This work was supported by R01NS075062-01A1. FACS sorting performed at UAB Comprehensive Flow Cytometry Core facility (P30 AR048311, P30 A1027767). Dr. Scott Philips from the UAB Neurobiology Core facility and Dr. Susan Nozell from UAB CDIB assisted with technical aspects of the luciferase assay.

所有动物均按照健康准则全国学院处理。动物护理和使用委员会在阿拉巴马大学伯明翰分校批准的动物使用。 1.使用荧光激活细胞从全脑组织排序星形胶质细胞（FACS）从富集的星形胶质细胞人口获得的RNA和DNA <ol><li>稳重动物用CO 2 1分钟，然后迅速斩首。在阿尔布开克等人解剖皮层，26。这是没有必要去除脑膜。 注:表示在S100β绿色荧光蛋白（星形胶质细胞标记物）子转基因大鼠由板仓等 27产生和利用的星形胶质细胞的FACS分选。 </li><li>使用根据制造商的协议在非无菌条件下用木瓜蛋白酶的解离试剂盒制备全脑匀浆。在水浴热激活的木瓜蛋白酶在37℃下进行10分钟。注意:木瓜蛋白酶溶液通过制造商提供，并包含L-半胱氨酸和EDTA（见表材料）。 <ol><li>切割或DREMEL空穴到50毫升锥形管的顶部，以允许管道携带95％O 2:5％的CO 2被送入一个封闭的圆锥管（图1A）。放置在10毫米培养皿含有解离媒体解剖皮层（MEM补充有20mM的葡萄糖和青霉素/链霉素（500U / ml）和平衡至95％O 2，5％CO 2），并使用干净的刀片剁碎组织成1- ×1毫米2件 。 </li></ol></li><li>用10ml手册的Pipetman至50ml锥形管含有木瓜蛋白酶溶液转移组织。让组织来履行，以尽量减少分离介质的结转额前下沉到移液管的底部。保持平衡至95％ 的 O 2的木瓜蛋白酶溶液:5％的CO 2经由该孵育期间表面气体交换。不要泡木瓜蛋白酶soluti上。孵育组织为在37℃水浴20分钟。 </li><li>孵育后在木瓜蛋白酶溶液，磨碎的组织的10倍以低速10ml的移液管。离心浑浊细胞悬浮液在1000×g离心5分钟，室温。 <ol><li>平衡经由表面气体交换的DNase /白蛋白抑制剂溶液（由制造商提供的），并在3ml的DNA酶/清蛋白抑制剂溶液重新悬浮沉淀的细胞。制备商业不连续密度梯度依照制造商的指令。 </li></ol></li><li>自旋不连续密度梯度，在1000×g离心6分钟。通过抽吸底部沉淀用吸管隔离管的底部离解的细胞。 </li><li>重悬离解的细胞在2-3毫升的DPBS用0.02％牛血清白蛋白和1毫克/毫升DNA酶或缓冲培养基优选HEPES。流式细胞仪（ 图2A），然后通过40微米的过滤器传递。保持细胞在冰上，直到排序。 <ol><li>执行流式细胞仪28。&lt;/ LI&gt; <li>沉淀细胞在1.5ml的离心管中，在2000×g离心5分钟，在4℃。 注意:丸粒化星形胶质细胞可以立即使用或保存在-80℃直至DNA提取。 </li></ol></li><li> RNA和DNA使用优选的分离方法29 30提取。通过分光光度计和生物分析仪31评估RNA和DNA的浓度和质量。 注意:利用仅高质量RNA和DNA在随后的步骤中，260个/ 280个= 2.0-2.2和1.8-1.9，分别。生物分析仪分析是必不可少的，以评估RNA降解或DNA片段。 RNA或DNA可以立即使用或储存于-80℃或-20℃，分别用于随后的研究。 </li></ol> 2.评估使用甲基化敏感高分辨率熔解分析（MS-HRMA）的基因的DNA甲基化状态<ol><li>进入首选网上甲基化地图软件感兴趣的基因序列，以识别任何CpG岛的基因关注32。 <ol><li>亚硫酸氢盐对设计的引物转化DNA序列33和扩增按照制造商的方案使用优选的DNA聚合酶。通过运行在100伏的1％琼脂糖凝胶的DNA 45分钟确认扩增产物的大小。商店引物储备浓度20μM的在-20℃。 </li></ol></li><li>亚硫酸氢盐转化500-1000毫微克的每个样品和甲基化DNA的标准范围从0-100％来自同一动物种类34的DNA。洗脱样本以提供20毫微克/微升的浓度。通过分光光度计31验证的亚硫酸氢盐转化的DNA浓度。 </li><li>设置20微升反应为根据表1和2使用优选的DNA聚合酶和引物在5微米浓度的MS-HRM扩增。运行所有的样品，包括流式细胞仪DNA和甲基化的标准，以一式三份。 </li><li>根据分析软件，设置前置和后置启动和停止参数周围的熔解曲线的过渡。组预先熔融开始和预熔融停止参数，使两者之间的差为0.2 - 0.5℃。设置后开始融化，后熔停止类似。提取峰值温度的差数据的每个样本。 </li><li>使用百分之甲基标准（y值）和它们的相应的平均峰温度差（x值）生成的线性回归方程（图3A-B，表3-4）。使用该线性回归方程来估算未知样品35的甲基化状态。 </li></ol> 3.通过使用荧光素酶测定的评估超甲基化的启动子活性<ol><li>确定目标基因（步骤2.1）的CpG岛。 PCR扩增感兴趣36和克隆区域luc2萤火虫萤光素酶报道基因的上游，以产生的CpG岛luc2质粒37。 </li><li>线性化30微克的通过限制的CpG岛luc2质粒酶消化38。验证网站的限制消化，避免使用首选刀具软件38双削减。热灭活根据制造商的协议如下消化在适当的温度和持续时间的酶。在线性化步骤是DNA的损失降到最低。 </li><li>使用的CpG甲基化酶（M.Sssl）O / N在30°C或将未经处理的，按照制造商的协议，除了以下调整甲醇线性质粒。 </li><li>执行50微升反应。 </li><li>使用5个单位的CpG甲基对甲基化700纳克线性化的质粒。 </li><li>运行反应O / N为13-19小时。 </li><li>以下的CpG甲基化酶反应，使用标准的，根据制造商的协议市售硅胶膜上清理试剂盒进行DNA的清理。 DNA的显著损耗发生以下的CpG甲基化酶反应，30-60％的损失。 </li><li>通过中Hpa II酶切验证质粒甲基化。 </l我&gt; <li>取1微克的甲基化或非甲基化DNA和限制在37℃下消化用的Hpa II 1小时。使用5-10个单位的Hpa II为每微克DNA。继中Hpa II消化，使用的是首选，市售的硅胶膜清洁套装进行DNA清理。运行在100V在TAE或TBE缓冲液的1％琼脂糖凝胶的DNA在两个CpG的甲基化和非甲基化的质粒45分钟进行可视化（图4B）。 </li><li>受试者都甲基化和非甲基化的质粒双重消化用合适的限制性酶释放的全长的luc 2（矢量）和CpG岛（插入） 片 38。以下，根据制造商的协议消化进行双酶切O / N在适当的温度和热灭活酶。在双酶切出现DNA浓度的损失降到最低。 </li><li>运行在1％琼脂糖凝胶双酶切质粒在100V 1小时，以允许分离ö˚F载体和插入（图4C）。注意事项。戴防护紫外线面罩，放置在桌面黑光DNA凝胶可视化的乐队。基于大小，消费甲基化和非甲基化的插入和非甲基化的载体用干净的手术刀。 </li><li>凝胶提取DNA用饱和酚，pH值6.6。简单地说，衡量含载体或插入DNA凝胶。用100微升苯酚每0.1克DNA凝胶。用玻璃Dounce匀浆均质DNA凝胶苯酚。 </li><li>加氯仿（以苯酚量的1/5），并摇动样品20秒。孵育的样品在RT 2-3分钟。离心15分钟以最大速度（16.1×1,000 XG）在4℃。 </li><li>除去水溶液，并添加3M乙酸钠和乙醇2.5X体积0.1X体积。孵育样品1小时，在-80℃下。孵育后，除去乙醇并于30μl优选缓冲液重悬的DNA。 DNA的显著损失发生的插入和载体以下隔离，30-50％LOSS。 </li><li>再结扎甲基化和非甲基化的插入物，使用T4 DNA连接酶38未甲基矢量。使用1:4的比例矢量的插入用于连接反应。根据制造商的说明书设置反应。使用50微升的总体积的反应和孵化O / N在-20°C或在冰上用盖在冰桶。 </li><li>连接后，使用的是首选，市售的硅胶膜清洁套装进行DNA清理。的DNA显著损失发生以下的连接反应，DNA的近似30-50％的损失。验证重新结扎通过在1％琼脂糖凝胶的DNA 45分钟运行，在100 V和评估的重新连接质粒（图4D）的浓度。 </li><li>染甲基化或非甲基化的质粒导入使用根据制造商的协议的市售的转染试剂D54细胞。种子D54细胞到12孔板，在一个0.14×10 6个细胞/孔。 </li><li>继24小时，转染细胞无线个相等的浓度，无论（1）非methyated CpG的luc2质粒+海肾或其他控制荧光素酶载体或（2）甲基化的CpG-luc2质粒+海肾或其他控制荧光素酶载体。 </li><li>让细胞进行双荧光素酶检测前转24小时。根据制造商的说明进行检测。执行用光度计读数。一式三份阅读每口井。 </li><li>计算萤火虫荧光素酶活性的比为海肾或其他控制的荧光素酶的活性。通过将甲基化荧光素酶活性非甲基化的荧光素酶控制的荧光素酶:规范化甲基化萤火虫:控制荧光素酶的活性，以非甲基化的萤火虫 </li></ol>

