Maize ear development determines the crop yield,and many important transcription factors(TFs)have been identified to function in this process.However,their transcriptional regulation mechanisms are still elusive.In th...Maize ear development determines the crop yield,and many important transcription factors(TFs)have been identified to function in this process.However,their transcriptional regulation mechanisms are still elusive.In this study,we generated the genome-wide DNA binding sites for 8 TFs which are known to function or highly expressed in inflorescence by applying the ts CUT&Tag method in maize leaf protoplast.We exposed a regulatory grid of 4 WUSCHEL-related homeobox(WOX)genes and revealed their potential regulatory mechanisms.In addition,a hierarchical regulation network for the determinacy and specification of maize inflorescence meristems were also constructed using the binding profiles of a floral development gene INDETERMINATE FLORAL APEX1(IFA1)and 3 MADS-box genes.Our study provides an in-depth understanding and new insights of the regulatory mechanisms during maize inflorescence development.展开更多
新兴的染色质靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用转座酶在目标蛋白结合的DNA附近进行切割并对切割下的DNA片段进行标签化,通过后续的二代测序可以快速鉴定蛋白质-DNA相互作用,极大的简化了染色质...新兴的染色质靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用转座酶在目标蛋白结合的DNA附近进行切割并对切割下的DNA片段进行标签化,通过后续的二代测序可以快速鉴定蛋白质-DNA相互作用,极大的简化了染色质免疫共沉淀测序(chromatin immunoprecipitation sequencing,ChIP-seq)的实验过程。CUT&Tag中转座酶完成标签化后需要DNA回收或其他后处理才能进行建库PCR,不同的回收方法对CUT&Tag结果有着显著的影响。通过建立生物素化转座体-链霉亲和素磁珠体系(streptavidin beads recovery CUT&Tag,srCUT&Tag),可以快速便捷地完成CUT&Tag的产物回收。本文在K562细胞中展开H3K4me3、RNA聚合酶Ⅱ(RNA polymeraseⅡ,RNAPⅡ)、转录因子CTCF和HMGA1的CUT&Tag实验,并利用现有的乙醇沉淀、片段分选(solid-phase reversible immobilization,SPRI)磁珠回收和直接PCR法,以及本研究建立的srCUT&Tag方法对产物进行回收。结果表明,从整体上看,SPRI磁珠回收和srCUT&Tag方法着较高的回收效率,而乙醇沉淀法则回收效率低下。在全部4种CUT&Tag产物回收过程中,SPRI磁珠回收均会损失大部分小于150 bp的产物片段。在CTCF和HMGA1 CUT&Tag产物的回收中,直接PCR法则损失了大部分大于300 bp的片段并与其他回收方法的结果有较大的差别。因此,srCUT&Tag能够比其他三种回收方法提供更多更完整的测序信息。综上所述,新建立srCUT&Tag回收方法相比现有的CUT&Tag产物回收方法能提高建库效率并得到更好的数据质量,为表观遗传学研究提供了更好的技术选择。展开更多
染色体靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用Tn5转座酶与Protein A/G的融合蛋白,引导Tn5酶至与靶蛋白结合的抗体附近,对靶蛋白结合的附近染色质区域进行切割,随后通过标签化处理对片段化染色质进行...染色体靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用Tn5转座酶与Protein A/G的融合蛋白,引导Tn5酶至与靶蛋白结合的抗体附近,对靶蛋白结合的附近染色质区域进行切割,随后通过标签化处理对片段化染色质进行文库制备,并利用高通量测序技术获取特定位点或蛋白质结合位置的染色质信息。CUT&Tag技术在蛋白质和DNA相互作用的研究领域起到了重大作用,不仅可以了解组蛋白修饰发生的位置,而且可以明确转录因子结合的区域。相较于传统的染色质免疫沉淀高通量测序(chromatin immunoprecipitation-sequencing,ChIP-seq)技术,CUT&Tag技术具有信噪比高、可重复性好、实验周期短、细胞投入量低等优点,在早期胚胎发育、干细胞、肿瘤以及表观遗传学等研究领域体现出巨大优势。本文将针对CUT&Tag在代谢组织细胞(以小鼠原代胰岛细胞为例)的具体操作步骤进行描述,以提供一种研究代谢细胞的表观遗传学方法。展开更多
Tn5是一种细菌转座子。经改造的Tn5能够高效地切割DNA,同时连接上特定的接头序列,因而广泛应用于高通量二代测序文库构建中。CUT&Tag(Cleavage Under Target&Tagmentation)是一种改进的研究蛋白质与DNA互作的技术,具有重复性好...Tn5是一种细菌转座子。经改造的Tn5能够高效地切割DNA,同时连接上特定的接头序列,因而广泛应用于高通量二代测序文库构建中。CUT&Tag(Cleavage Under Target&Tagmentation)是一种改进的研究蛋白质与DNA互作的技术,具有重复性好、信噪比高及操作简便等优点。该技术采用pA(Protein A)或pG(Protein G)与Tn5形成融合蛋白,定位于特定抗体(用于识别目标蛋白),利用Tn5的特性,在目标位点附近打断DNA的同时引入测序接头,随后提取DNA,再进行PCR扩增即可获得测序文库。但不同类型的抗体与pA或pG的亲和力不同,因此限制了部分抗体在CUT&Tag技术中的应用。为克服这一局限,该文构建了pG与Tn5的融合蛋白表达载体,通过原核表达及亲和纯化的方式获得pG-Tn5重组蛋白;并以RNA聚合酶Ⅱ(PolⅡ)特异性抗体PolⅡSer5P(小鼠IgG1型抗体和兔IgG型抗体)为例,在模式植物拟南芥(Arabidopsis thaliana)中评估pA-Tn5与pG-Tn5在不同类型抗体的CUT&Tag测序文库构建中的效果。结果表明,IgG1型抗体与p G-Tn5的亲和力更高,构建的文库质量更好,而IgG型抗体与2种酶的亲和力相当;同时,较低起始量的植物材料也能获得较好的效果,证明了CUT&Tag的应用优势。该研究优化了CUT&Tag技术,可为后续CUT&Tag实验中针对不同抗体时Tn5融合蛋白的选择提供参考。展开更多
