DEVELOPMENT OF SPLIT DAMID AS AN ALTERNATIVE METHODOLOGY TO CHROMATIN IMMUNOPRECI

开发分裂 DAMID 作为染色质免疫分析的替代方法

• 批准号: 7937828
• 负责人: RAPHAEL KOPAN
• 金额: $ 50万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937828
• 关键词: Adenine; Affect; American; Animal Model; Animals; Antibodies; Area; Bacterial DNA; Behavior; Behavioral; Binding; Binding Sites; Brain; C-terminal; CREB1 gene; Cell Differentiation process; Cells; Chimeric Proteins; Chromatin; Cocaine; Communities; DNA; DNA Binding; DNA-Binding Proteins; Dam methyltransferase; Development; Digestion; Disease; Drug abuse; EP300 gene; Emerging Technologies; Enzymes; Epigenetic Process; Eukaryota; Exposure to; Expression Library; Gene Targeting; Generations; Genes; Genetic; Genetic Transcription; Genome; Healed; Housekeeping; Housekeeping Gene; Human; In Vitro; Lead; Link; Malignant Neoplasms; Mediating; Mental Health; Mental disorders; Methodology; Methods; Methylation; Modification; Mutation; N-terminal; Neuronal Differentiation; Neurons; Neurosciences; Neurosciences Research; Noise; Notch Signaling Pathway; Organism; Pattern; Pharmaceutical Preparations; Process; Proteins; Psychotropic Drugs; Reagent; Recovery; Relative (related person); Research; Research Personnel; Resources; Sensitivity and Specificity; Signal Transduction; Site; Solutions; Staging; T-Lymphocyte; TP53 gene; Techniques; Technology; Testing; Time; TimeLine; Tissues; Transgenic Organisms; Work; abstracting; calmodulin-dependent protein kinase II; cell type; chromatin immunoprecipitation; embryonic stem cell; expression vector; healing; homologous recombination; improved; in vivo; interest; methyl group; nestin protein; new technology; notch protein; novel; overexpression; promoter; reconstitution; relating to nervous system; response; transcription factor; vector

DESCRIPTION (provided by applicant): Research Area: (06) Enabling Technologies Challenge topic: New technologies for neuroscience research (06-MH-103) Project Title: Development of Split-DamID as an alternative methodology to chromatin immunoprecipitation Project Abstract A wide variety of genetic or drug-induced mental health disorders have been linked to altered functions of transcription factors, be it drug-induced or caused by genetic defects. Although it is known that the expression patterns of many genes are altered in the brain after exposure to a psychotropic drug, ultimately we must determine which genes were directly activated by the relevant transcription factors to uncover the expression hierarchy and prioritize druggable targets. Progress towards identification of these "direct targets" has been slow since the traditional methods used to identify the immediate targets of a transcription factor (chromatin IP or ChIP) are expensive, labor-intensive and technically demanding. Most dauntingly, they require very good antibodies and a large amount of starting material, largely limiting their use to cells in culture. In order to provide an approach that avoids these issues, we have optimized the sensitivity and specificity of an alternative technology, DamID, for broad use in neuroscience. Our solution (Split-DamID) relies on our ability to successfully split the bacterial DNA adenine methyltransferase (Dam) into N-terminal (D) and C-terminal (AM) halves. Each half is inactive; however, when fused to interacting transcription factors, the halves will reconstitute enzymatic activity only at the binding sites where these transcription factors interact. The reconstituted Dam marks adenine (A) by addition of a methyl group (Am) in the sequence GAmTC found in proximity to most cognate binding sites of any transcription factor. It is important to note that this epigenetic modification is never found in eukaryotic DNA and appears to have no consequence to cellular or organism survival. This GAmTC mark allows isolation of the transcription factor bound DNA from total DNA through digestion with Dpn1, an enzyme that digests only DNA methylated at GAmTC sites, and adaptor mediated PCR. We have successfully reconstituted Dam activity when fused to Notch or Mef2c. Importantly, we have observed enrichment of known Notch targets over other DNA fragments in cells reconstituting split-Dam fused to Notch. Split-DamID has several key advantages: much improved signal to noise ratio compared with ChIP or DamID; adenine methylation indelibly marks DNA bound by the fusion protein, allowing identification of transient interactions; the recovery of marked DNA is independent of antibody availability; and the amplification of the marked DNA by adaptor mediated PCR makes it amenable to working with the endogenous expression level of the fusion protein and low amounts of starting material. Moreover, Spilt-DamID permits precise spatial and temporal control of Dam reconstitution. We propose to generate multiple Split-Dam pairs with mental health related factors (Notch, DeltaFosB, CREB, NF¿B, and Mef2) and common transcription co-activators (p300, others) to reconstitute Dam activity only at sites where transcription takes place. These targeting vectors, expression vectors, cells and animals will create a resource for investigators interested in identifying sites directly bound by transcription factors implicated in mental health in animal models exposed to psychotropic drugs or carrying a mutation known to affect behavior in humans. We will develop resources allowing investigators interested in brain function to mark and identify key target genes bound by transcription factors of interest (e.g., those involved in mental health, cancer, and other transcription-driven processes in neuroscience). They will have control over when the marking occur, enabling precise correlation of transcriptional activity and behavior. In contrast to the current technology, our novel method will work for any transcription factor and requires only a few animals since the method allows amplification of the recovered target sites. It should allow the rapid characterization of the vast array of transcription factors involved in mental health and facilitate identification of therapeutically relevant targets altered by disease or drug abuse. Additionally, all of the reagents used will be obtained from companies within the USA, and the resource will be made available to the community through American depositories.

