Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

1，ATP依赖的核小体重构的测定来测量ATP依赖的核小体重构活动，免疫纯化INO80或INO80 subcomplexes孵育与ATP和mononucleosomal衬底，它包含一个单核小体定位于216碱基对，32 P标记的DNA片段的一端。将反应产物再进行电泳在天然聚丙烯酰胺凝胶。 <ol><li>以产生32 P标记的，"601"的DNA片段，从放大的pGEM-3Z-601 4包含端定位的601核小体定位序列的216 bp的DNA片段，使用寡核苷酸5&#39;-ACAGGATGTATATATCTGACACGTGCCTGG和5&#39;-AATACTCAAGCTTGGATGCCTGCAG如正向和反向引物。 <ol><li>设置了100微升PCR反应，如表1所述。 </li><li>在96℃，followe 1分钟:使用以下程序进行PCR反应在热循环仪ð45秒，94℃，30秒，57℃，60秒，72℃，30个循环; 7分钟72℃的结局。 </li><li>后的PCR反应结束后，通过无核酸酶的离心柱两次通过该反应产物中除去未掺入的核苷酸。 </li><li>运行在1.2％琼脂糖凝胶上对5μl纯化的PCR产物，以确认PCR反应生成所要的〜216碱基对的DNA片段。 </li><li>稀释5微升纯化的PCR产物20倍，并使用UV分光光度计测量DNA浓度。平均产量〜40毫微克/微升。 </li><li>测量的1微升纯化的PCR产物在闪烁计数器中的放射活性。通过计算CPM / NG估计标记效率。一个成功的标记反应，预计产量601的DNA片段〜15,000 CPM / NG。 </li></ol></li><li>转让Hela细胞的核小体上的标记601的DNA采用连续稀释法5。 <ol><li>混合2皮摩尔的32 P-标记的601的DNA片段6微克的Hela核小体在50μl含1.0 M氯化钠，的10mM Tris-盐酸，pH值8.0，1mM EDTA中，0.1mM的PMSF和1mM的缓冲液（其制备如所述5） DTT和孵育在30℃下进行30分钟。 </li><li>依次稀释该混合物至0.8摩尔，0.6 M和0.4 M氯化钠的10毫摩尔Tris-盐酸（pH值8.0），1mM EDTA中，0.1mM的PMSF由稀释用12.5微升，20.8微升，41.6微升，分别和1mM DTT的，用30分钟温育在30℃下各稀释后。 </li><li>进一步稀释该混合物至0.2M，然后以0.1 M氯化钠加入125微升，然后加入250μl含有0.1％诺乃洗涤剂P-40，20％甘油，和200微克/毫升BSA的相同缓冲液中，用30分钟温育在30℃下的最后两个稀释度之间。储存在4℃的mononucleosome基板长达3个月。 </li></ol></li><li>在10μl反应体系进行ATP依赖的核小体滑动反应。在REAction组分列在表2中，由于最佳NaCl浓度为INO80核小体重构活性为〜50 mM氯化钠（未发表结果），调整NaCl在各反应至50mM的总浓度，考虑到的NaCl中含有的量核小体和酶制剂。 <ol><li>在开始建立测定法，浇铸天然聚丙烯酰胺凝胶（18×16厘米）。以制备含5％丙烯酰胺的单一凝胶（丙烯酰胺:双丙烯酰胺37.5:1），0.5×TBE（45毫摩尔Tris硼酸盐，1mM EDTA）中，0.01％的过硫酸铵（APS）和0.001％的N，N，N&#39;， N&#39;四甲基乙二胺（TEMED），混合在表3中所列的成分。允许凝胶聚合至少2小时，在室温。 </li><li>另一方面，对于将要执行的每个反应中，结合在预冷的硅化1.5 ml离心管〜20nM的INO80或INO80亚复合（其制备如所述3）的EB100缓冲量（ <str翁&gt;表4）足以得到4.75微升的体积。立即重新冻结在粉状干冰或液氮中任何剩余的含INO80级分。 </li><li>设置主鸡尾酒的配料的其余部分，由"X"的因素扩大（其中3 X =总数的反应）。需要一个单一的10微升反应的各成分的量被列于表5中 ;配方应根据待进行的反应的数目按比例增加。通过敲击管道或吹打上下拌匀，用的Pipetman和旋管几秒钟的台式离心。 </li><li>免除5.25微升主鸡尾酒的每个反应管设置在步骤1.3.2。吹打上下拌匀。通过将反应管于30℃的加热块或水浴启动反应，并孵育2小时。 </li><li>同时，准备"移除组合"包含竞争对手的DNA鸡尾酒与核小体，扩大利用X（反应+ 4 x =总数）的一个因素。