Knowledge of the transcription factor binding landscape(TFBL)is necessary to analyze gene regulatory networks for important agronomic traits.However,a low-cost and high-throughput in vivo chromatin profiling method is...Knowledge of the transcription factor binding landscape(TFBL)is necessary to analyze gene regulatory networks for important agronomic traits.However,a low-cost and high-throughput in vivo chromatin profiling method is still lacking in plants.Here,we developed a transient and simplified cleavage under targets and tagmentation(tsCUT&Tag)that combines transient expression of transcription factor proteins in protoplasts with a simplified CUT&Tag without nucleus extraction.Our tsCUT&Tag method provided higher data quality and signal resolution with lower sequencing depth compared with traditional ChIP-seq.Furthermore,we developed a strategy combining tsCUT&Tag with machine learning,which has great potential for profiling the TFBL across plant development.展开更多
染色体免疫共沉淀测序(Chromatin immunoprecipitation followed by sequencing,ChIP-seq)是研究DNA-蛋白质互作的有力工具,被广泛用于RNA聚合酶、转录因子和组蛋白修饰等在基因组上的精确定位。近年来,在ChIP-seq技术的基础上,科学家...染色体免疫共沉淀测序(Chromatin immunoprecipitation followed by sequencing,ChIP-seq)是研究DNA-蛋白质互作的有力工具,被广泛用于RNA聚合酶、转录因子和组蛋白修饰等在基因组上的精确定位。近年来,在ChIP-seq技术的基础上,科学家提出了一系列研究DNA-蛋白质互作的技术方法,提高了测序分辨率,降低了实验成本,极大推动了表观基因组学的发展。本文综述了多种DNA-蛋白质互作研究技术的原理及其应用场景,介绍了在单细胞水平上研究DNA-蛋白质互作的实现方法,并展望其未来发展的方向。展开更多
基金financially supported by the National Natural Science Foundation of China(32172026)the 111 Project Crop Genomics and Molecular Breeding(B20051)。
文摘Maize ear development determines the crop yield,and many important transcription factors(TFs)have been identified to function in this process.However,their transcriptional regulation mechanisms are still elusive.In this study,we generated the genome-wide DNA binding sites for 8 TFs which are known to function or highly expressed in inflorescence by applying the ts CUT&Tag method in maize leaf protoplast.We exposed a regulatory grid of 4 WUSCHEL-related homeobox(WOX)genes and revealed their potential regulatory mechanisms.In addition,a hierarchical regulation network for the determinacy and specification of maize inflorescence meristems were also constructed using the binding profiles of a floral development gene INDETERMINATE FLORAL APEX1(IFA1)and 3 MADS-box genes.Our study provides an in-depth understanding and new insights of the regulatory mechanisms during maize inflorescence development.
文摘染色体靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用Tn5转座酶与Protein A/G的融合蛋白,引导Tn5酶至与靶蛋白结合的抗体附近,对靶蛋白结合的附近染色质区域进行切割,随后通过标签化处理对片段化染色质进行文库制备,并利用高通量测序技术获取特定位点或蛋白质结合位置的染色质信息。CUT&Tag技术在蛋白质和DNA相互作用的研究领域起到了重大作用,不仅可以了解组蛋白修饰发生的位置,而且可以明确转录因子结合的区域。相较于传统的染色质免疫沉淀高通量测序(chromatin immunoprecipitation-sequencing,ChIP-seq)技术,CUT&Tag技术具有信噪比高、可重复性好、实验周期短、细胞投入量低等优点,在早期胚胎发育、干细胞、肿瘤以及表观遗传学等研究领域体现出巨大优势。本文将针对CUT&Tag在代谢组织细胞(以小鼠原代胰岛细胞为例)的具体操作步骤进行描述,以提供一种研究代谢细胞的表观遗传学方法。
基金supported by the National Natural Science Foundation of China(31922068)the Fundamental Research Funds for the Central Universities(ZK202101)the China Postdoctoral Science Foundation(2019M662666)。
文摘Knowledge of the transcription factor binding landscape(TFBL)is necessary to analyze gene regulatory networks for important agronomic traits.However,a low-cost and high-throughput in vivo chromatin profiling method is still lacking in plants.Here,we developed a transient and simplified cleavage under targets and tagmentation(tsCUT&Tag)that combines transient expression of transcription factor proteins in protoplasts with a simplified CUT&Tag without nucleus extraction.Our tsCUT&Tag method provided higher data quality and signal resolution with lower sequencing depth compared with traditional ChIP-seq.Furthermore,we developed a strategy combining tsCUT&Tag with machine learning,which has great potential for profiling the TFBL across plant development.
文摘染色体免疫共沉淀测序(Chromatin immunoprecipitation followed by sequencing,ChIP-seq)是研究DNA-蛋白质互作的有力工具,被广泛用于RNA聚合酶、转录因子和组蛋白修饰等在基因组上的精确定位。近年来,在ChIP-seq技术的基础上,科学家提出了一系列研究DNA-蛋白质互作的技术方法,提高了测序分辨率,降低了实验成本,极大推动了表观基因组学的发展。本文综述了多种DNA-蛋白质互作研究技术的原理及其应用场景,介绍了在单细胞水平上研究DNA-蛋白质互作的实现方法,并展望其未来发展的方向。
