<td>Section 7.2 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H+L), Horseradish peroxidase conjugate</td>
			<td>Invitrogen</td>
			<td>G21234</td>
			<td>Section 7.2 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Peroxidase Anti-Peroxidase (PAP) antibody produced in rabbit for the detection of TAP-tagged proteins</td>
			<td>Sigma Aldrich</td>
			<td>P1291-500UL</td>
			<td>Section 7.2 
			Storage: Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Rabbit IgG antibodies</td>
			<td>Sigma</td>
			<td>I5006-100MG</td>
			<td>Section 2 
			Storage: Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Primers (10 &#181;M)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ARS316: fwd 5&#39;- CGGCATTATCGTACACAACCT, rev 5&#39;- GTTCTTCGTTGCCTACATTTTCT</td>
			<td></td>
			<td></td>
			<td>Section 7.1</td>
		</tr>
		<tr>
			<td>K071 Spike-in plasmid DNA: fwd: 5&#39;-TTTTCGCTGCTTGTCCTTTT, rev 5&#39;- CATTTTCGTCCTCCCAACAT</td>
			<td></td>
			<td></td>
			<td>Section 7.1</td>
		</tr>
		<tr>
			<td>PCR fragment from yeast genomic DNA as a template &#160;for ARS316 amplification (for southern blot): fwd 5&#8217;- AAATTCTGCCCTTGATTCGT&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; rev 5&#8217;- TTTGTTTATCTCATCACTAAT</td>
			<td></td>
			<td></td>
			<td>Section 7.1</td>
		</tr>
		<tr>
			<td>PDC1: fwd 5&#39;- CATGATCAGATGGGGCTTCA, rev 5&#39;-ACCGGTGGTAGCGACTCTGT</td>
			<td></td>
			<td></td>
			<td>Section 7.1</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coffee grinder</td>
			<td>Gastroback</td>
			<td>42601</td>
			<td>Section 4</td>
		</tr>
		<tr>
			<td>Dewar flask</td>
			<td>NAL GENE</td>
			<td>4150-2000</td>
			<td>Section 3</td>
		</tr>
		<tr>
			<td>DynaMag TM-2 magnetic rack</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td>Section 4, 5 and 6</td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Hybaid Mini10</td>
			<td>Ri418</td>
			<td>Section 2</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td>Section 4 and 7.1</td>
		</tr>
		<tr>
			<td>UV-crosslinker</td>
			<td>Analytikjena</td>
			<td>95-0174-02</td>
			<td>Section 7.1</td>
		</tr>
	</tbody>
</table>

single-copy gene locus chromatin purification in saccharomyces cerevisiae

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。

~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...

Watch this Scientific Journal Video about Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol at JoVE.com

作者聚焦：使用高富集位点特异性染色质分离方案推进染色质研究并克服限制 

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 。

酿酒酵母的单拷贝基因位点染色质纯化

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

真核生物核非常拥挤，许多生物过程同时发生。我们的研究范围是了解所有这些过程如何在我们的基因组上协调，而不会发生重大事故，否则会导致基因组不稳定和人类疾病。该协议允许我们纯化处于天然状态的基因组的特定位点，从而允许对分离材料进行组成和功能表征，例如蛋白质DNA相互作用的测量。该协议能够对靶向位点进行高水平的富集，从而克服了目前位点特异性染色质分离的许多局限性。该材料尚未交联。这也允许许多下游功能分析，例如体外转录或体外复制。该协议使我们能够从酵母染色体中切除和纯化不同的复制来源，使我们能够进行蛋白质组学分析并确定新的起源相互作用染色质因子，从而推进DNA复制的研究领域。该协议能够使用纯化的原始材料进行体外复制研究，以检查染色质状态，组蛋白修饰，并对来自内源性染色体的天然核小体模板进行结构分析，从而推进染色质研究。

single-copy-gene-locus-chromatin-purification-saccharomyces

Research

JoVE Journal

Biochemistry

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

612 次观看.  Helmholtz Zentrum München. 真核生物核非常拥挤，许多生物过程同时发生。我们的研究范围是了解所有这些过程如何在我们的基因组上协调，而不会发生重大事故，否则会导致基因组不稳定和人类疾病。该协议允许我们纯化处于天然状态的基因组的特定位点，从而允许对分离材料进行组成和功能表征，例如蛋白质DNA相互作用的测量。该协议能够对靶向位点进行高水平的富集，从而克服了目前位点?...

视频: 作者聚焦：使用高富集位点特异性染色质分离方案推进染色质研究并克服限制 

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

格拉菲克斯用于检测 糖精 的瞬态相互作用器

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

贝克酵母糖精中线粒体基因组表达产品的标记和可视化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

萌芽酵母酿酒酵母中软骨蛋白定位的评估

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

<meta charset="utf8"></head><body>用于单分子研究的原位核小体重构

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

<meta charset="utf8"></head><body>作者聚焦：用于单分子技术的高效核小体重构

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

用于单分子相关力和荧光显微镜的原位核小体组装

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...