根据需要，制备1.5微升除去混在单一反应各成分的量被列于表6;配方应相应加大取决于要进行的测定的次数。 </li><li>加入1.5微升去除混合终止反应。拌匀，降速和孵化，在30℃下再30分钟。 </li><li>同时，在4℃下以100伏运行前在垂直电泳单元的非变性聚丙烯酰胺凝胶30分钟，用0.​​5×TBE为电泳缓冲液与下部腔室内部的磁力搅拌棒，以维持恒定的缓冲循环。 </li><li>加载样品，加入2.5微升含3×TBE，30％甘油，0.25％溴酚蓝，和0.25％二甲苯蓝FF加载染料。拌匀后短暂离心样品，并加载到使用装载提示凝胶。 </li><li>运行在200伏的凝胶为4.5小时，在4℃下用缓冲液循环。</li><li>为了检测信号，将凝胶转移到堆栈两片滤纸。包在滤纸上用保鲜膜上方的凝胶，然后将其暴露到存储荧光屏，在4℃下进行所需的时间。 </li><li>扫描用同位素成像扫描仪系统的屏幕，并使用合适的软件进行数据分析。 </li></ol></li></ol> 2 Mononucleosome结合测定为了分析给定INO80复杂，mononucleosomes的结合亲和力，执行使用步骤1.2生成的mononucleosomal基板的电泳迁移率变动分析（EMSA）。 <ol><li>建立了反应的混合所描述的核小体重塑的实验，但省略了反应的ATP和拆卸组合结合分析;孵育在30℃下进行30分钟。 </li><li>加入2.5微升上样染料的各反应混合物中，并适用于含有3.5％的丙烯酰胺（丙烯酰胺非变性聚丙烯酰胺凝胶:双37.5:1），1％甘油，0.5×TBE，0.01％APS，和0.001％TEMED。 </li><li>用0.5×TBE为电泳缓冲液，运行在200伏的凝胶2.5小时，在4℃用缓冲液循环和暴露到存储荧光屏。 </li></ol> 3，DNA和核小体依赖性ATP酶测定在含有20mM的Tris-HCl（pH为7.5），60 mM氯化钠，6.6毫米MgCl 2，0.8毫摩尔EDTA，0.015％诺乃洗涤剂P-40，2.5％甘油，0.1毫克/毫升BSA，5微升反应混合物中进行ATP酶测定法毫摩尔DTT，0.1mM的PMSF，2毫摩尔ATP，产品[α-32 P] ATP的2微居里（3000次/毫摩尔）。对于INO80复杂每个INO80复杂或金额进行检测设置三个平行反应，一个包含EB100缓冲区来衡量DNA-或核无关的ATP酶，一种含闭合环状质粒DNA（5,000个基点，约30纳米）来衡量DNA-依赖性ATP酶，和一种含有宫颈癌寡聚核小体（〜185 nm）的测定核小体依赖性ATP酶。成立于冰上的所有反应。 <oL&gt; <li>对于每个反应，结合10-50纳米的免疫纯化INO80或INO80 subcomplexes用的EB100缓冲器足以得到2.2微升的体积中预先冷却的润滑的1.5 ml离心管中的量。立即重新冻结在粉状干冰或液氮中的任何含INO80级分。 </li><li>设置主鸡尾酒。需要一个单一的反应的各成分的量被列于表7中 。向上扩展的配方通过用因子为3（X + 2）1，其中X =待测定INO80制剂的数量按比例放大。 </li><li>以制备"子鸡尾酒"包含缓冲器只，DNA或核小体，可免除2.5（X + 2）微升主鸡尾酒的成三个独立的管中。添加0.3（X + 2）微升无论是EB100的，闭合环状质粒DNA（1.5微克/微升），或宫颈癌寡（1.5微克/微升），搅拌均匀。 </li><li>分配2.8微升适当子鸡尾酒到含酶的反应管设置在日EP 3.1。吸管轻轻上下混用;避免产生气泡。 </li><li>以启动反应，将反应管转移到30℃的热块。 </li><li>后5，15，30，及温育60分钟后，发现0.5微升每个反应混合物到纤维素聚乙烯薄层色谱（TLC）板（20×10厘米）在一条直线上至少为1.5厘米的距离从底部边缘。立即返回反应管于30℃加热块，使多个时间点可以从一个单一的管。点样后，用干一击机的TLC板。 </li><li>传送的TLC板，以含有足够的0.375磷酸钾（pH值3.5），以使底0.5厘米TLC板上，以在溶液中被淹没的玻璃室中。 </li><li>覆盖所述腔室，并发展到液相的前沿到达TLC板的顶部。立即擦干板完全采用吹塑机。 </li><li>揭露干燥薄层板，在室温下存储荧光屏。扫描用同位素成像扫描仪系统的屏幕，并确定放射性ATP底物和ADP产物的量。 </li><li>计算出的ATP水解的量，用以下公式乘以％的ATP通过ATP的存在于起始反应混合物中的量水解:皮摩尔ATP水解= 10pmol的ATP在起始反应×[ADP /（ATP + ADP）] </li></ol>

