Name: an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling
Uploaded: 2017-12-18T14:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling at JoVE.com

我们感谢国家卫生研究院 (R01GM112003 YZ)、韦尔奇基金会 (BE-1913 YZ)、美国癌症学会 (RSG-16-215-01)、德克萨斯癌症预防和研究所 (YZ 至 RR140053) 提供的财政支持,RP170660 至 YZ), 美国心脏协会 (16IRG27250155 YH), 和约翰·邓恩基金会合作研究奖计划。

1. 细胞培养、质粒转染和化学诱导
<ol>
	<li>培养一个黏附细胞线 (例如, 人的胚胎肾脏 HEK293T 细胞) 在 Dulbecco 的修改过的老鹰的媒介 (DMEM), 补充与10% 热失活胎牛血清, 100 U/毫升青霉素和链霉素在37° c 与 5% CO2。</li>
	<li>染细胞与苹果酒质粒。
	<ol>
		<li>至少18小时前转染, 种子 0.3 x 106细胞在2.5 毫升适当 DMEM 每井在 6-井板和孵育细胞在37° c 过夜达到 60-80% 70-100。</li>
		<li>在使用前将转染试剂预热到室温下。</li>
		<li>在无菌管中放置250µL 无血清培养基。 注: 这一数额是为一个 6-井板 80% 70-100。如果需要, 按比例调整中等数量根据板材或细胞数字的大小。</li>
		<li>添加2.5 µg 的质粒编码苹果酒18或 TET2 的催化领域 (TET2CD 为阳性对照)12,13进入无血清培养基, 并由移轻轻混合。</li>
		<li>在稀释的 DNA 混合物中加入7.5 µL 转染试剂, 并由移轻轻混合。</li>
		<li>室温下孵育混合物20-30 分钟。</li>
		<li>改变5ml 介质, 将混合物 drop-wise 到井中, 轻轻摇动细胞培养板, 均匀地分配转染试剂和 DNA 混合物。</li>
		<li>在一夜之间孵育细胞和混合物, 然后用2ml 的新鲜完整的 DMEM 取代含有培养基的转染试剂。</li>
	</ol>
	</li>
	<li>化学诱导
	<ol>
		<li>稀释2毫米的雷帕霉素库存 (在亚甲基亚砜中溶解) 到20µM 在适当的完整培养基中的浓度。</li>
		<li>在2毫升培养基中加入20µL 稀释的雷帕霉素, 以达到 200 nM 的最终浓度。 注: 在孵育48小时后, 细胞可用于量化5hmC 代或任何其他下游应用。如果需要, 可以通过取出每3小时的细胞来监测感应效率, 如下所述, 并显示在图 1中。</li>
		<li>为了更好地量化5hmC 感应效率, 种子分离井每3小时监测细胞。</li>
	</ol>
	</li>
</ol>
2. 免疫对雷帕霉素诱导的5hmC 产量的定量
<ol>
	<li>5hmC 免疫荧光染色 注: 添加足够数量的固定液, 由4% 甲醛, 0.2% 表面活性剂 (如海卫 X-100) 在 PBS 与 3 N HCl, 以覆盖所有的细胞在板块。例如, 500 µL 的溶液对一个6井板的井来说就足够了。在室温下执行所有步骤。<ol>
		<li>为了修复细胞, 在室温下, 在 PBS 中添加4% 甲醛到雷帕霉素治疗的细胞15分钟。对于对照组, 使用无雷帕霉素治疗的 mCherry 苹果酒的细胞。不要鼓动。 警告: 甲醛是有毒的。穿戴适当的保护。</li>
		<li>丢弃定影液。在 PBS 中孵育0.2% 表面活性剂的固定细胞30分钟 permeabilize 细胞膜。</li>
		<li>丢弃性的解决方案。在化细胞中加入3的 HCl, 孵育15分钟以变性 DNA。</li>
		<li>丢弃 hcl. 将100毫米的三盐酸缓冲液 (ph 8.0) 添加到细胞中, 10 分钟用于中和 ph 值。</li>
		<li>丢弃所有解决方案。用 PBS 彻底冲洗细胞3-5 次。彻底删除 PBS。 注: 为每个洗涤步骤添加足够数量的 PBS。例如, 1 毫升的 PBS 是足够的6井板。</li>
		<li>通过在 PBS 中添加包含 1% BSA 和0.05% 的表面活性剂 (如吐温 20) 的阻塞缓冲单元, 在 1 h 中进行温和晃动。</li>
		<li>丢弃阻塞解决方案。将稀释的5hmC 抗体 (1:200) 添加到细胞中, 在室温或夜间在4° c 下孵育2小时。</li>
		<li>用 PBS 三次洗涤细胞15分钟。</li>
