这项工作得到德克萨斯大学MD安德森癌症中心，德克萨斯州休斯顿（启动基金会BB）的支持。

1.准备解决方案<ol><li> 为每个实验准备以下解决方案。 <ol><li>对于固定溶液 ，制备37％甲醛并将其储存在室温下。在1x磷酸盐缓冲盐水（PBS）中稀释至1.42％的最终浓度。 注意:甲醛被分类为刺激性，腐蚀性，致突变性，致畸性和致癌性。不要摄取不要吸入气体/烟雾/蒸气/喷雾。在通风不足的情况下，佩戴合适的呼吸设备。避免与皮肤和眼睛接触。远离不兼容的物质，如氧化剂，还原剂，酸，碱和水分。 </li><li>对于甘氨酸止痛溶液，在1×PBS溶液中制备0.125M甘氨酸并将其储存在室温下。 </li><li>对于细胞刮擦溶液，在dH 2 O中制备1×PBS溶液并将其储存在冰上。直接使用前，加入蛋白酶抑制剂苯甲磺酰氟（PMSF）t最终浓度为0.5mM。 小心:PMSF分类为腐蚀性和毒性;穿防护服，并在通风的地方使用。 </li><li>在使用细胞裂解缓冲液（20mM Tris-HCl; pH 8.0,85mM KCl和0.5％NP40）之前，直接加入1X蛋白酶抑制剂混合物（PIC; 100X储存液在二甲基亚砜（DMSO）中:104mM 4-（ 2-氨基乙基）苯磺酰氟化物盐酸盐（AEBSF），80μM抑肽酶，4mM bestatin，1.4mM E-64,2mM亮抑酶肽和1.5mM胃酶抑素A）和0.5mM PMSF。 </li><li>在使用细胞裂解缓冲液（50mM Tris-Cl; pH8.0,10mM乙二胺四乙酸（EDTA）和1％SDS）之前，直接加入1X PIC和0.5mM PMSF。 </li></ol></li></ol>细胞固定和剪切<ol><li>在10厘米的组织培养板上将细胞生长至70-80％汇合。根据实验目标刺激细胞。 </li><li>取出组织培养基，加入5 mL固定液，孵育细胞在摇床上在室温下10分钟。 </li><li>取出固定液并用10mL冰冷的1×PBS洗涤细胞两次。 </li><li>取出1×PBS，加入5 mL甘氨酸止痛溶液，并在摇床上室温孵育细胞5 min。 </li><li>取出甘氨酸止血溶液，并用10ml冰冷的1×PBS洗涤细胞两次。 </li><li>取出1×PBS，加入2mL细胞刮擦溶液，并用细胞刮刀收集细胞。将收获的细胞转移到冰上的锥形管中。 </li><li>在600×g和4℃下将样品离心10分钟。 </li><li>取出上清液，将沉淀重悬于1 mL 1X细胞裂解缓冲液中，并在冰上孵育30 min。或者，将颗粒快速冻结在液氮中并将其储存在-80℃。 </li><li>一旦将沉淀重新悬浮在细胞裂解缓冲液中，将细胞悬浮液转移到冰冷的均质器中，并使用B型瘟疫e用于细胞破坏。 </li><li>将细胞悬浮液转移至微量离心管，并以​​2,400 xg和4℃离心样品10分钟。 </li><li>弃去上清液，将沉淀物重悬于350μL核消化缓冲液中，并在37℃下孵育样品5分钟。 </li><li>加入微球菌核酸酶（1U /μL）并通过涡旋混合。在37℃下孵育样品5-20分钟;通过反演每2分钟混合样品。 注意:时间和酶浓度在细胞系之间不同，并且可能需要在次优酶剪切的情况下进行优化。 </li><li> 或者，通过超声处理使用市售的超声波器实现染色质剪切。 注意:超声波条件将需要优化。以下使用300μL的样品体积和25％的样品处理能力提供优化示例协议。 <ol><li>在步骤2.10之后，丢弃上清液并重悬在1.0mL商业剪切缓冲液中的核颗粒。 </li><li>将等分试样将300μL再悬浮的核颗粒沉积到三个1.7mL微量离心管中。将它们放在冰上。 </li><li>使用3种不同的条件，以25％功率剪切固定染色质的3个等分试样，以确定最佳超声处理条件: 每个20秒的5个脉冲，每个脉冲之间在冰上休息30秒。 10秒脉冲20秒，每个脉冲之间在冰上休息30秒。 每个20秒的20个脉冲，每个脉冲之间在冰上休息30秒。 </li><li>继续执行步骤2.15，如下所述。 </li></ol></li><li>在与微球菌核酸酶孵育后，加入7μL冰冷的0.5M EDTA终止反应。 </li><li>用剪切的DNA在16,200 xg和4℃下离心10分钟。 </li><li>将剪切的含DNA上清液转移到新鲜的微量离心管中。将等分试样放入一个单独的微量离心管中，以确认是否存在成功剪切DNA（第3节）。立即使用剩余容量进行免疫沉淀（第4部分）或将其储存在-80°C。 </li></ol> 3.确认剪切效率<ol><li>向步骤2.16的剪切染色质的50μL等分试样中加入150μL无核酸酶的dH 2 O和10μL5 M NaCl。 </li><li>在65℃下孵育样品4小时以逆转交联。 </li><li>加入2μLRNase A（10μg/μL），37℃孵育15 min。 </li><li>加入10μL蛋白酶K（0.5μg/μL），并将样品在42℃孵育90分钟。 </li><li> 为了纯化DNA，进行苯酚/氯仿萃取12 。 <ol><li>加入无核酸酶的水至最终体积为200μL。向样品中加入200μL苯酚:氯仿:异戊醇（25:24:1）并旋涡20秒。 </li><li>室内离心机以16,000 x g的速度进行5分钟。仔细取出上部水相并将其转移到新鲜管中。在移液时请确保不要携带任何苯酚。 </li><li>加入0.1体积的3M乙酸钠（NH 4 OAc），pH5.2，并用手指轻拍管子进行混合。加入2.5体积的冰冷的100％乙醇，并通过涡旋混合5秒。将样品O / N在-20℃下孵育，或在-80℃下孵育至少1小时。 </li><li>在4℃和16,000×g离心样品30分钟以沉淀cDNA。 </li><li>仔细取出上清液，不会破坏沉淀物。加入1 mL RT，70％乙醇，并倒置多次。 </li><li>在4℃和16,000×g离心样品2分钟。仔细清除上清液。 </li><li>在室温下干燥沉淀5-10分钟。将沉淀重悬于30μL的dH 2 O. </li></ol></li><li>使用5和10μL等分的纯化DNA进行凝胶电泳，并用1％TAE进行凝胶电泳琼脂糖凝胶。 注意:成功的染色质剪切导致200-1,500 bp的DNA模式。如果剪切不成功，则通过将剪切步骤（步骤2.12）的孵育时间修改为5,10和15分钟来确定特定细胞的最佳剪切条件。 </li><li>使用剩余样品分析样品的DNA浓度。反向计算剪切染色质样品中的DNA浓度（步骤2.16）;每个治疗组使用相同的DNA量进行免疫沉淀（第4部分）。 </li></ol> 4.免疫沉淀<ol><li>将10-25μg剪切的交联染色质（步骤2.16）与25μLChIP级蛋白G磁珠，10μL免疫沉淀稀释缓冲液（储备溶液:0.01％SDS，1.1％Triton X-100,16.7mM Tris-HCl; pH 8.0,1.2mM EDTA和167mM NaCl）和1μLPIC。 </li><li>孵育4小时后，加入1-10μg抗体（抗体浓缩物取决于抗体的亲和力;应首先根据制造商的建议使用并根据需要进行实验优化）至1.5 mL微量离心管，并加入dH 2 O至终体积为100μL。 注意:代替上述直接免疫沉淀法，可以通过首先将染色质与抗体结合并随后加入蛋白G磁珠来使用间接免疫沉淀法。 </li><li>在4℃下将样品在端对端旋转器上孵育4-12小时。 </li></ol>洗脱<ol><li>将管放在磁性支架上。一旦磁珠聚集在管的侧面，小心地取出并丢弃上清液。 </li><li>用800μL洗涤缓冲液1（0.1％SDS，1％Triton X-100,2mM EDTA pH8,20mM Tris-HCl; pH8和150mM NaCl）洗涤珠珠三次;通过反转管多次混合。将管放在磁性支架上a一旦磁珠集中在管的侧面，请小心地取出并丢弃洗涤缓冲液。 </li><li>用800μL洗涤缓冲液2（0.1％SDS，1％Triton X-100,2mM EDTA; pH8,20mM Tris-HCl pH8和500mM NaCl）洗涤珠子一次;通过反转管多次混合。一旦磁珠在管的侧面聚集，将管放在磁性支架上，小心地取出并丢弃洗涤缓冲液。 </li><li>将珠子悬浮在50μL洗脱缓冲液（1％SDS和100mM NaHCO 3 ）中，并将样品在端到端旋转器上在室温下孵育15分钟。 </li><li>将管放置在磁性支架上，一旦磁珠收集在管的侧面，将上清液转移到新管中。 </li><li>加入6μL5 M NaCl，反向交联，加入2μL蛋白酶K（0.5μg/μL）。混合样品后，在65℃下孵育样品2小时。 </li><li>为了纯化DNA，请执行一次nol /氯仿提取12 （步骤3.5），随后将沉淀重悬于30μL的dH 2 O. 注意:样品可以直接分析蛋白质结合位点（第5部分），或者可以保存在-20°C。 </li></ol>结合位点分析<ol><li>使用PCR或qRT-PCR对已知核酸序列内的特异性结合位点进行分析。对于引物设计，遵循常规PCR或qRT-PCR方案。设计引物以合成一个150-400bp的桥连蛋白结合位点的扩增子（ 图1 ）。 </li><li>使用四种不同的DNA模板设置PCR，以确保特异性:i）用感兴趣的抗体免疫沉淀的ChIP的DNA，ii）用非特异性IgG抗体免疫沉淀的ChIP的DNA，iii）输入DNA，和iv）H 2 O作为对PCR进行阴性对照以排除污染。在途径刺激的情况下使用针对免疫沉淀蛋白的已知结合位点特异性的不同的引物组选择第五模板。 注意:在c-KIT配体启动子分析的情况下，使用常规PCR来证明c-KIT配体启动子区域内的SBE上的TGF-β诱导的SMAD结合。作为阳性对照，对作为TGF-β诱导的SMAD结合的已知靶标的纤溶酶原激活物抑制剂-1（PAI-1）进行PCR。 </li></ol>

