Name: analysis of the c-kit ligand promoter using chromatin immunoprecipitation
Uploaded: 2017-06-27T15:00:00
Description: Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

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...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+human+transcription+factor+targets+by+coupling+chromatin+immunoprecipitation+and+CpG+island+microarray+analysis.">Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis.</a> GenesDev. 16 (2), 235-244 (2002).</li><li>Massague, J., Seoane, J., Wotton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad+transcription+factors.">Smad transcription factors.</a> Genes Dev. 19 (23), 2783-2810 (2005).</li><li>Rojas, 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+Positive+TGF-beta/c-KIT+Feedback+Loop+Drives+Tumor+Progression+in+Advanced+Primary+Liver+Cancer.">A Positive TGF-beta/c-KIT Feedback Loop Drives Tumor Progression in Advanced Primary Liver Cancer.</a> Neoplasia. 18 (6), 371-386 (2016).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nucleic+acids+by+extraction+with+phenol:chloroform.">Purification of nucleic acids by extraction with phenol:chloroform.</a> CSH Protoc. 2006 (1), (2006).</li><li>Dennler, 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=Direct+binding+of+Smad3+and+Smad4+to+critical+TGF+beta-inducible+elements+in+the+promoter+of+human+plasminogen+activator+inhibitor-type+1+gene.">Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.</a> EMBO J. 17 (11), 3091-3100 (1998).</li><li>Shi, 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=Crystal+structure+of+a+Smad+MH1+domain+bound+to+DNA:+insights+on+DNA+binding+in+TGF-beta+signaling.">Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling.</a> Cell. 94 (5), 585-594 (1998).</li><li>Seidel, H. 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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?...

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...

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. ...

Research

JoVE Journal

Genetics

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

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

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

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

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 Protocol for Low-abundance 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 Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

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

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

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

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

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, ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

使用染色质免疫沉淀和高通量测序（ChIP-seq）发现沙门氏菌生物膜中的CsgD监管目标

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...