The authors would like to acknowledge Dr. Bence Daniel, Matt Peloquin, Dr. Endre Barta, Dr. Balint L Balint and members of the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by grants from the Hungarian Scientific Research Fund (OTKA K100196 and K111941) and co-financed by the European Social Fund and the European Regional Development Fund and Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-I/9.

1.基于芯片序列分析选择增强<ol><li>从http://ngsdebftp.med.unideb.hu/bioinformatics/下载RXR芯片起原始数据FASTQ文件（mm_ES_RXR_24h_ATRA.fastq.gz） </li><li>下载并提取对准所需的BWA索引文件（在我们的例子: Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz 注:关于生物信息学分析的步骤参观https://github.com/ahorvath/Bioinformatics_scripts获取更多信息，并下载下面使用的脚本。 </li><li>对准例子FASTQ文件MM10基因组（使用脚本:perform_alignment.sh）。这将创建一个.bam文件和比对统计数据的文件夹中。不结盟RXR芯片起的数据（mm_ES_RXR_24h_ATRA.bam），以及相应的索引文件（.bai）都可以在这里: http://ngsdebftp.med.unideb.hu/bioinformatics/ 注:BWA是一个软件包，对齐RELAtively短序列（ 例如，芯片起的结果），以一序列数据库，如鼠标参照基因组（ 例如，MM10）13。 </li><li>润峰主叫和从头主题分析脚本（callpeaks.sh）。使用.bam文件作为输入。该脚本是基于荷马findPeaks 14。 注意:输出文件给我们关于峰的总数，富集的图案，背景的百分比，并与基序的靶序列等相关信息。 （ 图1）。通常几个主题中列出了它们的重要性顺序。 </li><li>重新映射排名第一的基序（NR半`AGGTCA`）（使用脚本:remap_motif.sh） 注意:作为结果，脚本生成.bed文件，该文件的ChIP-峰覆盖包含感兴趣的转录因子的规范结合位点的基因组区域，将显示。 </li><li>下载并可视化对准RXR芯片起读取结果（mm_ES_RXR_24h_ATRA.bam（也下载.bai））和AGGTCA基序发生（mm_ES_RXR_24h_ATRA_homerpeaks_motif1_mm10s_200_remaped_mbed.bed）通过结合基因组浏览器（IGV）15，以确定推定RAR / RXR结合位点（ 图2和图3）。 注:各种芯片起的数据整合，将有助于更精确地预测好的候选增强16,17。最近的研究表明，活性增强剂通常富集P300和H3K4me1和H3K27ac相关，并位于开放染色质区域，从而显示DNase的我超敏反应还建议18-21。 </li><li>使用在IGV 15" 定义的感兴趣区域 "，以获得200 -所选择的基因组区域的400bp的序列和用于随后的引物设计的序列。 </li></ol> 2.报告基因检测注:萤光素/萤光素酶系统用于作为一个非常敏感的报告基因检测的转录调控。取决于增强子活性萤光素酶是产生将催化萤光素氧化成氧化萤光素导致bioluminescense，它可以被检测到。作为第一步骤，确定推定的增强子序列应当亚克隆到报告载体（ 例如，TK萤光素酶22的pGL3或NanoLuc）。 <ol><li> 引物设计 <ol><li>设计PCR引物200扩增-通过引物3假定增强地区的400基点（可在这里:http://bioinfo.ut.ee/primer3/）23 </li><li>复制 - 粘贴IGV基因组序列，其中包括假定的结合位点为:引物（见步骤1.7）。集产品尺寸范围250 - 在引物3 300基点。 </li><li>验证使用UCSC基因组浏览器的PCR引物在硅片 PCR工具（https://genome.ucsc.edu/cgi-bin/hgPcr）24 注意:此协议在后面的步骤用HindIII和BamHI限制性内切酶进行克隆。检查是否由UCSC 的In-硅片的PCR工具预测将包含AAGCTT或GGATCC限制性位点的PCR扩增序列（ 例如，http://nc2.neb.com/NEBcutter2/）。 </li><li>获得从商业供应商所需的引物。 </li></ol></li><li> 从基因组DNA增强子序列的PCR扩增 <ol><li>从净化主要细胞（ 如初级胚胎成纤维细胞）的模板基因组DNA。使用基因组DNA的分离商业工具并按照制造商的说明。 注:基因组DNA的高品质是成功的PCR扩增至关重要。如果PCR是不从基因组DNA容易适当BAC克隆都可以使用。 </li><li>制备含有100ng的基因组DNA，5μl的10×缓冲液，3微升用MgSO 4（25毫摩尔），5微升的dNTP（2mM的各），1.5微升正向引物（10μM），1.5微升启引物50微升的PCR反应（10μM） ，1微升DNA聚合酶 <li>设置PCR循环（2分钟95℃（20秒95℃，10秒58 - 65℃，18秒70℃）X25重复，2分钟70°C，永远4℃）。设置退火温度为审议底漆的预计值的Tm值。 </li><li>净化与商业PCR纯化试剂盒PCR产物，并按照制造商的说明。 </li><li>通过用引物重复PCR如上使用，但对它们的5添加突出端引入用于克隆的限制性位点&#39;末端（ 例如，正向:5&#39;-ATAT AAGCTT XXXXXXXXXXXXXXXXXXXX-3&#39;（HindⅢ位），版本:5&#39;-TATA GGATCC XXXXXXXXXXXXXXXXXXXX -3&#39;（BamHⅠ位））。 </li><li>运行5微升在琼脂糖凝胶上对PCR产物的检查非特异性PCR产物。 注:如果检测到更多的频段，从而使整个PCR产物和由商业凝胶提取试剂盒净化合适的频段。 </li></o升&gt; </li><li> 限制 注意:要插入克隆TK启动22纯化的PCR产物和载体（ 例如，TK-吕克）的上游侧应用HindIII和BamHI消化。 <ol><li>制备含1微克纯化的PCR产物，1微升的HindIII（20U /微升），1微升的BamHI（20U /微升）和5微升10X缓冲液50微升反应混合物。 </li><li>制备250μl的反应混合物含有5μg的向量（TK吕克，或替代报告载体），5微升的HindIII（20U /微升），5微升的BamHI（20U /微升），5微升的热敏碱性磷酸酶（1U /微升）和25微升10X缓冲。 注:AP治疗载体的大大降低单消化的载体的自身连接，从而所述背景。 </li><li>孵育4小时，反应混合物在37℃，然后纯化消化的PCR产物和用市售的PCR纯化试剂盒的载体。 </li></ol></li><li> Ligatioñ <ol><li>设置用于连接在PCR管包括2微升10×T4 DNA连接酶缓冲液，10毫微克TK-吕克矢量，50纳克刀片（消化的PCR产物），1微升T4 DNA连接酶（400单位/微升）和无核酸酶水，反应多达20微升。 注:准备一个阴性对照，其中在反应中不包含插入片段。 </li><li>轻轻吹打起来并混合反应下来，后短暂离心使用mini-离心机的PCR管，然后在室温孵育10分钟。 </li><li>热灭活在65℃下10分钟，然后冰浴，并使用5微升用于转化的产物进50微升感受态细胞（ 例如，DH5α）。 </li><li>使用标准的热休克过程转化细菌，拿起殖民地和隔离质粒DNA与商业套件。通过之前的下一步测序验证所有的结构。 </li></ol></li><li> ES细胞的转染 <ol><li>使用48孔板。加入200微升0.1％明胶SOLUT离子/孔30分钟前，细胞电镀。 </li><li>板胚胎干细胞（ES细胞）转染的前一天。使用无饲养ES细胞和板以每孔3×10 4个细胞在250μl的ES介质/孔的密度。 </li><li>制备质粒混合物（每个组合计算了8个孔，4未处理和4视黄酸处理的） 混合1:1250纳克TK-吕克空（阴性对照），950纳克β半乳糖苷酶编码向量（的βGal）， 混合2 :1.250纳克TK-吕克的Hoxa1增强（阳性对照），950毫微克的βGal， 混合3:1.250纳克TK-吕克PRMT8增强剂（感兴趣区域），950毫微克的βGal。 注:ES细胞表达所需的转录因子（RAR / RXR）。离开井未转染的过程中的荧光素酶和β半乳糖苷酶的测量后，确定基值。 </li><li>转染的品质降低血清培养基添加到每个质粒混合高达106微升总体积（足够用于8孔）。 </li><li>加入4.5微升ES质量染离子试剂通过移液15倍上下仔细混合。在室温下孵育10分钟的转染混合物。 </li><li>加入13微升转染混合物，以每口井的细胞，吹打调匀。将细胞放回孵化器（37°C）O /配体治疗N前。 </li><li>从细胞中吸小心取出媒体，并添加250微升新鲜培养基/孔含有0.01 - 1μM全反式维甲酸（RA）作为最终的浓度或DMSO作为溶媒。孵育细胞24 - 48小时。 注:维甲酸对光敏感。 </li></ol></li><li> 细胞裂解液的制备 <ol><li>稀释5倍裂解缓冲液（1.25毫升中含0.5摩尔Tris pH值= 7.8，加入1ml的1M二硫苏糖醇（DTT）（以H 2 O），将10毫升的0.1M的EDTA的pH = 8.0，将50ml甘油，加入5ml的Triton X-100，无菌加水至100毫升）至1x使用无菌水，加入20微升的1M DTT / 10毫升并在使用前平衡至室温。