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作者宣称他们没有竞争的经济利益。

在本报告中，我们证明了TGF-β1诱导的SMAD2与c-KIT配体启动子内的SBE和TGF-β1诱导的STAT3与TGF-β1配体基因内识别序列的结合的结合。我们使用染色质免疫沉淀显示细胞因子诱导的两种转录因子的结合。 染色质免疫沉淀是一个有力的工具，用于证明目标蛋白与DNA的直接结合，以表征诱导蛋白质与DNA结合的刺激物，并表征蛋白质结合的DNA序列。后一信息可以帮助鉴定由特定目的蛋白质调控的基因，并通过使用片上芯片，ChIP-seq或克隆策略7,8,9来实现 。 ChIP与证明DNA-蛋白结合的其他方法的主要优点之一，如EMSA或DNase I足迹，是在ChIP技术中，绑定在体内被捕获，而其他的则是在体外进行的 。因此，ChIP提供了对细胞环境中DNA-蛋白结合的洞察，而其他技术则代表了一个孤立的系统。 然而，ChIP分析是复杂的，涉及多个可能影响结果的步骤，需要优化和体验。对于成功的ChIP而言，关键的第一步是交联步骤（步骤2.2）。紫外线交联是不可逆的，因此不适合于ChIP。甲醛交联是优选的，但甲醛浓度和交联时间都可以影响染色质剪切和抗原沉淀的效率。通常，降低甲醛浓度（1％w / v）和较短的交联时间（5-10分钟）是优选的，因为它们提高了剪切效率。但是甲醛效率不高在蛋白质 - 蛋白质交联中，因此对于不直接与DNA结合的蛋白质不是最佳的。对于这种情况，可以以两步法进行ChIP，其中首先使用交联剂例如戊二酸二琥珀酰亚胺酯进行蛋白质 - 蛋白质交联，然后进行甲醛介导的DNA-蛋白质交联17 。 下一个关键步骤是染色质剪切。在我们的研究中，我们使用微生物核酸酶进行酶剪切。当超声处理会破坏DNA-蛋白结合时，酶切剪切对非交联的天然ChIP（N-ChIP）特别有用。 N-ChIP主要用于组蛋白和组蛋白修饰剂18的分析。虽然微球菌核酸酶被认为是相对非特异性的内切核酸外切酶，但是已经显示引发序列特异性切割19 。这可能导致所产生的序列依赖性偏差具有某些基因座20的超表达。超声处理产生随机大小的DNA片段，没有序列偏差，并且通常优选用于交联ChIP（X-ChIP）。然而，超声处理条件必须根据每个细胞或组织类型和超声波仪器的经验来确定，并且所得的DNA片段通常大于使用酶剪切16 。另外，由于蛋白质变性和降解，样品的超声处理或乳化可导致抗体表位的丧失。 免疫沉淀步骤是可以大大影响ChIP结果的另一个关键步骤。琼脂糖珠非特异性地结合DNA，并且添加的珠的数量的变化可以影响特定的信号与背景比。因此，当添加到DNA-蛋白质样品中时，保持琼脂糖珠"浆液"充分悬浮是很重要的。关于用于i的抗体一般来说，应该使用ChIP级抗体，如果不能使用这些抗体，则应优先使用免疫沉淀级抗体。由于特异性表位可以在交联过程中被掩蔽，所以多克隆抗体是有利的，因为它们识别几个表位。加入抗体的量应超过沉淀因子，因此应根据每个因子/抗体的经验来确定。此外，由于达到抗体结合平衡的动力学对于每种抗体不同，因此必须确定每种抗体的最佳培养条件。 几个控件可以并且应该包括在实验设置中。在评估特异性DNA-蛋白结合反应的诱导的情况下，重要的是在最终分析（ 即 PCR或qRT-PCR）中包括输入DNA对照以证明相等的模板DNA量。需要进行抗体控制确认感兴趣的蛋白质的免疫沉淀的特异性。通常同种型匹配的免疫球蛋白非常适合作为阴性对照，但也可以使用琼脂糖珠。偶尔使用阳性对照来确认ChIP的功能性实验流程，并且经常使用抗组蛋白抗体用于此目的。