CN111849918A - 一种研究鸡SSCs中受组蛋白甲基化调控的靶基因的方法 - Google Patents
一种研究鸡SSCs中受组蛋白甲基化调控的靶基因的方法 Download PDFInfo
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Abstract
一种鸡SSCs中受组蛋白甲基化调控的靶基因的方法，属于生物技术领域。该方法尝试将生物信息学和实验验证相结合，RNA‑seq和CHIP‑seq相结合，筛选出在鸡SSCs中受组蛋白甲基化调控的关键靶基因。采用本发明对组蛋白甲基化调控的靶基因进行的研究简单可靠，同时考虑到靶基因的保守性，为拓宽组蛋白甲基化在SSCs上的研究提供理论依据。
Description
技术领域
本发明涉及一种鸡SSCs中受组蛋白甲基化调控的靶基因的方法，属于生物技术领域。
背景技术
在小鼠中，已发现H3K4me3、H3K9me3和H3K27me3同时存在于SSCs中，但三者修饰具有特异的调控方式，H3K4me3激活基因表达，H3K9me3和H3K27me3则抑制基因表达；过表达H3K4脱甲基酶Lsd1,将破坏精子发生过程中组蛋白甲基化水平，影响后代健康。然而目前，对组蛋白甲基化在鸡精原干细胞中转录调控机制的研究还未有报道，许多未知的受组蛋白甲基化调控的靶基因还有待挖掘。
精子的质量和数量对人类和动物受精和胚胎发育至关重要。而精原干细胞是保证精子正常发生的基础。精原干细胞在生殖过程中不断自我更新，以备作为减数分裂分化为精子的原材料。鸡作为研究鸟类的模型，目前，仅有鸡上发现的FGF8、Wnt5a和C1EIS等基因促进胚胎干细胞向SSCs的分化研究，除基因表达的调控外，SSCs的增殖还受表观遗传的调控。然而目前，对组蛋白甲基化在鸡精原干细胞中转录调控机制的研究还未有报道。
发明内容
本发明的目的是针对上述现有技术的不足，提供一种鸡SSCs中受组蛋白甲基化调控的靶基因的方法，该方法尝试将生物信息学和实验验证相结合，RNA-seq和CHIP-seq相结合，筛选出在鸡SSCs中受组蛋白甲基化调控的关键靶基因。
本发明的技术方案如下：
一种鸡SSCs中受组蛋白甲基化调控的靶基因的方法，包括：
利用组蛋白甲基化酶Mll2和组蛋白去甲基化酶的Lsd1的干扰载体转染SSCs细胞，并以未处理组作为对照，进行RNA-seq测序；
通过然后利用生物信息学对干扰Mll2/Lsd1后出现的差异基因进行GO注释和KEGG富集分析；
联合组蛋白甲基化在SSCs中的CHP-seq数据和RNA-seq数据进行VENNY分析；
对筛选到的候选基因进行qRT-pCR检测候选基因在鸡组织中的表达以及在SSCs中干扰Mll2和Lsd1后的表达。将鸡睾丸中高表达，受Mll2/Lsd1调控的差异基因和人睾丸中高表达的基因联合分析，筛选出分别受组蛋白甲基化酶Mll2和组蛋白去甲基化酶Lsd1调控的关键靶基因。
采用本发明对组蛋白甲基化调控的靶基因进行的研究简单可靠，同时考虑到靶基因的保守性，为拓宽组蛋白甲基化在SSCs上的研究提供理论依据。
本发明的有益效果如下：
本发明的方法简单可行，流程清晰，逻辑清楚，操作可行性高，在普通实验室就能完成。通过对测序结果的联合分析、验证，筛选出受组蛋白甲基化调控的靶基因，实验者可将其应用于小鼠的研究中，为研究哺乳动物组蛋白甲基化调控精原干细胞形成、分化提供参考，应用广泛。
具体实施方式
慢病毒干扰载体感染鸡精原干细胞
用慢病毒shRNA-Mll2和shRNA-Lsd1感染鸡精原干细胞(MOI=15)，病毒液中添加6 µg/mL polybrene。感染48h后，吸除含慢病毒的培养基，换为新鲜的精原干细胞培养基。继续培养24~48h后，收集RNA样品。将三次独立试验的RNA样品混合后，送至北京诺禾致源科技股份有限公司进行转录本分析，使用Agilent 2100 bioanalyzer对纯化后的细胞总RNA进行质量检测和测序。
干扰Mll2和Lsd1后测序结果的主成分分析和热图分析
对测序分析得到的数据进行主成分分析，发现干扰Mll2/Lsd1后，基因表达谱出现差异。热图分析发现，干扰Mll2/Lsd1，存在大量差异基因。以上结果说明Mll2/Lsd1对SSCs形成的调控作用，可能与基因表达变化有关。对测序结果进行分析，相比Blank，干扰Mll2后，共出现17431个差异基因，根据log2（Fold Change）>1或<-1，P <0.05进行筛选，发现共456个差异显著的基因，159个基因下调，包括97个已知基因和62个新基因；297个基因上调，包括214个已知基因和83个新基因。干扰Lsd1后，共出现17335个差异基因，其中含有395个差异显著的基因，其中212个基因上调，包括121个已知基因和91个新基因；183个下调，包括65个已知基因和118个新基因。
干扰Mll2/Lsd1后，差异基因的筛选