		<li>在细胞中加入稀释的荧光 (FITC)-共轭二级抗体 (1:200), 孵育1小时。</li>
		<li>用 PBS 三次清洗细胞, 去除残留的抗体。</li>
		<li>添加 250 ng/毫升 4, 6-diamidino-2-吲 (DAPI), 以染色核为5分钟. 去除染色液。</li>
		<li>安装幻灯片或玻璃底培养皿与 immunostained 细胞共焦成像。有代表性的结果显示在图 1B中。</li>
	</ol>
	</li>
	<li>流式细胞仪 注: 使用96井圆底板进行以下染色程序。通常, 150 µL 的试剂对每一个井都足够。<ol>
		<li>重转染和雷帕霉素诱导的细胞在外地资产管制署缓冲区 (PBS 与 1% BSA, 2 毫米 EDTA)。对于对照组, 使用无雷帕霉素治疗的苹果酒表达细胞。</li>
		<li>按照步骤2.1 中的说明修复和 permeabilize 单元格。</li>
		<li>将2的 HCl 添加到细胞中, 以变性10分钟的 DNA。</li>
		<li>放弃 HCl. 中和并阻止如步骤2.1 中所述的单元格。</li>
		<li>将稀释的 anti-5hmC 抗体 (1:200) 添加到细胞中, 在室温或夜间在4° c 下孵育1小时。</li>
		<li>用三倍的缓冲液清洗细胞。彻底删除外地资产管制的缓冲区。</li>
		<li>添加稀释的荧光 (FITC)-共轭二级抗体 (1:400) 用于荧光活化细胞分类 (磁场) 分析, 并在室温下孵育1小时。</li>
		<li>用三倍的缓冲液清洗细胞。</li>
		<li>样品已准备好进行外地资产管制分析。有代表性的结果显示在图 1C-d中。</li>
	</ol>
	</li>
</ol>
3. 斑点杂交法定量分析5hmC 和 5-嘧啶 (5mC) 量
<ol>
	<li>使用商业 dna 提取试剂盒将基因组 dna 从转染和雷帕霉素诱导的细胞中分离出来。对于对照组, 使用无雷帕霉素治疗的苹果酒转染细胞。</li>
	<li>将真空烘箱加热至80° c, pre-soak 硝化纤维素膜在双 H2O, 然后在6x 盐水-柠檬酸钠 (SSC) 缓冲中10分钟。</li>
	<li>负载60µL 等量的 DNA (总共2µg 在 TE 缓冲) 的 96-井 PCR 板。添加30µL 的 TE 缓冲器 (10 毫米三盐酸, 1 毫米 EDTA, pH 8.0) 到其余的井。</li>
	<li>通过将1行上30µL 的解决方案转移到2行的同一列位置, 使两个折叠串行稀释垂直于96井板。再次混合, 并转移30µL 的解决方案, 以井在随后的行, 直到到达边界。</li></ol>删除最后30µL。
	<li>每井加20µL 1 米氢氧化钠和10毫米 EDTA, 密封板并将混合物加热10分钟, 在95° c 完全变性 DNA 复式。</li>
	<li>立即冷却冰上的样品, 加50µL ice-cold 2 米的乙酸铵 (pH 7.0), 并在冰上孵育10分钟。 注: 步骤 3.7-3.10 涉及使用真空。</li>
	<li>用浸膜组装斑点杂交装置, 用全真空去除残留的缓冲器。</li>
	<li>通过添加200µL 的 TE 缓冲器的每一个井的斑点杂交设备清洗膜。打开真空以卸下 TE 缓冲器。</li>
	<li>加载的变性 DNA (在以前的步骤 3.1-3.6) 每井的斑点杂交设备。打开真空以消除溶液。</li>
	<li>通过添加200µL 的 2x SSC 来冲洗膜, 并完全使用真空去除残留的溶液。</li>
	<li>拆卸点印迹装置, 取出膜, 用20毫升 2x SSC 冲洗整个膜。</li>
	<li>空气干燥膜20分钟。</li>
	<li>将膜在80° c 下真空烘烤2小时。</li>
	<li>将堵塞液 (5% 脱脂牛奶 TBST) 添加到膜上, 室温下孵育1小时。</li>
	<li>丢弃阻塞解决方案。在膜上加入稀释的 5hmC (1:5、000) 或 5mC (1:1、000) 抗体, 在4° c 的夜间孵育。</li>
	<li>用 TBST 冲洗膜三次, 10 分钟去除残余抗体。彻底删除 TBST。</li>
	<li>添加 hrp 共轭二级抗体 (1:10,000 用于兔 hrp (5 hmc) 或 1:3, 000 用于 anti-mouse hrp (5 mc)) 到膜上, 在室温下孵育 1 h。</li>
	<li>用 TBST 冲洗膜三次, 10 分钟。</li>
	<li>添加 ECL 西部印迹基板的膜, 以可视化的5hmC 信号使用化学发光检测器。有代表性的结果显示在图 1E中。</li>