The authors have no disclosures.

This protocol describes the isolation of an enriched population of astrocytes via FACS as well as a variety of techniques that allow for both correlative and causative studies between DNA methylation and gene expression. These techniques, used in isolation or in combination, are particularly useful for laboratories that work with tissue of high cellular heterogeneity or are interested in the DNA methylation status of a particular gene or gene region versus global DNA methylation changes. One relatively unique challenge i...

Epigenetics is the study of chemical modifications that can affect the transcriptional activity of the genome. Essentially, without a change in the DNA sequence, epigenetic modifications such as DNA methylation, histone acetylation, and histone methylation are sufficient to reversibly alter patterns of gene expression 1. DNA methylation, a potent regulator of gene expression, is the most well characterized epigenetic modification. DNA methylation is the covalent attachment of methyl groups on the C5 position of a cytosine, typically the cytosine of a cytosine-guanine dinucleotide, also known as a CpG site. Areas that contain a high density of CpG sites are known as CpG islands (CGIs). CGIs are frequently associated with transcriptional start sites (TSS) and gene promoters 1-3. Thus, while changes in DNA methylation at CGIs are not always concomitant with changes in cellular expression or function, changes in DNA methylation at CGIs can exert powerful regulation on transcriptional activity 2. 
Historically, DNA methylation was observed to be essential in embryogenesis, imprinting, and development, with little changes in the levels of DNA methylation occurring in post-mitotic cells (with the exception of alterations in cancer-related genes) 4,5. However, the field of neuroepigenetics has highlighted an important non-developmental role for DNA methylation. Specifically, cognitive epigenetics has redefined DNA methylation as a highly plastic mechanism integral in mediating both the transcriptional activation and repression of genes essential for the process of learning and memory 6. Apart from cognitive epigenetics, studies modeling ischemic injury and neuropathic pain characterize DNA methylation as a labile mechanism that responds rapidly to a variety of CNS insults 7-9. In regards to astrocytes, several lines of evidence suggest DNA methylation plays an important role in astrogliogenesis. Fan et al., found that conditional KO of DNMT1 in neural progenitor cells (NPCs) resulted in precocious development of astrocytes concordant with a global state of hypomethylation 10. Additionally, Perisic et al., concluded differential levels of DNA methylation of the GLT-1 promoter mediated differential levels of expression of the glutamate transporter in the cortex and cerebellum, emphasizing a role in DNA methylation in establishing brain-region specific patterns of astrocytic gene expression 11. Overall, numerous studies underscore the dynamic and labile nature of DNA methylation in the CNS as environment, drugs, and injury have all been shown to change DNA methylation and often, gene expression 4,9. Together, these neuroepigenetic studies point to DNA methylation as a feasible therapeutic target with the potential to mitigate a variety of CNS pathologies.
As the field of epigenetics expands its understanding of the role of DNA methylation in neurodevelopment and disease, the challenge of moving DNA methylation towards a therapeutic target is performing not only correlative, but causative studies that define specific gene targets and sites. Additionally, surveying changes in DNA methylation specific to brain region and cell type remains an ongoing and time worthy challenge unique to the field of neuroepigenetics. This protocol utilizes a variety of techniques including fluorescence-activated cell sorting (FACS) of astrocytes, methylation-sensitive high resolution melt analysis (MS-HRM), and a methylation luciferase assay to investigate the DNA methylation status of KCNJ10, a gene that encodes for Kir4.1. Kir4.1 is a glial specific potassium channel that demonstrates both brain region and cell specific patterns of expression in the CNS 12-16. Kir4.1 expression increases moving from rostral to caudal CNS regions, with the highest expression occurring in the spinal cord 15. Although the channel is expressed in ependymal cells, oligodendrocytes and their precursor cells, Kir4.1 is predominantly expressed in astrocytes and thought to be essential for maintaining homeostatic levels of potassium as well as supporting glutamate uptake by setting the astrocytic resting membrane potential at a hyperpolarized -80mV 12,16-19. Importantly, the expression of Kir4.1 is non-static both during development and following multiple forms of CNS injury 20-25. We wished to examine the epigenetic regulation of this channel, specifically in astrocytes during development. The techniques utilized offer gene-specific and targeted CpG site analyses that provide causal evidence for a role of DNA methylation in regulating KCNJ10 gene expression. These techniques can be applied to other genes.

