强化酵母单杂交筛网识别与人 dna 序列结合的转录因子
Summary
在这里, 我们提出了一个增强的酵母单杂交筛选方案, 以确定转录因子 (tf), 可以绑定到人类 dna 区域感兴趣。该方法采用高通量筛选管道, 可以在一次实验中询问 tf 和 gt;1,000 的结合。
Abstract
识别调节每个人类基因的转录因子 (tf) 集是一项艰巨的任务, 需要整合许多实验和计算方法。其中一种方法是酵母单杂交 (y1h) 检测, 在该方法中, 利用报告基因在酵母细胞核环境中测试 tf 和 dna 区域之间的相互作用。y1h 检测涉及两个组成部分: “dna 诱饵” (例如, 促进剂、增强剂、消音器等) 和 “tf-猎物”, 可用于记者基因激活的筛选。大多数用于执行 y1h 屏幕的已发布协议都是基于将 tf-猎物库或阵列转换为 dna 诱饵酵母菌株。在这里, 我们描述了一个被称为增强 y1h (ey1h) 检测的管道, 在这种情况下, tf-dna 相互作用通过将 dna 诱饵菌株与使用高密度阵列 (hda) 机器人平台排列的 tf-猎物菌株进行交配来进行测试, 该平台允许在 1 536个测试中进行筛选菌落格式。这使得吞吐量大幅增加 (60个 dna 诱饵序列对 & gt;1,000 tf 每个研究人员需要两周时间) 和可重复性。我们通过针对 1, 086个人 tf 的阵列测试人类启动子序列来说明不同类型的预期结果, 以及屏幕过程中可能出现的问题以及如何进行故障排除的示例。
Introduction
生物医学领域的一个核心问题是确定调节每一个人基因的机制。转录是控制基因表达水平的第一步, 它是由每个基因特有的转录因子 (tf) 集调节的。考虑到人类编码 & gt;1,500 tf1,2, 确定一整套控制每个基因表达的 tf 仍然是一个开放的挑战。
两种类型的方法可用于映射 tf-dna 相互作用: tf 为中心的方法3和以 dna 为中心的方法 3 (图 1 a)。在以 tf 为中心的方法中, 研究了与基因组 dna 区域结合或确定其 dna 结合特异性的感兴趣的 tf。这些方法包括染色质免疫沉淀 (chip), 然后是高通量测序、蛋白质结合微阵列和 selex4,5,6。在以 dna 为中心的方法中, 研究了感兴趣的 dna 序列, 以确定与 dna 序列结合的 tf 集。这种方法应用最广泛的是酵母单杂交 (y1h) 检测, 其中 tf 和 dna 区域之间的相互作用在酵母细胞核的环境中使用报告基因7,8,9进行测试。
y1h 检测涉及两个组成部分: “dna 诱饵” (例如, 促进剂、增强剂、消音器等) 和 “tf-猎物”, 可用于对报告基因激活9、10 (图 1 b) 进行筛选。dna 诱饵是克隆的上游两个报告基因 (lacz和his3) 和两个 dna 诱饵:: 记者结构被集成到酵母基因组中, 产生染色质的 ‘ dna 诱饵菌株 ‘。tf-猎物编码在表达 tf 融合到酵母 gal4 tf 的激活域 (ad) 的质粒中, 被引入 dna 诱饵菌株中, 用于 tf-dna 的相互作用。如果 tf-猎物与 dna 诱饵序列结合, 那么 tf-猎物中存在的 ad 将导致两个记者基因的激活。因此, 可以选择具有正相互作用的细胞在缺乏组氨酸的板材上生长, 并克服竞争性抑制剂, 3-氨基-1, 2, 4-三唑 (3-at), 并可视化为蓝色菌落在 x-ger 的存在。由于使用了强效酵母 gal4 ad, y1h 检测可以检测涉及转录激活剂和抑制剂的相互作用。此外, 考虑到 tf-preys 是由强酵母启动子 (adh1) 表达的, 即使是内源性表达水平较低的 tf 也可以检测到相互作用, 而 chip 11,12很难检测到这些 tf.
