JoVE Journal > Genetics
Summary
Abstract
Introduction
Protocol
Results
Discussion
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Acknowledgements
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유전학

果蝇细胞中的核内高C

Published: September 15, 2021
doi:
10.3791/62106
, , ,
1Departamento de Genética Molecular, Instituto de Fisiología Celular,Universidad Nacional Autónoma de México

Summary

基因组在核空间中组织成不同的结构，可以通过染色体构象捕获技术揭示出来。核内Hi-C方法提供了在果蝇细胞系中全基因组的染色质相互作用集合，生成接触图，可以在限制片段水平下以兆基分辨率进行探索。

Abstract

基因组被组织成拓扑关联域（TADs），由隔离域之间相互作用的边界划分。在德罗索菲拉，TAD形成和边界背后的机制仍在调查中。此处描述的核内 Hi-C 方法的应用有助于解剖 TAD 边界下隔离 诺奇 基因的建筑蛋白 （AP） 结合位点的功能。导致 APs 损失的域界的基因改造会导致 TAD 融合、转录缺陷和远程拓扑变化。这些结果提供了证据，证明遗传元素对果蝇的域界形成和基因表达控制的贡献。在这里，详细描述了核内Hi-C方法，它提供了重要的检查点，以评估实验的质量以及协议。还显示所需的测序读数和有效的 Hi-C 对，以分析不同基因组尺度的基因组相互作用。CRISPR/Cas9介质的遗传编辑调节元素和高分辨率的基因组相互作用分析使用这种核内Hi-C协议可以是一个强大的组合，研究遗传元素的结构功能。

Introduction

在真核生物中，基因组被分割成染色体，在相间第1阶段占据核空间的特定区域。形成染色体的染色质可分为两种主要状态：一种是转录允许的可访问染色质，另一种是转录抑制的紧凑型染色质。这些染色质状态在核空间中分离和很少混合，在核2中形成两个不同的隔间。在亚兆基尺度上，边界分隔高频染色质相互作用的领域，称为TAD，标记染色体组织3，4，5。在哺乳动物中，TAD边界被凝聚力和CCTC结合因子（CTCF）6、7、8所占据。凝固素复合物挤出色度素，并停止在CTCF结合点，在基因组序列的收敛方向处置，形成稳定的染色质循环9，10，13，14。CTCF DNA结合部位点在CTCF和凝聚蛋白丰度的边界上发生基因破坏或减少，导致调控元素之间的异常相互作用、TAD形成的丧失和基因表达放松管制9、10、11、13、14。

在德罗索菲拉， TAD 之间的边界由多个 AP 占据，包括边界元素相关因子 32 kDa （BEAF-32）、Motif 1 结合蛋白 （M1BP）、中生代蛋白 190 （CP190）、毛翼抑制剂 （SuHW） 和 CTCF，并富含活性组蛋白修饰和聚合酶 II 16、17、18。有人建议，在德罗索菲拉，TADs出现的结果是转录13，17，19，和独立的AP在边界形成和绝缘属性的确切作用仍在调查之中。因此，德罗索菲拉的域名是否是类似转录状态区域聚合的唯一结果，或者包括反恐委员会在内的 AP 是否有助于边界形成，仍有待充分说明。通过开发染色体构象捕获技术以及下一代测序，可以探索高分辨率基因组接触。Hi-C 协议首先描述为”解决方案“2的粘合步骤，以避免染色质片段之间的虚假粘合产品。然而，一些研究指出，数据中的有用信号来自在部分赖化核形成的结结产品，这些结核不在解决方案20、21中。

然后，作为单细胞Hi-C实验22的一部分，对协议进行了修改，以在细胞核内进行粘合。核内Hi-C协议随后被纳入细胞群Hi-C，在全基因组距离内产生更一致的覆盖，并产生技术噪声较少的数据23，24。该协议，在这里详细描述，是基于人口在细胞核Hi-C协议23，24，并用于调查遗传去除DNA结合图案的CTCF和M1BP的后果，从领域边界在D罗索菲拉25。结果表明，改变边界上对 APs 的 DNA 结合图案对 Notch 域的形成、Notch点周围区域更大的拓扑缺陷以及基因表达放松管制有严重的影响。这表明，域界的遗传元素对于维持25号果蝇的基因组拓扑学和基因表达非常重要。