<ol>
	<li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chromatin+remodeling+complexes.">The biology of chromatin remodeling complexes.</a> Annual Review of Biochemistry. 78, 273-304 (2009).</li><li>Narlikar, G. J., Sundaramoorthy, R., Owen-Hughes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+ATP-dependent+chromatin-remodeling+enzymes.">Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes.</a> Cell. 154 (3), 490-503 (2013).</li><li>Chen, L., Ooi, S. K., Conaway, J. W., Conaway, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+purification+of+human+INO80+chromatin+remodeling+complexes+and+subcomplexes.">Generation and purification of human INO80 chromatin remodeling complexes and subcomplexes.</a> , (2013).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> J. Mol. Biol. 276 (1), (1006).</li><li>Owen-Hughes, 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=Analysis+of+nucleosome+disruption+by+ATP-driven+chromatin+remodeling+complexes.">Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes.</a> Methods Mol. Biol. 119, 319-331 (1999).</li><li>Udugama, M., Sabri, A., Bartholomew, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INO80+ATP-dependent+chromatin+remodeling+complex+is+a+nucleosome+spacing+factor.">The INO80 ATP-dependent chromatin remodeling complex is a nucleosome spacing factor.</a> Mol. Cell Biol. 31 (4), 662-673 (2011).</li><li>Jin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+chromatin+remodeling+complex+with+similarities+to+the+yeast+INO80+complex.">A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex.</a> Journal of Biological Chemistry. 280 (50), 41207-41212 (1074).</li><li>Chen, 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=Subunit+organization+of+the+human+INO80+chromatin+remodeling+complex:+an+evolutionarily+conserved+core+complex+catalyzes+ATP-dependent+nucleosome+remodeling.">Subunit organization of the human INO80 chromatin remodeling complex: an evolutionarily conserved core complex catalyzes ATP-dependent nucleosome remodeling.</a> Journal of Biological Chemistry. 286 (13), 11283-11289 (2011).</li><li>Hamiche, A., Sandaltzopoulos, R., Gdula, D. A., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-dependent+histone+octamer+sliding+mediated+by+the+chromatin+remodeling+complex+NURF.">ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF.</a> Cell. 97 (7), 833-842 (1999).</li><li>Polach, K. J., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+enzymes+as+probes+of+nucleosome+stability+and+dynamics.">Restriction enzymes as probes of nucleosome stability and dynamics.</a> Methods Enzymol. 304, 278-298 (1999).</li><li>Anderson, J. D., Thastrom, A., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+access+of+proteins+to+buried+nucleosomal+DNA+target+sites+occurs+via+a+mechanism+that+is+distinct+from+nucleosome+translocation,+Mol.Cell+Biol.">Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation, Mol.Cell Biol.</a> 22 (20), 7147-7157 (2002).</li><li>Saha, A., Wittmeyer, J., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodeling+through+directional+DNA+translocation+from+an+internal+nucleosomal+site.">Chromatin remodeling through directional DNA translocation from an internal nucleosomal site.</a> Nature Structural and Molecular Biology. 12 (9), 747-755 (2005).</li><li>Gottschalk, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribosyl)ation+directs+recruitment+and+activation+of+an+ATP-dependent+chromatin+remodeler,+Proc.Natl.Acad.Sci.U.S.A.">Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler, Proc.Natl.Acad.Sci.U.S.A.</a> 106 (33), 13770-13774 (2009).</li><li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+ISWI+involves+inhibitory+modules+antagonized+by+nucleosomal+epitopes.">Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes.</a> Nature. 492 (7428), 280-284 (2012).</li><li>Brune, M., Hunter, J. L., Corrie, J. E. T., Direct Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Measurement+of+Rapid+Inorganic+Phosphate+Release+Using+a+Novel+Fluorescent+Probe+and+Its+Application+to+Actomyosin+Subfragment+1+ATPase,+Biochemistry.">Real-Time Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Application to Actomyosin Subfragment 1 ATPase, Biochemistry.</a> 33 (27), 8262-8271 (1994).</li><li>Luk, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+histone+replacement+by+SWR1+requires+dual+activation+with+histone+H2A.Z+and+canonical+nucleosome.">Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome.</a> Cell. 143 (5), 725-736 (2010).</li></ol>