本研究分别对干扰MLL2和LSD1后进行差异基因分析。首先分析空白组和Mll2/Lsd1干扰组差异基因的相关性。接着对干扰Mll2后下调的基因/干扰Lsd1上调的基因进行GO注释和KEGG分析，筛选出与细胞分化、增殖、发育等过程的差异基因。热图分析显示不同处理中差异基因的表达。然后结合前期CHIP-seq在SSCs上的H3K4me2富集差异基因进行VENNY分析，筛选出既受Mll2/Lsd1调控又存在H3K4me2富集的差异基因。这里，以空白组和Mll2干扰组为例进行分析如下：
相关性分析结果显示，Blank和干扰Mll2后的基因表达呈高度相关（R=0.9747）。对干扰Mll2后下调的基因进行GO注释发现，基因富集于negative regulation of cell growth，regulation of MAPK cascade，steroid hydroxylase activity等生物学过程。BP、CC、MF中12条与细胞分化、增殖、发育等过程相关的条目含72个差异基因，包括TGFB2、WNT11、KIT、JAK2基因，热图分析显示其在不同处理中的表达。结合前期CHIP-seq分析的差异富集基因进行venny分析，发现17个基因存在H3K4me2的富集且受Mll2的调控。结合CHIP-seq和RNA-seq分析，筛选出7个基因，其中Kit和Jak2为H3K4me2富集的基因，同时受Mll2调节；Mmp2、Sycp3、Tdrd5、Pank2和Piwil1与精子发生相关。以上结果初步说明Mll2可能通过提高H3K4me2促进SSCs自我更新和分化相关基因的表达。
和联合分析确定受H3K4甲基化调控的关键靶基因
利用qRT-PCR检测候选关键基因在鸡不同组织（心、肝、脾、肺、肾、睾丸、卵巢）中的表达，筛选出在睾丸中高表达的差异基因，说明其具有组织特异性。接着在SSCs中干扰Mll2/Lsd1，利用qRT-PCR检测差异基因在不同处理中的表达。将得到鸡睾丸中高表达的差异基因，受Mll2/Lsd1调控的差异基因以及人睾丸中高表达的差异基因进行联合分析，筛选出受Mll2/Lsd1调控的且存在H3K4me2富集的关键靶基因。以空白组和Mll2干扰组为例进行分析如下：
qRT-PCR检测发现受Mll2调控的7个差异基因中Sycp3、Tdrd5、Piwil1在睾丸中高表达；干扰Mll2后，抑制Jak2、Sycp3、Tdrd5、Pank2和Piwil1的表达；结合鸡睾丸中、人睾丸中高表达和受Mll2/Lsd1调控的差异基因联合分析发现，Sycp3、Tdrd5和Piwil1是受Mll2调控的SSCs关键基因。
Claims (4)
1.一种研究鸡SSCs中受组蛋白甲基化调控的靶基因的方法，其特征是，包括以下步骤：
1）慢病毒干扰载体感染鸡精原干细胞
利用组蛋白甲基化酶Mll2和组蛋白去甲基化酶的Lsd1的干扰载体转染SSCs细胞，并以未处理组作为对照，进行RNA-seq测序；
2）干扰Mll2和Lsd1后测序结果分析
首先对测序结果进行主成分分析和差异基因表达的热图分析，然后利用生物信息学对干扰Mll2/Lsd1后出现的差异基因进行GO注释和KEGG富集分析；
3）干扰Mll2/Lsd1后，差异基因的筛选
联合SSCs中组蛋白甲基化的CHP-seq数据和干扰Mll2/Lsd1后的RNA-seq数据进行VENNY分析；
4）qRT-PCR和联合分析确定受H3K4甲基化调控的关键靶基因
对筛选到的候选基因进行qRT-pCR检测候选基因在鸡组织中的表达以及在SSCs中干扰Mll2和Lsd1后的表达；将鸡睾丸中高表达，受Mll2/Lsd1调控的差异基因和人睾丸中高表达的基因联合分析，筛选出分别受组蛋白甲基化酶Mll2和组蛋白去甲基化酶Lsd1调控的关键靶基因。
2.根据权利要求1所述的一种研究鸡SSCs中受组蛋白甲基化调控的靶基因的方法，其特征是，步骤1）中，用慢病毒shRNA-Mll2和shRNA-Lsd1感染鸡精原干细胞，病毒液中添加6µg/mL polybrene；感染48h后，吸除含慢病毒的培养基，换为新鲜的精原干细胞培养基；继续培养24~48h后，收集RNA样品；将三次独立试验的RNA样品混合后，对纯化后的细胞总RNA进行质量检测和测序。
3.根据权利要求2所述的一种研究鸡SSCs中受组蛋白甲基化调控的靶基因的方法，其特征是，步骤2）中，对测序分析得到的数据进行主成分分析，发现干扰Mll2/Lsd1后，基因表达谱出现差异；热图分析发现，干扰Mll2/Lsd1，存在大量差异基因。
4.根据权利要求3所述的一种研究鸡SSCs中受组蛋白甲基化调控的靶基因的方法，其特征是，步骤3）中，分别对干扰MLL2和LSD1后进行差异基因分析；首先分析空白组和Mll2/Lsd1干扰组差异基因的相关性；对干扰Mll2后下调的基因/干扰Lsd1上调的基因进行GO注释和KEGG分析，筛选出与细胞分化、增殖、发育过程的差异基因；热图分析显示不同处理中差异基因的表达；然后结合前期CHIP-seq在SSCs上的H3K4me2富集差异基因进行VENNY分析，筛选出既受Mll2/Lsd1调控又存在H3K4me2富集的差异基因。
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