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作者声明没有竞争的金融利益。

在这里, 我们已经说明了使用的工程 split-TET2 酶, 以实现时间控制的 DNA 化。在发现了 5-methycytosine 酶的春节系列后, 许多研究已经进行了破译的生物功能的春节蛋白及其主要催化产品 5hmC1,2,3, 4,5,6,78,</su...

dna 甲基化, 主要是指添加一个甲基组的碳5位胞嘧啶形成 5-嘧啶 (5mC), 是由 dna 甲基转移 (DNMTs) 催化。5mC 作为哺乳动物基因组中的主要后生标记, 通常信号为转录抑制, X 染色体失活和转沉默1。DNA 甲基化的逆转是由十-精灵易位 (春节) 蛋白家族介导的。春节酶属于铁 (II) 和 2-二依赖性 dioxygenases, 催化连续氧化5mC 至 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) 和 5-carboxycytosine (5caC)。由春节介导的5mC 氧化对哺乳动物的基因组施加了额外的后生控制层。春节的发现激发了人们对表观遗传学领域的浓厚兴趣, 揭示了春节蛋白质及其主要催化产品5hmC 的生物学功能。5hmC不仅是在春节介导的活性 DNA 甲基2,3,4, 但也作为一个稳定的后生标记5,6,7,,8.尽管 dna 化与基因表达和 dna 化的异常变化密切相关, 但与一些人类疾病有关9,10,11, 因果关系在 dna 的后生修饰和表型之间往往仍然具有挑战性的建立, 这可以部分归因于缺乏可靠的工具, 以准确添加或删除 dna 修改在定义的时间和空间基因组决议.
在这里, 我们报告了使用化学诱导的基因重塑工具 (苹果酒) 克服的障碍, 研究的因果关系 DNA 化和基因转录。该设计的假设是, TET2 (TET2CD) 的催化领域可以分为两个非活性片段时, 表达的哺乳动物细胞和它的酶功能可以恢复通过采取化学诱导二方法 (图 1A)。为了建立一个 split-TET2CD 系统, 我们选择了六个 TET2CD, 由一个胱氨酸丰富的地区和双β螺旋 (DSBH) 褶皱组成, 根据报告的 TET2-DNA 复杂的晶体结构, 缺乏低复杂度区域12。一种合成的基因编码雷帕霉素诱导的二模块 FK506 结合蛋白 12 (FKBP12) 和 FKBP 雷帕霉素结合域 (FRB) 的哺乳动物目标的雷帕霉素14,15, 连同 self-cleaving 肽 T2A多肽序列16,17, 分别插入到 TET2CD 中的选定拆分站点中。选择 TET2 作为我们的工程目标是基于以下考虑。首先, TET2 的体细胞突变与伴随减少的 DNA 化在人类疾病中经常被观察到, 包括髓系疾病和癌症10, 它提供了有关敏感站点的有用信息, 以避免插入.第二, TET2 催化域的很大一部分, 特别是低复杂度区域, 对于酶函数12是可有可无的, 从而使我们能够为诱导的后生修饰创造一个最小的 split-TET2。在筛查超过15构造后, 在哺乳动物系统中选择了一种最高的雷帕霉素诱导还原酶活性的结构, 并被指定为苹果酒18。我们在此描述的使用 mCherry 标签苹果酒, 以实现诱导 DNA 化和表观遗传重塑与雷帕霉素, 并提出三方法验证苹果酒介导的5hmC 生产在一个模型蜂窝系统 HEK293T。