描述（应用程序提供）：研究领域：（06）启用技术挑战主题：神经科学研究的新技术（06-MH-103）项目标题：开发分裂damid作为一种替代方法，用于染色质免疫沉淀项目摘要摘要摘要的遗传或​​药物诱导的多种遗传性疾病的表达方式已与该遗传性疾病的表达方式相关，但它是指的转化，虽然它的转化是它的作用。暴露于精神药物后的大脑中会改变，最终我们必须确定哪些基因被相关转录因子直接激活，以发现表达层次结构并优先考虑可药物的靶标。由于用于识别转录因子（染色质IP或芯片）的直接目标的传统方法，鉴定这些“直接目标”的进展一直很慢。最令人生畏的是，它们需要非常好的抗体和大量的起始物质，在很大程度上将其用于培养细胞的用途。为了提供避免这些问题的方法，我们优化了替代技术Damid的敏感性和特异性，以在神经科学中进行广泛使用。我们的溶液（分木）取决于我们成功将细菌DNA腺嘌呤甲基转移酶（DAM）分为N末端（D）和C末端（AM）两半的能力。每半都是不活动的；但是，当与相互作用的转录因子融合时，一半仅在这些转录因子相互作用的结合位点重新构建酶促活性。通过在与任何转录因子的大多数同源结合位点的序列GAMTC中添加甲基（AM），通过添加甲基（AM）来标记腺嘌呤（A）。重要的是要注意，这种表观遗传修饰在真核DNA中从未发现，并且似乎对细胞或生物体的生存没有影响。该GAMTC标记允许从总DNA通过DPN1消化分离转录因子DNA，该酶是一种仅在GAMTC位点消化的DNA甲基化的酶，以及适配器介导的PCR。与Notch或MEF2C融合时，我们已经成功地重新建立了大坝活动。重要的是，我们已经观察到已知的Notch靶标超过了与Notch融合的分裂DAM的细胞中其他DNA片段相比。分离命中具有多个关键优势：与芯片或DAMID相比，信号与噪声比大大提高；腺嘌呤甲基化纵隔地标记了由融合蛋白结合的DNA，从而鉴定了瞬时相互作用。标记DNA的恢复与抗体可用性无关。通过衔接子介导的PCR对标记的DNA进行扩增使得与融合蛋白的内源性表达水平和低量的起始材料一起工作。此外，Spilt-Damid允许精确的空间和临时控制大坝重建。我们建议生成与心理健康相关因素（Notch，Deltafosb，Creb，Nf¿B和Mef2）和共同转录共激活因子（P300，其他）的多个分裂配对，以重新建立在转录发生的地点的大坝活动。这些靶向矢量，表达媒介，细胞和动物将为有兴趣识别直接识别的位点的研究者创造资源，该地点受到暴露于精神药物的动物模型中在心理健康中实施的转录因子或携带已知会影响人类行为的突变。我们将开发资源，使对大脑功能感兴趣的研究人员可以标记和确定受感兴趣的转录因子绑定的关键靶基因（例如，在神经科学中参与精神健康，癌症和其他转录驱动过程的基因）。他们将控制标记何时发生，从而实现转录活性和行为的精确相关性。与当前的技术相反，我们的新方法将适用于任何转录因子，仅需要几个动物，因为该方法可以放大恢复的目标位点。它应该允许快速表征精神健康涉及的大量转录因素，并促进对疾病或药物滥用改变的历史相关目标的识别。此外，所有使用的试剂将从美国境内的公司那里获得，该资源将通过美国存放者提供给社区。