制备出10毫升在测定当天的48孔板中。 </li&gt; <li>从细胞中吸小心取出介质。用1X PBS冲洗细胞一次。 200μl的1×裂解缓冲液/孔直接添加到细胞中。 </li><li>在RT（化学裂解）摇动2小时。冻结在平板上的细胞裂解物在-80℃（机械裂解）。裂解物可以储存在-80℃几天。 </li><li>解冻的裂解。完成解冻后，使用电子多道移液器转移细胞裂解物至96孔板中。转移80微升细胞溶胞产物的96孔透明板β半乳糖苷酶测定法和40微升细胞裂解物，以白色板为萤光素酶测定。避免形成任何气泡。 </li></ol></li><li> β半乳糖苷酶测定法 注:β-半乳糖苷酶或替代性的第二记者应该被用作内部对照，以占非特异性的实验变异。 <ol><li>准备β半乳糖苷酶底物缓冲液80.0克Na 2 HPO 4•7H 2 O，3.8克和 NaH 2 PO 4•H 2 0，30.0克氯化钾，将1.0g硫酸镁4•7H 2 O，使多达1000毫升无菌水。调节pH至7.4。过滤消毒（0.2微米），并存储在4℃ </li><li>混合1毫升β半乳糖苷酶底物缓冲液与4毫克ONPG（邻 - 硝基苯基β-D-半乳糖苷酶）。使用前添加3.5微升每1毫升缓冲液2-巯基乙醇（βME）的。 100μl的β半乳糖苷酶底物缓冲液添加到80μl的细胞裂解物。 </li><li>在37℃孵育反应，直到昏黄色开发。阅读在OD 420处的吸光度的酶标仪和出口数据。 注:时间取决于转染效率，通常是超过30分钟不再。 </li></ol></li><li> 萤光素酶检测 <ol><li>准备50毫升萤光素底物试剂:15.1毫克D-荧光素盐，82.7毫克ATP盐，185毫克硫酸镁4•7H 2 O，加至50ml聚丙烯管，然后加入1.5毫升1M的HEPES缓冲液，48​​.5毫升ATP免费的水，旋涡，确保所有的化合物完全溶解。保持5 - - 10毫升分装，在-80℃。 </li><li>温馨5毫升萤光素底物试剂，以RT在开始之前。吸移管将100μl底物试剂到含有40微升细胞溶胞产物的白色板的每个孔中，并立即用发光计数器机器测量信号。 </li><li>从一式三份转染至少三个独立的实验获得的活动。 </li><li>首先计算每个井的基部标准化荧光素酶活性和碱规范化β半乳糖苷酶值减去用于从测得的各值的非转染的细胞测得的平均值。然后计算萤光素酶/β半乳糖苷酶的活性的比值。 </li></ol></li></ol> 3.增强RNA的表征注:增强活动的一个更直接的指标已经从最近的基因组-W涌现该确定的许多短的非编码RNA，从50至2000个核苷酸，这是从增强剂转录大小不等，和被称为增强剂的RNA（eRNAs）IDE研究16,25,26（ 图4）。尔娜感应与相邻的外显子编码基因的诱导高度相关。因此，与信号相关 ​​的增强子活性可以在体内通过比较用RT-qPCR的各种条件之间尔娜生产定量。 <ol><li> 指引引物设计用于检测尔娜用RT-qPCR的 <ol><li>选择尔娜测量基因组区域是至少1.5 - 2 kb的距离注释转录起始位点。 注:重要的是，高含量的整个基因内增强剂的mRNA转录防止尔娜的准确定量的有义方向，在基因内增强剂反义eRNAs是可检测的，并且在基因外增强剂水平eRNAs相似。 </li><li>作为一般规则，使用地区200 - 1,000 bp的往复m为转录因子结合的任一对有义或反义链（ 图4和图6），用于引物设计的兴趣点的中心。 注意:如果GRO-以次，DNase的以次，H3K4me1，H3K4me3的或H3K27ac芯片起数据可从所需细胞类型和条件下，它可以用于更准确的引物设计。 eRNAs从特征在于高水平的H3K4me1和ME3的推定增强子区转录。 eRNAs的表达与活化蛋白增强标记H3K27ac的富集正相关。 </li><li>设计PCR引物eRNAs的基于染料的荧光RT-qPCR的测量由引物3（http://bioinfo.ut.ee/primer3/）23生成用于引物设计为每反义序列（反向有义链的互补）: http://www.bioinformatics.org/sms/rev_comp.html。插入200 - 引物设计300bp的有义或反义序列的。 </li></ol></li><li> 尔娜转录的测量 </s仲&gt; <ol><li>根据制造商的建议从处理的细胞与商业酸性苯酚/基于氯仿提取试剂分离总RNA。 </li><li>使用DNA酶I，以逆转录反应使用它们之前，对待分离的RNA。根据逆转录现有的制造商的建议，灭活脱氧核糖核酸酶。 </li><li>测量RNA浓度的DNase I处理后，用每RT反应1000纳克。使用高品质的逆转录酶。 注:由于大量eRNAs可能无法聚腺苷酸化，反转录需要使用随机引物。 </li><li>检测用标准程序逆转录尔娜用RT-qPCR的。测量用于归一非治疗依赖性的mRNA（如 36B4，PPIA，GAPDH，Actb的）。 </li></ol></li></ol>