<table><tbody><tr><td>Papain Dissociation System</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td></td></tr><tr><td>AllPrep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td></td></tr><tr><td>Methyl Primer</td><td>Applied Biosystems</td><td>online</td><td>software to localize CpG Islands</td></tr><tr><td>EZ DNA methylation Kit</td><td>Zymo Research</td><td>D5001</td><td></td></tr><tr><td>Rat Premixed Calibration Standard</td><td>EpiGenDx</td><td>80-8060R-Premix</td><td></td></tr><tr><td>CpG Methylase (M.Sssl)</td><td>Zymo Research</td><td>E2010</td><td></td></tr><tr><td>QIAquick Gel Extraction&amp;nbsp;</td><td>QIagen</td><td>28704</td><td>Used for gel extraction and DNA cleanup</td></tr><tr><td>Restriction enzymes</td><td>New England BioLabs</td><td></td><td></td></tr><tr><td>NEB cutter</td><td>New England BioLabs</td><td>online</td><td>verify restriction digest sites</td></tr><tr><td>Dual Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>Luc2 vector, pGL4.10</td><td>Promega</td><td>E6651</td><td></td></tr><tr><td>renilla vector, pGL4.74</td><td>Promega</td><td>E2241</td><td></td></tr><tr><td>TD-20/20 Luminometer</td><td>Turner Designs</td><td></td><td></td></tr><tr><td>Lipofectamine LTX and Plus Reagent</td><td>Life Technologies</td><td>A12621</td><td></td></tr><tr><td>Phenol, saturated pH 6.6/6.9</td><td>Fisher Scientific</td><td>BP 17501-100</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophtometer</td><td>ThermoScientific</td><td></td><td></td></tr><tr><td>MeltDoctor Master Mix</td><td>Life Technologies</td><td>4415440</td><td></td></tr><tr><td>High Resolution Melt (HRM) Software v2.0</td><td>Life Technologies</td><td>4397808</td><td></td></tr><tr><td>AB SDS software v2.3</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB High Resolution Melting Getting Started Guide&amp;nbsp;</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB 7900HT Fast Real-Time System</td><td>Life Technologies</td><td></td><td></td></tr></tbody></table>

correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

经由的eGFP-S100β转基因动物27的 FACS分选被收购的星形胶质细胞的富集群。由于降低年龄P0-P40细胞和年龄超过出生后第50天（P50）动物中分离分子的分子，动物的质量是最适合这样的实验。皮质组织被用于这些实验。从两个到六个动物皮质合并在一起。流式细胞仪在UAB综合流式细胞仪核心设施进行。在Becton Dickinson公司FacsAria II进行排序。采用488 nm激光获得绿色荧光蛋白的激励;没有补偿是必?...

Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

correlating-gene-specific-dna-methylation-changes-with-expression

Research

JoVE Journal

Biology

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

7.9K 次观看.  University of Alabama at Birmingham. DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression. 

视频: Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

科研

遗传学

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

大鼠甲基 Seq 平台识别与应力接触相关的表观变化

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

生物化学

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

神经科学

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

发育生物学

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性

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