大多数已发布的执行 y1h 检测的协议都是基于将 tf 前置入酵母 dna 诱饵菌株, 方法是转换集合 tf-猎物库, 然后进行选择、群体选择和排序, 以识别相互作用的 tf, 或者通过改变单个克隆8,9。这些都是耗时的协议, 限制了每个研究人员可以测试的 dna 序列的数量。最近对 y1h 检测的改进被称为增强 y1h (ey1h), 通过使用高密度阵列 (hda) 机器人平台将酵母 dna-诱饵菌株与酵母 dna-诱饵菌株组合在一起, 每个菌株都表达了不同的表达, 从而显著提高了筛选吞吐量tf-猎物 10,13 (图 1c)。这些屏幕采用 1, 536个菌落格式, 只需三个盘子即可测试大多数人体的 tf, 这些 tf 为四元。此外, 考虑到 tf-dna 相互作用是以对等的方式进行测试的, 这种方法允许比较 dna 诱饵 (如两个非编码单核苷酸变种) 之间和不同 tf 或 tf 变种之间的相互作用11,12 ,14。
利用 ey1h 检测, 我们已经描述了迄今为止最大的人类和以 dna为中心的 tf-dna 相互作用网络。特别是, 我们确定了246个人类发展促进者和 283个 tf12之间的 2 2230 种互动。此外, 我们还使用 ey1h 检测发现了改变后的 tf 结合到109种与遗传性疾病 (如发育畸形、癌症和神经紊乱) 相关的单核苷酸非编码变异。最近, 我们使用 ey1h 来描述一个网络, 该网络由 2, 576 ·c. elegans基因启动子和 366个 tf11之间的 21, 714个相互作用组成。这个网络有助于揭示数十种线虫的功能作用。
产生 dna 诱饵污渍和评估背景记者活动水平的协议已在其他地方报道过 15、16、17。在这里, 我们描述了一个 ey1h 管道, 它可以用来筛选任何人类基因组 dna 区域, 以对抗 1, 086个人类 tf 阵列。一旦产生酵母 dna-诱饵菌株, 并在相应的板块上发现 tf-猎物阵列, 整个协议可以在两周内执行 (表 1)。更重要的是, 该协议可以并行化, 以便单个研究人员可以同时筛选 60个 dna 诱饵序列。为了证明该协议, 我们筛选了两个细胞因子基因 ccl15 和 il17f 的启动子。此外, 我们还显示了失败屏幕的结果, 以说明在执行 ay1h 检测时可能出现的问题类型以及如何对其进行故障排除。
Protocol
Representative Results
Discussion
与以前的库筛选或基于转换的排列筛选方法相比, 这里描述的机器人 ay1h 交配筛选方法大大提高了识别绑定到感兴趣的 dna 区域的 tf 集的吞吐量。此外, 由 ey1h 检测检测到的 tf-dna 相互作用是高度可重复的, 因为检测到的90% 的相互作用对每个 tf 测试的所有四个菌落都呈阳性, 90% 的相互作用在同一酵母 dna-诱饵菌株的独立屏幕上重新检测,12,<sup class=…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院 [r35-gm128625 至 j. i. f. b.] 的支持。
Materials
| 3-Amino-1,2,4-triazole (3AT) ~95 % TLC | Sigma | A8056-100G | Competitive inhibitor for products of HIS3 gene |
| Adenine sulfate (hemisulfate), dihydrate | US Biologicals | A0865 | Required for proper yeast growth |
| Agar High Gel Strength – Bacteriological grade | American International Chemical | AGHGUP | Nutritive media for yeast growth |
| Ammonium Sulfate | US Biologicals | A1450 | Nitrogen source in synthetic yeast media |
| D+ Glucose Anhydrous | US Biologicals | G3050 | Required for yeast growth |
| Drop-Out Mix minus His, Leu, Tryp and Uracil, adenine rich w/o yeast nitrogen base | US Biologicals | D9540-02 | Synthetic complete media required for yeast growth |
| edge Multiparameter pH Meter | Hanna Instruments | HI2020-01 | To measure pH of selective media |
| Flat Toothpicks 750ct | Diamond | To streak yeasts on petridishes | |
| Glass Beads | Walter Stern | 100C | To spreak yeast when making lawns |
| Glycerol ≥99% | Millipore Sigma | G9012-1L | Required to make frozen yeast stocks |
| L-Histidine | US Biologicals | H5100 | For yeast growth selection in selective media |
| L-Leucine | US Biologicals | L2020-05 | For yeast growth selection in selective media |
| L-Tryptophan | Sigma | T-0254 | For yeast growth selection in selective media |
| N,N-Dimethylformamide | Sigma | 319937-1L | To make X-gal solution |
| Omnipense Elite | Wheaton | W375030-A | For dispensing accurate volumes of media into Singer plates |
| Peptone, Bacteriological | American International Chemical | PEBAUP | Protein source required for yeast growth |
| Petri Dish, 150×15 mm | VWR | 10753-950 | For growing yeast baits for screening |
| PlusPlates | Singer Instruments | PLU-003 | To make rectangular agar plates to use with Singer Robot |
| Precision Low Temperature BOD Refrigerated Incubator | ThermoFisher Scientific | PR205745R | To incubate yeast plates at constant temperature |
| RePads 1,536 short | Singer Instruments | REP-005 | To transfer the TF-prey array, mate yeast, and transfer yeast to diploid selection and readout plates |
| RePads 384 short | Singer Instruments | REP-004 | To transfer TF-prey array from 384 to 1,536 colony format |
| RePads 96 long | Singer Instruments | REP-001 | To transfer TF-prey array from glycerol stock to agar plate |
| RePads 96 short | Singer Instruments | REP-002 | To transfer TF-prey array from 96 to 384 colony format |
| Singer HDA RoToR robot | Singer Instruments | For transfering yeast in high-throughput manner | |
| Sodium Hydroxide (Pellets/Certified ACS) | Fisher | S318-1 | For adjusting pH of selective media |
| Sodium Phosphate dibasic heptahydrate | Santa Cruz Biotechnology | sc-203402C | Required for LacZ reporter activity on X-gal |
| Sodium Phosphate monobasic monohydrate | Santa Cruz Biotechnology | sc-202342B | Required for LacZ reporter activity on X-gal |
| Uracil | Sigma | U0750-100G | For yeast growth selection in selective media |
| X-gal (5-Bromo-4-chloro-3-indoxyl-beta-Dgalactopyranoside) | Gold Biotechnology | X4281C100 | β-galactosidase turns colorless X-gal blue to detect protein-DNA interaction |
| Yeast Extract | US Biologicals | Y2010 | Nutritious medium for growth and propagation of yeast |
| Yeast Nitrogen Base (powder) w/o AA, carbohydrate and w/o AS | US Biologicals | Y2030 | Required for vigorous yeast growth |
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