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L., Poliska, S., Ruhl, R., Nagy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+retinoic+acid+receptor+signaling+coordinates+lineage+commitment+of+spontaneously+differentiating+mouse+embryonic+stem+cells+in+embryoid+bodies.">Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies.</a> FEBS Lett. 584, 3123-3130 (2010).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25, 1754-1760 (2009).</li><li>Heinz, 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=Simple+combinations+of+lineage-determining+transcription+factors+prime+cis-regulatory+elements+required+for+macrophage+and+B+cell+identities.">Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities.</a> Mol Cell. 38, 576-589 (2010).</li><li>Robinson, J. 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=Integrative+genomics+viewer.">Integrative genomics viewer.</a> Nat Biotechnol. 29, 24-26 (2011).</li><li>Daniel, B., 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=The+active+enhancer+network+operated+by+liganded+RXR+supports+angiogenic+activity+in+macrophages.">The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages.</a> Genes Dev. 28, 1562-1577 (2013).</li><li>Zhu, Y., 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=Predicting+enhancer+transcription+and+activity+from+chromatin+modifications.">Predicting enhancer transcription and activity from chromatin modifications.</a> Nucleic Acids Res. 41, 10032-10043 (2013).</li><li>Visel, 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=ChIP-seq+accurately+predicts+tissue-specific+activity+of+enhancers.">ChIP-seq accurately predicts tissue-specific activity of enhancers.</a> Nature. 457, 854-858 (2009).</li><li>Heintzman, N. D., 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=Distinct+and+predictive+chromatin+signatures+of+transcriptional+promoters+and+enhancers+in+the+human+genome.">Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.</a> Nat Genet. 39, 311-318 (2007).</li><li>Rada-Iglesias, 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=A+unique+chromatin+signature+uncovers+early+developmental+enhancers+in+humans.">A unique chromatin signature uncovers early developmental enhancers in humans.</a> Nature. 470, 279-283 (2010).</li><li>Bonn, 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=Tissue-specific+analysis+of+chromatin+state+identifies+temporal+signatures+of+enhancer+activity+during+embryonic+development.">Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.</a> Nat Genet. 44, 148-156 (2012).</li><li>Hollenberg, S. M., Giguere, V., Segui, P., Evans, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+DNA-binding+and+transcriptional+activation+functions+in+the+human+glucocorticoid+receptor.">Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor.</a> Cell. 49, 39-46 (1987).</li><li>Untergasser, 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=Primer3--new+capabilities+and+interfaces.">Primer3--new capabilities and interfaces.</a> Nucleic Acids Res. 40, e115 (2012).</li><li>Rhead, B., 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=The+UCSC+Genome+Browser+database:+update+2010.">The UCSC Genome Browser database: update 2010.</a> Nucleic Acids Res. 38, D613-D619 (2010).</li><li>Kim, T. K., 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=Widespread+transcription+at+neuronal+activity-regulated+enhancers.">Widespread transcription at neuronal activity-regulated enhancers.</a> Nature. 465, 182-187 (2010).</li><li>Wang, D., 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=Reprogramming+transcription+by+distinct+classes+of+enhancers+functionally+defined+by+eRNA.">Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA.</a> Nature. 474, 390-394 (2011).</li><li>Simandi, Z., 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=PRMT1+and+PRMT8+regulate+retinoic+acid-dependent+neuronal+differentiation+with+implications+to+neuropathology.">PRMT1 and PRMT8 regulate retinoic acid-dependent neuronal differentiation with implications to neuropathology.</a> Stem Cells. 33, 726-741 (2014).</li><li>Zhou, H. Y., 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=A+Sox2+distal+enhancer+cluster+regulates+embryonic+stem+cell+differentiation+potential.">A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.</a> Genes Dev. 28, 2699-2711 (2014).</li></ol>

