DEVELOPMENT OF SPLIT DAMID AS AN ALTERNATIVE METHODOLOGY TO CHROMATIN IMMUNOPRECI

描述(由申请人提供)：研究领域：(06)使能技术挑战主题：神经科学研究的新技术(06-MH-103)项目标题：开发Split-DamID作为染色质免疫沉淀项目的替代方法摘要各种各样的遗传或药物引起的精神健康障碍与转录因子的功能改变有关，无论是药物引起的还是由遗传缺陷引起的。虽然已知许多基因在接触精神药物后在大脑中的表达模式会发生变化，但最终我们必须确定哪些基因直接被相关转录因子激活，以揭示表达层次并优先选择可用药的靶点。由于用于识别转录因子(染色质IP或芯片)的直接靶标的传统方法昂贵、劳动密集型和技术要求高，因此识别这些“直接靶标”的进展缓慢。最令人望而生畏的是，它们需要非常好的抗体和大量的起始材料，这在很大程度上限制了它们在培养细胞中的使用。为了提供一种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，以便在神经科学中广泛使用。我们的解决方案(Split-DamID)依赖于我们成功地将细菌DNA腺嘌呤甲基转移酶(DAM)分成N端(D)和C端(AM)的能力。每一半都是不活跃的；然而，当与相互作用的转录因子融合时，这一半将只在这些转录因子相互作用的结合部位重建酶活性。重组的Dam通过在GAMTC序列中添加甲基(AM)来标记腺嘌呤(A)，GAMTC在任何转录因子的大多数同源结合位点附近发现。值得注意的是，这种表观遗传修饰在真核DNA中从未发现，似乎对细胞或有机体的生存没有任何影响。这种GAmTC标记允许通过Dpn1(一种只消化在GAmTC位点甲基化的DNA的酶)和适配器介导的PCR从总DNA中分离出转录因子结合的DNA。当与Notch或MEF2C融合时，我们成功地重建了Dam的活性。重要的是，我们观察到在重组裂开-Dam融合到Notch的细胞中，已知的Notch靶标比其他DNA片段更丰富。Split-DamID有几个关键的优点：与CHIP或DamID相比，信噪比大大提高；腺嘌呤甲基化不可磨灭地标记与融合蛋白结合的DNA，允许识别瞬时相互作用；标记的DNA的恢复不依赖于抗体的可获得性；通过接头介导的PCR扩增标记的DNA使其能够与融合蛋白的内源表达水平和少量的起始物质一起工作。此外，Spilt-DamID允许对大坝重建进行精确的空间和时间控制。我们建议用与心理健康相关的因子(Notch、DeltaFosB、CREB、NFB和MEF2)和共同的转录共激活因子(p300等)生成多个分裂-Dam对，以仅在发生转录的位置重建Dam活性。这些靶向载体、表达载体、细胞和动物将为研究人员创造一个资源，这些研究人员有兴趣在暴露于精神药物或携带已知影响人类行为的突变的动物模型中识别与精神健康有关的转录因子直接结合的位置。我们将开发资源，允许对大脑功能感兴趣的研究人员标记和识别由感兴趣的转录因子结合的关键目标基因(例如，那些涉及精神健康、癌症和神经科学中其他转录驱动过程的基因)。他们将控制标记发生的时间，使转录活动和行为能够精确关联。与目前的技术不同，我们的新方法适用于任何转录因子，并且只需要几个动物，因为该方法允许扩增恢复的靶点。它应该能够快速表征与精神健康有关的大量转录因子，并有助于识别因疾病或药物滥用而改变的治疗相关靶点。此外，所有使用的试剂都将从美国境内的公司获得，资源将通过美国的托管机构提供给社区。

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.