<table><tbody><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668027</td><td></td></tr><tr><td>Rapamycin</td><td>Sigma-Aldrich</td><td>37094</td><td></td></tr><tr><td>Paraformaldehyde, 16% Solution</td><td>VWR</td><td>100503-916</td><td></td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>0694-1L</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Amresco</td><td>0369-500ML</td><td></td></tr><tr><td>Albumin, Bovine</td><td>Amresco</td><td>0332-100G</td><td></td></tr><tr><td>Tween 20</td><td>Amresco</td><td>0777-1L</td><td></td></tr><tr><td>5-Hydroxymethylcytosine (5-hmC) antibody (pAb)</td><td>Active Motif</td><td>39769</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-11008</td><td></td></tr><tr><td>4,6-Diamidino-2-phenylindolde (DAPI)</td><td>Biotium</td><td>40043</td><td></td></tr><tr><td>SVAC1E SHEL LAB Economy Vacuum Oven, 0.6 Cu.Ft. (17 L)</td><td>SHEL LAB</td><td>SVAC1E</td><td></td></tr><tr><td>UltraPure SSC, 20X</td><td>Thermo Fisher Scientific</td><td>15557036</td><td></td></tr><tr><td>Bio-Dot Apparatus</td><td>BIO-RAD</td><td>1706545</td><td></td></tr><tr><td>Anti-5-methylcytosine (5mC) Antibody, clone 33D3</td><td>Millipore</td><td>MABE146</td><td></td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7076S</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7074S</td><td></td></tr><tr><td>West-Q Pico Dura ECL Solution</td><td>Gendepot</td><td>W3653-100</td><td></td></tr></tbody></table>

an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling

DNA 甲基化是哺乳动物基因组中一种稳定且可遗传的表观遗传修饰, 参与调控基因表达以控制细胞功能。dna 甲基化的逆转, 或 dna 甲基, 是介导的十-十一易位 (春节) 蛋白家族的 dioxygenases。尽管人们广泛报道, 异常 DNA 甲基化和甲基与发育缺陷和癌症有关, 但这些表观遗传变化如何直接导致基因表达或疾病进展的改变仍然不清楚, 主要是由于缺乏可靠的工具来精确地在定义的时间和空间分辨率下在基因组中添加或删除 DNA 修饰。为了克服这一障碍, 我们设计了一种 split-TET2 酶, 使时间控制 5-嘧啶 (5mC) 氧化, 并随后重塑哺乳动物细胞中后生状态的简单添加化学物质。在这里, 我们介绍了引进一种化学诱导基因重塑工具 (苹果酒) 的方法, 基于工程化的 split-TET2 酶, 进入哺乳动物细胞和量化的化学诱导生产的 5-hydroxymethylcytosine (5hmC) 与免疫, 流式细胞仪或斑点杂交法。这种化学诱导基因重塑工具将发现广泛使用在审讯蜂窝系统不改变遗传密码, 以及在探索 epigenotype−phenotype 关系的各种生物系统。

化学诱导 5 mc 到-5 hmc 转换可以通过免疫在单细胞水平, 流式细胞仪分析转染 (mCherry 阳性) 细胞的数量, 或更定量斑点杂交检测, 如图 1所示。TET2 的催化领域, 或 TET2CD, 在整个研究中作为一个积极的控制使用。参见参考资料 18-20, 进一步详细说明雷帕霉素诱导的5hmC 和染色质可达性变化的全基因组图谱。
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Watch this Scientific Journal Video about An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling at JoVE.com

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

用于化学诱导的 DNA 化和表观遗传重塑的基因工程 Split-TET2 酶

介绍了将工程化的 split-TET2 酶 (苹果酒) 引入哺乳动物细胞中用于化学诱导 DNA 化和后生重塑的详细协议。

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

an-engineered-split-tet2-enzyme-for-chemical-inducible-dna

Research

JoVE Journal

Chemistry

6.6K 次观看.  Texas A&M University. 介绍了将工程化的 split-TET2 酶 (苹果酒) 引入哺乳动物细胞中用于化学诱导 DNA 化和后生重塑的详细协议。

视频: An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

科研

神经科学

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

生物化学

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

遗传学

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

具有更严格特异性的限制内核内核酶的体外定向进化

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

生物工程

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

生物学

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

用于化学诱导的 DNA 化和表观遗传重塑的基因工程 Split-TET2 酶

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BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

具有更严格特异性的限制内核内核酶的体外定向进化

用化学表观修饰因子重定向细胞机械抑制基因转录

DNA甲基化：亚硫酸氢修改和分析

DNA系留RNA聚合酶用于可编程体外转录和分子计算

从基因组DNA选择性捕获的5 - 羟甲基

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

用于化学诱导的 DNA 化和表观遗传重塑的基因工程 Split-TET2 酶

摘要

探索更多视频

此视频中的章节

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

具有更严格特异性的限制内核内核酶的体外定向进化

用化学表观修饰因子重定向细胞机械抑制基因转录

DNA甲基化：亚硫酸氢修改和分析

DNA系留RNA聚合酶用于可编程体外转录和分子计算

从基因组DNA选择性捕获的5 - 羟甲基

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择