The authors have nothing to disclose.

In recent years, advances in sequencing technology have allowed large-scale predictions of enhancers in many cell types and tissues 7-9. The workflow described above allows one to perform primary characterization of candidate enhancers chosen based on ChIP-seq data. The detailed steps and notes will help anyone to set up a routine enhancer validation in the lab.
The most critical step in the luciferase reporter assay is the transfection efficiency. It is recommended to include a GFP...

Development of a multicellular organism requires precisely regulated expression of thousands of genes across developing tissues. Regulation of gene expression is accomplished in large part by enhancers. Enhancers are short non-coding DNA elements that can be bound with transcription factors (TFs) and act from a distance to activate transcription of a target gene1. Enhancers are generally cis-acting and most frequently found just upstream of the transcription start site (TSS), but recent studies also described examples where enhancers were found much further upstream, on the 3' of the gene or even within the introns and exons2. 
There are hundreds of thousands of potential enhancers in the vertebrate genomes1. Recent methods based on chromatin immunoprecipitation (ChIP) provide high-throughput data of the whole genome that can be used for enhancer analysis3-9. Though data obtained by ChIP-seq experiments greatly increases the likelihood to identify cell and tissue-specific enhancers, it is important to keep in mind that detected binding sites do not necessarily identify direct DNA binding and/or functional enhancers. Thus, further functional analysis of newly identified enhancers is indispensable. In this work, we present a basic three-step process of putative active enhancer identification and validation. This includes: 1) selection of putative transcription factor binding sites by bioinformatics analysis of ChIP-seq data, 2) cloning and validation of these regulatory sequences in reporter constructs, and 3) measurement of enhancer RNA (eRNA). 
Exposure of embryonic stem (ES) cells to retinoic acid (RA) is frequently used to promote neural differentiation of the pluripotent cells 10. RA exerts its effects by binding to RA receptors (RAR&#945;, &#946;, &#947;) and retinoid X receptors (RXR&#945;, &#946;, &#947;). RARs and RXRs in a form of heterodimer bind to DNA motifs called RA-response elements, that is typically arranged as direct repeats of AGGTCA sequence (called as half site) and regulate transcription. Ligand-treatment experiments allowed the identification of several retinoic acid regulated genes in ES cells 11,12. However, enhancer elements for many of these genes has not been described yet. To demonstrate how the here-described workflow can be used for enhancer identification and validation we show step-by-step the selection and characterization of two retinoic acid-dependent enhancers in embryonic stem cells.