Protocol

1. 固定 从 1000 万施耐德的 2 号线加 （S2R+） 细胞开始，在施耐德介质中准备 17.5 mL 的细胞悬浮，其中含有 10% 的胎儿牛血清 （FBS） 在室温下 （RT）。 加入无甲醇甲醛，最终浓度为2%。混合和孵育10分钟在RT，小心混合每分钟。注：甲醛是一种危险化学品。遵守相应的健康和安全法规，并在烟罩中工作。 通过添加甘氨酸来抑制反应，实现最终浓度为 0.125 M 和混合。在 Rt 孵?…

Representative Results

下面描述的是一个成功的高C协议的结果（见图1A中的高C协议工作流程摘要）。在核中Hi-C实验期间，有几个质量控制检查点。样品在 （UD） 和 （D） 染色素限制步骤之前和之后以及连合 （L） 之后收集。十字线被逆转，DNA被净化，运行在阿加罗斯凝胶上。当与Mbo I的限制成功时，观察到200-1000个基点的涂片（图1B）。分子的预期大小取决于选择的限制酶?…

Discussion

这里介绍的核内Hi-C方法允许以高分辨率详细探索果蝇基因组拓扑学，提供不同基因组尺度的基因组相互作用的视图，从促进剂和增强剂等调控元素之间的染色质循环到TAD和大隔间识别25。同样的技术也有效地应用于哺乳动物组织与一些修改33。例如，当处理组织而不是单细胞悬浮时，组织通过 70 μm 过滤器进行筛选，裂解步骤在使用 Dounce 同质化器对材料进行均?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了联阿特派团技术创新和研究支助方案（PAPIIT）赠款编号为207319和科学技术国家委员会（CONACyT-FORDECyT）赠款编号303068的支持。A.E.-L.是国家科学技术委员会（CONACyT）CVU号码968128支持的硕士生。

Materials

16% (vol/vol) paraformaldehyde solution Agar Scientific R1026
Biotin-14-dATP Invitrogen CA1524-016
ClaI enzyme NEB R0197S
COVARIS Ultrasonicator Covaris LE220-M220
Cut Smart NEB B72002S
Dulbecco's Modified Eagle Medium (DMEM) 1x Life Technologies 41965-039
Dynabeads MyOne Streptabidin C1 Invitrogen 65002
Fetal bovine serum (FBS) sterile filtered Sigma F9665
Klenow Dna PolI large fragment NEB M0210L
Klenow exo(-) NEB M0210S
Ligation Buffer NEB B020S
MboI enzyme NEB R0147M
NP40-Igepal SIGMA CA-420 Non-ionic surfactant for addition in lysis buffer
PE adapter 1.0 Illumina 5'-P-GATCGGAAGAGCGGTTCAGCAG
GAATGCCGAG-3'
PE adapter 2.0 Illumina 5'-ACACTCTTTCCCTACACGACGCT
CTTCCGATCT-3'
PE PCR primer 1.0 Illumina 5'-AATGATACGGCGACCACCGAGAT
CTACACTCTTTCCCTACACGACG
CTCTTCCGATCT-3'
PE PCR primer 2.0 Illumina 5'-CAAGCAGAAGACGGCATACGAG
ATCGGTCTCGGCATTCCTGCTGA
ACCGCTCTTCCGATCT-3'
Phenol: Chloroform:Isoamyl Alcohol 25:24:1 SIGMA P2069
Primer 1 (known interaction, Figure 2A) Sigma 5'-TCGCGGTAATTTTGCGTTTGA-3'
Primer 2 (known interactions, Figure 2A) Sigma 5'-CCTCCCTGCCAAAACGTTTT-3'
Protease inhibitor cocktail tablet Roche 4693132001
Proteinase K Roche 3115879001
Qubit ThermoFisher Q33327
RNAse Roche 10109142001
SPRI Beads Beckman B23318
T4 DNA ligase Invitrogen 15224-025
T4 DNA polymerase NEB M0203S
T4 polynucleotide kinase (PNK)  NEB M0201L
TaqPhusion NEB M0530S DNA polymerase
Triton X-100 Non-ionic surfactant for quenching of SDS
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Tags

Hi-CChromatin ConformationGenome OrganizationChromosomal InteractionsDrosophila CellsNuclear ClumpsFormaldehyde CrosslinkingGlycine QuenchingCell LysisRestriction Enzyme DigestionSDS PermeabilizationTriton TreatmentQuality ControlSequencing

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Esquivel-López, A., Arzate-Mejía, R., Pérez-Molina, R., Furlan-Magaril, M. In-Nucleus Hi-C in Drosophila Cells. J. Vis. Exp. (175), e62106, doi:10.3791/62106 (2021).
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