The authors declare that they have no competing financial interests.

以确保核小体重构和ATP酶活性，我们观察到在分析依赖INO80复合物的催化活性，并且不污染重塑和/或ATP酶，我们经常测定核小体重构和INO80复合物的催化活性的版本的ATP酶活性，纯化使用野生型INO80同样地进行平行。阴性对照反应缺少ATP也应测定核小体重构的活性时，以测试污染ATP和/或ATP的独立重塑活动的存在，这可能会复杂化的比较INO80复合物或subcomplexes不同制剂的活性实验解释执行。催化INO80?...

SNF2家庭染色质重塑复合物包括中央SNF2般的ATP酶亚基1,2。一些SNF2状ATP酶的功能单亚基酶，而其他用作较大的多亚基复合物的催化亚基。阐明的分子机制，其中每一个染色质重塑复合物有助于其活动亚基的要求来执行解剖重建过程的生化分析的能力。 ATP依赖的核小体重构的人类INO80复合物和其他染色质重塑酶，可以设想为，与重塑酶，以核小体结合启动一个多步骤的过程，随后激活其DNA和/或核小体依赖性ATP酶，易位于核小体的DNA，和核小体1,2的最终位置改变重塑酶。理解ATP依赖的染色质重塑过程r的分子细节equires重塑反应的染色质重塑复合物在反应中的每个步骤的各个子单元的贡献成单个步骤和定义的清扫。这样的分析需要使用定义的底物分子在体外进行分析核小体重塑和其他活动的能力。 在以前的JOVE协议中，我们描述的程序来生成INO80染色质重塑复合物及其亚与定义亚基组成3。在这里，我们提出了3生化检测，使核小体结合，DNA和核小体的激活ATP酶，而这种复合物相关的核小体重塑活动的定量分析。