<table><tbody><tr><td>KOD DNA polymerase</td><td>Merck Millipore</td><td>71085-3</td><td>for PCR amplification of enhancer from gDNA</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue kit&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>for genomic DNA isolation</td></tr><tr><td>QIAquick PCR Purification kit</td><td>Qiagen</td><td>28106</td><td>for PCR product purification</td></tr><tr><td>Gel extraction kit&amp;nbsp;</td><td>Qiagen</td><td>28706</td><td>for gel extraction if there are more PCR product</td></tr><tr><td>HindIII</td><td>NEB</td><td>R3104L</td><td>restriction enzyme</td></tr><tr><td>BamHI</td><td>NEB</td><td>R3136L</td><td>restriction enzyme</td></tr><tr><td>FastAP</td><td>Thermo Scientific</td><td>EF0651</td><td>release of 5&#039;- and 3&#039;-phosphate groups from DNA</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202</td><td>for ligation</td></tr><tr><td>QIAprep Spin Miniprep kit</td><td>Qiagen</td><td>27106</td><td>for plasmid isolation</td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966-021</td><td>ES media</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td>ES media</td></tr><tr><td>MEM Non-Essential Amino Acid</td><td>Sigma</td><td>M7145</td><td>ES media</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td>ES media</td></tr><tr><td>Beta Mercaptoethanol</td><td>Sigma</td><td>M6250</td><td>ES media</td></tr><tr><td>FuGENE HD&amp;nbsp;</td><td>Promega</td><td>E2311</td><td>transfection reagent</td></tr><tr><td>Opti-MEM&amp;reg; I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-062</td><td>for transfection</td></tr><tr><td>All-trans retinoic acid</td><td>Sigma</td><td>R2625</td><td>ligand, for activation of RAR/RXR</td></tr><tr><td>96-well clear plate</td><td>Greiner</td><td>655101</td><td>for Beta galactosidase assay</td></tr><tr><td>96-well white plate</td><td>Greiner</td><td>655075</td><td>for Luciferase assay</td></tr><tr><td>D-luciferin, potassium salt</td><td>Goldbio.com</td><td>115144-35-9</td><td>for Luciferase assay</td></tr><tr><td>ATP salt</td><td>Sigma</td><td>A7699-1G</td><td>for Luciferase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Luciferase assay</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-25G</td><td>for Luciferase assay</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>431478-50G</td><td>for Beta galactosidase assay</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S9638-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541-500G</td><td>for Beta galactosidase assay</td></tr><tr><td>ONPG (o-nitrophenyl-&amp;beta;-D-galactosidase)</td><td>Sigma</td><td>N1127-1G</td><td>for Beta galactosidase assay</td></tr><tr><td>TRIzol&amp;reg;</td><td>Life Technologies</td><td>15596-026</td><td>RNA isolation</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Life Technologies</td><td>4368814</td><td>reverse transcription of eRNA</td></tr><tr><td>Rnase-free Dnase</td><td>Promega</td><td>M6101</td><td>Dnase treatment</td></tr><tr><td>SsoFast Eva Green</td><td>BioRad</td><td>750000105</td><td>RT-qPCR mastermix</td></tr><tr><td>CFX384 Touch&amp;trade; Real-Time PCR Detection System</td><td>BioRad</td><td></td><td>qPCR machine</td></tr><tr><td>BioTek Synergy 4 microplate reader</td><td>BioTek</td><td></td><td>luminescent counter</td></tr></tbody></table>

prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation

胚胎发育是涉及到许多基因的激活和镇压一个多步骤的过程。在基因组中的增强子元件是公知的细胞分化过程中以促进基因表达的组织和细胞类型特异性调节。因此，他们的鉴定和进一步调查，以了解细胞的命运是如何决定的重要。基因表达数据（ 例如，微阵列或RNA-SEQ）和染色质免疫沉淀（ChIP）为基础的全基因组研究（芯片起）结果的整合使得这些调控区域大规模鉴定。然而，细胞类型特异性增强剂的功能验证进一步要求在体外和体内实验程序。在这里，我们描述了活性增强剂如何可以识别和实验验证。通过芯片起数据分析，2）克隆和EXPER 1）鉴定调节区:此协议提供了一步一步的工作流程，其包括在报道实验所识别的基因组序列的推定的调节潜力imental验证，和3）测定增强子活性的体内通过测量增强RNA转录水平。所提出的协议是不够详细，帮助任何人设立这个工作流程中的实验室。重要的是，该协议可以很容易地适应，并在任何细胞模型系统中使用。

我们使用了泛特异性RXR抗体以鉴定的全基因组其类风湿性关节炎调节基因在它们的靠近受体富集。从用视黄酸处理的ES细胞获得RXR芯片起数据的生物信息学分析揭示了核受体半位点（AGGTCA）下的RXR占用位点（ 图1）的富集。使用生物信息学算法我们映射回的基序，检索结果为半位点的RXR芯片起数据（ 图2）。这种分析有助于我们准确地识别，用规范...

Watch this Scientific Journal Video about Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation at JoVE.com

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

prediction-validation-gene-regulatory-elements-activated-during

Research

JoVE Journal

Developmental Biology

8.2K 次观看.  Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona. In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

视频: Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

科研

发育生物学

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神经科学

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

生物学

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

Source: Ababon, M. R., et al. <a href="https://app.jove.com/v/54443">Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for assessing retinoic acid activity in mouse embryos. Upon isolating cells from the mouse embryos, the cells are co-cultured with retinoic acid (RA) reporter cells. Embryonic cells produce RA, which induces beta-galactosidase expression in reporter cells, indicating RA activity in the embryonic cells.

在共培养物中使用报告细胞研究小鼠胚胎细胞产生视黄酸

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Solution and Media Preparation

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