Name: an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling
Uploaded: 2017-12-18T14:00:00
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...

<ol>
	<li>Li, E., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+mammals.">DNA methylation in mammals.</a> Cold Spring Harbor Perspectv Biol. 6 (5), a019133 (2014).</li><li>Tahiliani, M., 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=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324 (5929), 930-935 (2009).</li><li>He, Y. F., 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=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333 (6047), 1303-1307 (2011).</li><li>Ito, S., 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=Tet+proteins+can+convert+5-methylcytosine+to+5-formylcytosine+and+5-carboxylcytosine.">Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.</a> Science. 333 (6047), 1300-1303 (2011).</li><li>Spruijt, C. G., 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=Dynamic+readers+for+5-(hydroxy)methylcytosine+and+its+oxidized+derivatives.">Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.</a> Cell. 152 (5), 1146-1159 (2013).</li><li>Lio, C. W., 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=Tet2+and+Tet3+cooperate+with+B-lineage+transcription+factors+to+regulate+DNA+modification+and+chromatin+accessibility.">Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility.</a> eLife. 5, e18290 (2016).</li><li>Kellinger, M. W., 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=5-formylcytosine+and+5-carboxylcytosine+reduce+the+rate+and+substrate+specificity+of+RNA+polymerase+II+transcription.">5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription.</a> Nat Struct Mol Biol. 19 (8), 831-833 (2012).</li><li>Su, M., 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=5-Formylcytosine+Could+Be+a+Semipermanent+Base+in+Specific+Genome+Sites.">5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites.</a> Angew Chem Int Ed Engl. 55 (39), 11797-11800 (2016).</li><li>Ko, M., 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=Impaired+hydroxylation+of+5-methylcytosine+in+myeloid+cancers+with+mutant+TET2.">Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.</a> Nature. 468 (7325), 839-843 (2010).</li><li>Huang, Y., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connections+between+TET+proteins+and+aberrant+DNA+modification+in+cancer.">Connections between TET proteins and aberrant DNA modification in cancer.</a> Trends Genet. 30 (10), 464-474 (2014).</li><li>Lu, X., Zhao, B. S., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TET+family+proteins:+oxidation+activity,+interacting+molecules,+and+functions+in+diseases.">TET family proteins: oxidation activity, interacting molecules, and functions in diseases.</a> Chem Rev. 115 (6), 2225-2239 (2015).</li><li>Hu, L., 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=Crystal+structure+of+TET2-DNA+complex:+insight+into+TET-mediated+5mC+oxidation.">Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation.</a> Cell. 155 (7), 1545-1555 (2013).</li><li>Hashimoto, H., 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=Structure+of+a+Naegleria+Tet-like+dioxygenase+in+complex+with+5-methylcytosine+DNA.">Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.</a> Nature. 506 (7488), 391-395 (2014).</li><li>Banaszynski, L. A., Liu, C. W., Wandless, T. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+FKBP.rapamycin.FRB+ternary+complex.">Characterization of the FKBP.rapamycin.FRB ternary complex.</a> J Am Chem Soc. 127 (13), 4715-4721 (2005).</li><li>Clackson, 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=Redesigning+an+FKBP-ligand+interface+to+generate+chemical+dimerizers+with+novel+specificity.">Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.</a> Proc Natl Acad Sci U S A. 95 (18), 10437-10442 (1998).</li><li>Ibrahimi, A., 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=Highly+efficient+multicistronic+lentiviral+vectors+with+peptide+2A+sequences.">Highly efficient multicistronic lentiviral vectors with peptide 2A sequences.</a> Hum Gene Ther. 20 (8), 845-860 (2009).</li><li>Kim, J. H., 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=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PLoS One. 6 (4), e18556 (2011).</li><li>Lee, M., 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=Engineered+Split-TET2+Enzyme+for+Inducible+Epigenetic+Remodeling.">Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling.</a> J Am Chem Soc. 139 (13), 4659-4662 (2017).</li><li>Huang, Y., Pastor, W. A., Zepeda-Mart&#237;nez, J. A., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-CMS+technique+for+genome-wide+mapping+of+5-hydroxymethylcytosine.">The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.</a> Nat Protoc. 7 (10), 1897-1908 (2012).</li><li>Buenrostro, J. D., Wu, B., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATAC-seq:+A+Method+for+Assaying+Chromatin+Accessibility+Genome-Wide.">ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide.</a> Curr Protoc Mol Biol. 109 (21.29), 1-9 (2015).</li></ol>

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 ...

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