在刺激实验中，优选的阳性对照是目的蛋白质的免疫沉淀，随后对蛋白质在刺激后结合的已知DNA区域进行序列分析。在我们的代表性结果中，PAI-1启动子中的SBE被用作TGF-β1诱导的SMAD结合的已知靶标。在没有刺激蛋白结合的实验中，已知不是目的蛋白质靶标的DNA序列可用于随后的DNA分析。关于DNA分析，重要的是包括没有模板DNA的反应以排除污染。 ChIP是直接证明目标蛋白与基因结合的优良技术。但是，这不是功能研究。在感兴趣的蛋白质被认为具有调节作用的情况下，还必须进行功能测定，例如基于报告基因的测定（ 例如荧光素酶）。在这些方案中，感兴趣的基因被克隆在报告基因的调节位置。感兴趣的基因导致报告基因的表达，如果它具有调节功能。为了进一步确认特定目的蛋白质的调节作用，可以产生敲低或敲入的细胞，其中DNA结合蛋白在遗传上是沉默的。作为另外的对照，调节基因中的蛋白质结合位点可以突变以防止感兴趣的蛋白质的结合。在后两种实验设计中，细胞刺激不会导致标记g的表达烯。

在真核生物核中，DNA与组蛋白和非组蛋白相互作用，并被凝聚成染色质。在生理，病理生理和细胞生物学环境中，细胞功能在空间上暂时受染色质协调基因表达控制。 DNA-蛋白质相互作用在细胞过程的调节中起重要作用，如DNA复制，重组和修复以及蛋白质表达。因此，DNA-蛋白质相互作用的分析是评估基因表达和细胞功能不可缺少的工具。 存在几种用于评估体外 DNA-蛋白质相互作用的技术 ，例如电泳（凝胶）移动性测定（EMSA）和DNA酶I印迹1,2 。然而，这些技术不分析染色质和细胞环境中的蛋白质 - DNA相互作用。 ChIP我sa技术捕获与其特异性DNA结合位点结合的蛋白质，从而有助于鉴定染色质环境中的DNA-蛋白质相互作用。该技术最初由Gimour和Lis开发，用于评估RNA聚合酶II与大肠杆菌和果蝇中的特异性基因的结合3,4 。通过固定DNA-蛋白复合物，然后进行染色质提取并将DNA剪切到约200个碱基对（bp）片段中。随后，通过免疫沉淀分离感兴趣的DNA结合蛋白。在DNA-蛋白质交联反转后，纯化和分析DNA。可以使用几种方法来分析蛋白质结合位点，并且取决于靶基因5内的蛋白质结合位点的核酸序列。在DNA序列已知的情况下，标准Pol可以使用侧翼于已知结合位点的特异性引物对应用ymerase链反应（PCR）。定量实时PCR（qRT-PCR）也可以使用6 。在序列未知的情况下，ChIP可以与DNA微阵列（片上芯片），DNA测序（ChIP-seq）或克隆技术7,8,9组合 。 TGF-β途径具有很强的肿瘤抑制功能，是细胞分化的关键途径。它通过TGF-β1配体与其同源受体复合物的结合而被激活，导致SMAD2 / 3转录因子的丝氨酸磷酸化。在与常见介体SMAD4结合后，SMAD复合物转移到细胞核并与靶基因启动子区域内的SBE结合，其中它调控控制细胞周期，细胞凋亡和细胞分化的基因上。 TGF-β刺激的转录反应是细胞类型和上下文特异性10 。最近，我们描述了TGF-β和c-KIT途径之间的正反馈回路11 。在该模型中，TGF-β1激活的SMAD2结合c-KIT配体启动子并诱导其表达和分泌。随后，c-KIT配体以自动和对位方式激活c-KIT受体。 c-KIT受体活化通过JAK1 / 2导致STAT3 Tyr 705-磷酸化。 STAT3激活和核易位后，STAT3结合TGF-β1配体基因并调节其表达。 在这里，我们证明ChIP分析对于识别SMAD2与c-KIT受体配体启动子的结合以及鉴定信号转导子（和）转录3（STAT3与TGF-β1-基因）。