<table><tbody><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>10x PCR reaction buffer&amp;nbsp;</td><td>Roche Applied Science&amp;nbsp;</td><td>11435094001</td><td></td></tr><tr><td>Roche Taq DNA Polymerase</td><td>Roche Applied Science&amp;nbsp;</td><td>11435094001</td><td></td></tr><tr><td>NucAway Nuclease-free Spin Columns&amp;nbsp;</td><td>Ambion</td><td>Cat. # AM10070</td><td></td></tr><tr><td>ultrapure ATP&amp;nbsp;</td><td>USB/Affymetrix</td><td>77241 25 UM</td><td></td></tr><tr><td>bovine serum albumin&amp;nbsp;</td><td>Sigma</td><td>A9418&amp;nbsp;</td><td></td></tr><tr><td>40% Acrylamide/Bis 37.5:1</td><td>Amresco</td><td>0254-500ML</td><td></td></tr><tr><td>Sonicated salmon sperm DNAs&amp;nbsp;</td><td>GE Healthcare</td><td>27-4565-01</td><td></td></tr><tr><td>10% ammonium persulfate (APS)</td><td>Thermo Scientific</td><td>17874</td><td></td></tr><tr><td>benzonase&amp;nbsp;</td><td>Novagen</td><td>Cat. No. 70664</td><td></td></tr><tr><td>dCTP, [&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P]- 6,000 Ci/mmol</td><td>PerkinElmer</td><td>BLU013Z250UC</td><td></td></tr><tr><td>PCR thermal cycler PTC 200</td><td>MJ Research</td><td>PTC 200</td><td></td></tr><tr><td>Hoefer vertical electrophoresis unit</td><td>Hoefer</td><td>SE600X-15-1.5</td><td></td></tr><tr><td>lubricated 1.5 ml microcentrifuge tubes&amp;nbsp;</td><td>Costar</td><td>3207</td><td></td></tr><tr><td>Storage Phosphor Screen&amp;nbsp;</td><td>Molecular Dynamics</td><td>63-0034-79</td><td></td></tr><tr><td>3MM filter paper</td><td>Whatman&amp;nbsp;</td><td>28458-005</td><td>VWR</td></tr><tr><td>Typhoon PhosphorImager&amp;nbsp;</td><td>GE Healthcare</td><td>8600</td><td></td></tr><tr><td>ImageQuant software</td><td>GE Healthcare</td><td>ver2003.02</td><td></td></tr><tr><td>TLC Glass Plates, PEI-Cellulose F</td><td>Millipore</td><td>5725-7</td><td></td></tr><tr><td>Immobilon-FL Transfer Membrane 7 x 8.4</td><td>Millipore</td><td>IPFL07810</td><td></td></tr><tr><td>General purpose survey meter with end-window or pancake GM (Geiger-Mueller) probe</td><td>Biodex</td><td>Model 14C</td><td></td></tr></tbody></table>

biochemical assays for analyzing activities of atp-dependent chromatin remodeling enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

该图显示用于表征INO80活动生化分析，包括核小体的滑动（ 图1）和结合（ 图2）测定法和DNA-或核小体依赖性ATP酶测定（ 图3）的代表性结果。 在图1所示的实验中比较了完整INO80复合物的能力，通过FLAG-IES2或FLAG-INO80E纯化的INO80 subcomplexes或者通过FLAG-Ino80ΔN或Ino80ΔNΔHSA精制，催化mononucleosomes汇集了216 bp的，放射性标记的DNA片段?...

Watch this Scientific Journal Video about Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes at JoVE.com

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

生化分析的分析ATP依赖的染色质重塑酶活性的影响

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

biochemical-assays-for-analyzing-activities-atp-dependent-chromatin

Research

JoVE Journal

Biology

14.7K 次观看.  Stowers Institute for Medical Research. Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

视频: Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

科研

遗传学

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

生物化学

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

癌症研究

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

CD光谱学研究DNA-蛋白质相互作用

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

体外 使用分析、下拉和伴侣测定表征组蛋白伴侣

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

DNA帘法破译组蛋白组装的分子机制

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

生物学

开发基于点击的检测方法，用于筛选 HAT1 酶活性

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

作者聚焦:开发用于 HAT1 抑制剂筛选的乙酰点击检测

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

生化分析的分析ATP依赖的染色质重塑酶活性的影响

摘要

探索更多视频

此视频中的章节

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

核糖体组蛋白的体外泛化和二次基质测定

验证组蛋白乙酰转移酶抑制剂的检测

CD光谱学研究DNA-蛋白质相互作用

体外使用分析、下拉和伴侣测定表征组蛋白伴侣

DNA帘法破译组蛋白组装的分子机制

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

生化分析的分析ATP依赖的染色质重塑酶活性的影响

摘要

探索更多视频

此视频中的章节

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

核糖体组蛋白的体外泛化和二次基质测定

验证组蛋白乙酰转移酶抑制剂的检测

CD光谱学研究DNA-蛋白质相互作用

体外 使用分析、下拉和伴侣测定表征组蛋白伴侣

DNA帘法破译组蛋白组装的分子机制

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

体外使用分析、下拉和伴侣测定表征组蛋白伴侣