CUTANA CUT&RUN 2022重要文献精选

CUT&RUN（Cleavage Under Targets and Release Using Nuclease）是一项用于在细胞天然染色质环境下检测蛋白质-DNA相互作用的强大技术。在CUT&RUN中，蛋白A和蛋白G与微球菌核酸酶(pAG-MNase)融合，并选择性地切割抗体标记的染色质，经过剪切后的片段从细胞中分离出来，纯化，最后通过NGS进行分析。作为ChIP的高效替代方案，CUT&RUN克服了传统ChIP-seq分析法的许多缺点。

与ChIP-seq相比CUT&RUN的优势 

● 需要的细胞数量较少：CUTANA™ CUT&RUN分析只需要5,000个细胞即可生成高分辨率的结果。

● 操作步骤简单：CUTANA™ CUT&RUN在3天内即可完成从细胞到文库的建立。还适用于多道移液器和8联排管，提高了分析的重复性和通量。

● 测序成本降低：只需要300 – 800万个测序读段，高通量测序可以检测更多样本。

● 减少实验中需要优化的步骤：CUT&RUN跳过了ChIP-seq中最具挑战性的部分(包括交联、染色质片段化和免疫沉淀(IP))，只需要较少的优化步骤。

EpiCypher可提供包括CUTANA CUT&RUN Assay Kit和 Library Prep Kit，以及一系列不断扩大的经过CUT&RUN验证的抗体产品。SNAP-CUTANA™ K-MetStat Panel提供了一个基本的检测控制，可用于抗体验证，实验流程的优化，并作为实验成功与否的直接衡量标准。下面重点介绍了一些研究，展示了CUTANA CUT&RUN在不同的研究领域的应用。希望这些文献能够为CUT&RUN如何应用于您的项目提供思路。

1. Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer–promoter interactions

Wang et al. Nucleic Acids Research, 2022. PMID: 35849129

细胞类型：Transfected HeLa cells and HEK293T cells

目标蛋白：H3K27ac

主要内容：argeted modification of the epigenome represents a promising strategy for precision medicine, since it allows activation of genes without disrupting DNA sequence. CRISPR/Cas9 has been cleverly modified for this purpose by fusing a nuclease-defective Cas9 (dCas9) with transcriptional activation domains, such as histone lysine acetyltransferases CBP or p300. Here, Wang et al. used CUT&RUN to help analyze local and genome-wide changes in chromatin structure when using different dCas9 activation systems. In agreement with previous studies, they found that dCas9 activators were highly variable, with results varying by the type of dCas9 fusion protein, cell type, and genomic target. They also discovered that targeting dCas9 activators to enhancers can induce reciprocal epigenomic changes at target promoters, leading to increased gene expression.

上榜理由：虽然dCas9激活系统提供了特定的基因激活，但表观基因组变化也是可能的。CUT&RUN做为一种快速可靠的染色质分析检测方法，可帮助分析使用不同dCas9激活系统时，染色质结构的局部和全基因组变化。

2. Histone H3 proline 16 hydroxylation regulates mammalian gene expression

Liu et al. Nature Genetics, 2022. PMID: 36347944

细胞类型：MDA-MB-231 cells (hypoxia-sensitive breast cancer cells) and 293T cells; includes experiments with and without knockdown of EGLN2 using the inducible CRISPR v2 system

目标蛋白：H3P16oh, EGLN2, KDM5A, H3K4me3

主要内容：Low oxygen (hypoxia) induces transcriptional changes that drive tumor growth and increase cancer severity. Although multiple studies show that chromatin is responsive to hypoxia and plays a role in these processes, additional information is needed to provide a cohesive mechanism. Here, Liu et al. defined a novel histone PTM directly linked to hypoxia: prolyl (proline) hydroxylation on histone H3, proline 16. As part of this work, the authors used CUTANA CUT&RUN to validate H3P16oh antibodies, characterize its enrichment, and determine its function during hypoxia. Their experiments revealed substantial crosstalk between H3P16oh and H3K4me3, establishing H3P16oh as a hypoxia-sensitive regulator of gene expression and cell proliferation in breast cancer cell lines.

上榜理由：这篇文章为如何使用CUTANA CUT&RUN分析法来研究新的PTMs提供了重要思路。Li等人还展示了组蛋白PTMs如何调节疾病中的染色质结构和基因表达，为表观遗传学靶向药物开发提供了支持。

3. Mapping cis-regulatory elements in human neurons links psychiatric disease heritability and activity-regulated transcriptional programs

Sanchez-Priego et al. Cell Reports, 2022. PMID: 35649373

细胞类型： Excitatory glutamatergic neurons and inhibitory GABAergic neurons derived from human pluripotent stem cells, with and without stimulation (membrane depolarization)

目标蛋白：H3K27ac (0, 30, 90 min), FOS (0, 2 hr)

主要内容：Genetic risk variants for psychiatric diseases are concentrated in cis-regulatory DNA, suggesting roles in cell-type specific gene expression. However, due to the inherent challenges of studying human brain tissues, these genomic regions remain largely unexplored. In this paper, Sanchez-Priego et al. generated large amounts of excitatory and inhibitory neurons using human pluripotent stem cells. They profiled cells using a variety of techniques, including CUT&RUN, ATAC-seq, and RNA-seq, to identify putative cis-regulatory elements (i.e. enhancers) associated with activity-dependent gene expression. To support the relevance of their results to human disease, the authors compared the list of candidate enhancers to a large database of psychiatric-disease risk variants, which revealed significant links to schizophrenia, ADHD, and bipolar disorder.

上榜理由：Sanchez-Priego等人没有只针对个体变异进行研究，而是采取了整体法，他们使用多种技术，包括CUT&RUN, ATAC-seq和RNA-seq对细胞进行分析，定义疾病相关细胞类型中的表观基因组元素，并与不同数据库进行比较引用。

4. Acute depletion of human core nucleoporin reveals direct roles in transcription control but dispensability for 3D genome organization

Zhu et al. Cell Reports, 2022. PMID: 36323253

细胞类型：HCT116 cell line (all targets), HeLa cell line (NUP93 only)

目标蛋白：NUP93, NUP35, NUP205, BRD4, SEC13

主要内容：The nuclear pore complex acts as the gateway to the nucleus in eukaryotic cells and is composed of ~30 different nucleoporin proteins (NUPs). There is ample evidence that the nuclear pore complex helps regulate 3D chromatin organization and transcription. However, the functions of NUP subunits in human cells are not defined. Here, Zhu et al. studied NUPs using multiple techniques, including CUTANA CUT&RUN, Hi-C, PRO-seq, and CRISPR/dCas9 tethering. CUT&RUN showed that NUP proteins were generally bound to active chromatin regions. NUP93 specifically associated with promoters and enhancers and directly regulated transcription (similar to Brown et al. and Ibarra et al.). Strikingly, the authors found that core NUP proteins were not required for 3D chromatin architecture, in contrast to leading hypotheses in the field.

上榜理由：CUTANA CUT&RUN的一个关键优势是能够在自然条件下定位大分子复合物的亚基，而不需要交联。值得注意的是，在哺乳动物细胞中研究NUP蛋白和核孔复合物一直具有挑战性，而利用CUT&RUN分析技术更能够快速的助力这项研究。

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