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">HepG2 cells</td>
			<td style="width:193px;">ATCC</td>
			<td style="width:119px;">HB-8065</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Hep3B cells</td>
			<td style="width:193px;">ATCC</td>
			<td>HB-8064</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">TGF-&#946;1</td>
			<td>R&#38;D Systems</td>
			<td>101-B1</td>
			<td>Used at a concentration of 10 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-SMAD2 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">5339</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-STAT3 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">4904</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">ChIP-IT Protein G Magnetic Beads</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">53033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Protease Inhibitor Cocktail</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">37490</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Micrococcal Nuclease</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">10011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GGAAGAGGATAAAGGACAAGCTG-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCAGCCAGCCACGTGATTGTC-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-CACTGATGTTAATGTTCAGC-3&#8217;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GCTCTAATTTAAACCTGGAGC-3&#8217;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GAGAGAGACGTGAGTGGCATGTT-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TAGCTTTCTCTGCCTTGGTCTCCCC-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GTACTGGGGGAGGAGCGGCATC-3&#8217;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCCACTGTCTGGAGAGAGGTGTGTC-3&#8217;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

analysis of the c-kit ligand promoter using chromatin immunoprecipitation

多重细胞过程，包括DNA复制和修复，DNA重组和基因表达，需要蛋白质和DNA之间的相互作用。因此，DNA-蛋白相互作用调节多种生理，病理生理和生物功能，如细胞分化，细胞增殖，细胞周期控制，染色体稳定性，表观遗传基因调控和细胞转化。在真核细胞中，DNA与组蛋白和非组蛋白蛋白相互作用，并被浓缩成染色质。可以使用几种技术工具来分析DNA-蛋白质相互作用，例如电泳（凝胶）移动性测定（EMSA）和DNA酶I足迹。然而，这些技术在体外分析蛋白质 - DNA相互作用，而不是在细胞环境中。染色质免疫沉淀（ChIP）是一种在其特定DNA结合位点捕获蛋白质的技术，从而允许鉴定DNA-蛋白质相互作用在染色质环境中。通过固定DNA-蛋白质相互作用，然后免疫沉淀所关注的蛋白质来完成。随后，表征蛋白质结合的基因组位点。在这里，我们描述和讨论ChIP并证明其分析价值，用于鉴定转录生长因子-β（TGF-β）诱导的转录因子SMAD2与酪氨酸 - 酪氨酸激酶启动子区域中SMAD结合元件（SBE）的结合，蛋白激酶试剂盒（c-KIT）受体配体干细胞因子（SCF）。

TGF-β1配体与其同源受体复合物的结合导致SMAD2 / 3转录因子的丝氨酸磷酸化，其后与其共同介质SMAD4结合。 SMAD复合体移位到细胞核。 TGF-β可以直接通过SMAD与目标基因调节区内的SBE结合或间接通过转录激活子或阻遏物的SMAD调节表达调节基因转录，其随后调节感兴趣的基因10的表达。为了测试c-KIT配体SCF是否通过直接TGF-β1诱导的SMAD2与其启动子的结合转录调节，我们分析了SCF基因的SCF启动子，2.4-kb，5&#39;-侧翼区域SMAD2结合基序，5&#39;-AGAC-3&#39;（SBE） 13,14 。我们确定了7个推定的SBE upsSCF起始密码子的ream（ 图1A ）。为了证实TGF-β1诱导的SMAD2与SCF启动子的结合，进行了这里描述的ChIP测定。 TGF-β1处理导致SMAD2与人肝癌细胞系HepG2和Hep3B细胞中的SCF启动子结合（ 图1B ）。在没有TGF-β1刺激的情况下，没有观察到SMAD结合。作为TGF-β1诱导的SMAD激活的阳性对照，进行了PAI-1的ChIP。 PAI-1启动子是TGF-β/ SMAD13的已知靶标。为了证实SMAD2免疫沉淀的特异性，使用非特异性IgG抗体;在用IgG免疫沉淀后没有观察到SMAD2与SBE的结合。 为了进一步证明ChIP技术，我们分析了STAT3共有结合基序5&#39;端的TGF-β配体基因的5&#39;侧翼区-TT（N4）AA-3&#39;和5&#39;-TT（N5）AA-3&#39;15。我们在位置-4384 / -4373（STB-1）和-5365 / -5357（STB-2）（ 图2A ）处鉴定了TGFB起始密码子上游两个假定的STAT3结合位点。为了证实TGF-β1诱导的STAT3与TGF-β配体基因的结合，我们使用TGF-β1处理的HepG2和Hep3B细胞进行ChIP测定。 TGF-β1处理导致STAT3与TGFB基因的第二推定的STAT3结合位点（STB-2）结合，但不与第一个（STB-1）结合（ 图2B ）。在这个例子中，正向STAT3与STB-2的结合作为内部阳性对照。与上述ChIP实验相似，使用IgG免疫沉淀后，STAT3与STB-2的结合未见。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55689/55689fig1.jpg" /> 图1 <str<html> ong&gt;:TGF-β诱导的SMAD2与SCF启动子的结合。 （ A ） SCF启动子的示意图，推定的SBE显示为盒（白盒= AGAC）及其与起始密码子的相对位置。 ChIP引物位置如下所示，其与SCF起始密码子和PCR产物大小的相对位置。 （ B ）SMAD2与SCF启动子结合的ChIP。 TGF-β1处理和未处理的HepG2和Hep3B细胞的染色质 - 蛋白复合物用抗SMAD2抗体进行免疫沉淀。使用SCF特异性引物进行PCR。左侧显示使用输入DNA的PCR结果。在中间，用非特异性IgG免疫沉淀后的PCR结果显示用于确认SMAD2免疫沉淀特异性。如下图所示，SMAD2与PAI-1结合的ChIP作为阳性对照。ge.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55689/55689fig2.jpg" /> 图2 :TGF-β诱导的STAT3与TGF-β基因的结合。 （ A ）TGF-β基因示意图，其中假定的STAT3结合位点显示为具有与起始密码子相对位置的灰色框。 ChIP引物位置以其与TGF-β起始密码子的相对位置和下面所示的PCR产物大小显示。 （ B ）TGF-β1诱导的STAT3与TGF-β基因结合的ChIP。 TGF-β1处理和未处理的HepG2和Hep3B细胞的染色质 - 蛋白复合物用抗STAT3抗体进行免疫沉淀。使用TGF-β特异性引物进行PCR。左侧显示使用输入DNA的PCR结果。在中间，PCR显示用非特异性IgG免疫沉淀后的结果证实STAT3免疫沉淀特异性。上图显示了使用特异于STAT3结合位点1（STB-1）的引物的PCR结果，下图显示STAT3结合STAT3结合位点2（STB-2）的结果。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55689/55689fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

DNA-蛋白质的相互作用对多种生物过程至关重要。在评估细胞功能时，DNA-蛋白质相互作用的分析对于了解基因调控是必不可少的。染色质免疫沉淀（ChIP）是分析体内这种相互作用的有力工具。

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

analysis-c-kit-ligand-promoter-using-chromatin

Nanyang Technological University

Research

JoVE Journal

Genetics

7.7K 次观看.  The University of Texas MD Anderson Cancer Center.  DNA-蛋白质的相互作用对多种生物过程至关重要。在评估细胞功能时，DNA-蛋白质相互作用的分析对于了解基因调控是必不可少的。染色质免疫沉淀（ChIP）是分析体内这种相互作用的有力工具。 

视频: Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

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

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

小鼠棕色脂肪组织的染色质免疫沉淀

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer&#39;s disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

利用染色质配置的计算分析，将阿尔茨海默氏病变异体映射到其目标基因

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, ...