自转录活性调节区测序(self-transcribing active regulatory region sequencing,STARR-seq)是一种可发现并同时验证全基因组增强子活性的高通量测序方法。其原理为:将待验证序列插入质粒载体并电转入细胞中,该序列在作为增强子提高靶...自转录活性调节区测序(self-transcribing active regulatory region sequencing,STARR-seq)是一种可发现并同时验证全基因组增强子活性的高通量测序方法。其原理为:将待验证序列插入质粒载体并电转入细胞中,该序列在作为增强子提高靶基因转录的同时,其本身也作为靶基因被增强转录。通过对转录组进行测序,并对比未插入片段的测序结果,可获得增强子在基因组位置及活性的信息。在传统增强子研究方法中,通过对染色质开放区域和转录活性区域进行测序以预测增强子,但只能逐一验证预测结果,无法高通量验证增强子活性。STARR-seq技术解决了上述缺陷,可在对全基因组增强子高通量挖掘的同时,对其活性进行可靠的验证。自STARR-seq技术发明以来,已被广泛运用于不同物种与细胞中的增强子发现及活性验证研究。本文对传统增强子预测方法以及STARR-seq技术的基本原理、发展历史和具体运用进行了介绍,并对其发展前景进行展望,以期为后续增强子相关领域研究人员提供参考。展开更多
Background:The pig is an economically important livestock species and is a widely applied large animal model in medical research.Enhancers are critical regulatory elements that have fundamental functions in evolution,...Background:The pig is an economically important livestock species and is a widely applied large animal model in medical research.Enhancers are critical regulatory elements that have fundamental functions in evolution,development and disease.Genome-wide quantification of functional enhancers in the pig is needed.Results:We performed self-transcribing active regulatory region sequencing(STARR-seq)in the porcine kidney epithelial PK15 and testicular ST cell lines,and reliably identified 2576 functional enhancers.Most of these enhancers were located in repetitive sequences and were enriched within silent and lowly expressed genes.Enhancers poorly overlapped with chromatin accessibility regions and were highly enriched in chromatin with the repressive histone modification H3K9me3,which is different from predicted pig enhancers detected using ChIP-seq for H3K27ac or/and H3K4me1 modified histones.This suggests that most pig enhancers identified with STARR-seq are endogenously repressed at the chromatin level and may function during cell type-specific development or at specific developmental stages.Additionally,the PPP3CA gene is associated with the loin muscle area trait and the QKI gene is associated with alkaline phosphatase activity that may be regulated by distal functional enhancers.Conclusions:In summary,we generated the first functional enhancer map in PK15 and ST cells for the pig genome and highlight its potential roles in pig breeding.展开更多
Abscisic acid(ABA)-responsive elements(ABREs)are the major cis-regulatory elements in ABA-induced gene expression.However,the impact of sequence variations on ABRE function is not yet well-understood.Here,we used synt...Abscisic acid(ABA)-responsive elements(ABREs)are the major cis-regulatory elements in ABA-induced gene expression.However,the impact of sequence variations on ABRE function is not yet well-understood.Here,we used synthetic STARR-seq to quantitatively assess the effects of singlenucleotide substitutions on ABRE activity.Our results revealed that the nucleotide substitutions in both the ACGT-core and ACGT-flank regions affected transcriptional strength.Alterations in the ACGT-core sequence had a more significant impact on ABRE activity than changes in the flanking region.Interestingly,we observed that the ACGT-flank variants with high activity exhibited a strong sequence preference in the downstream region,whereas the highly active core variants were diverse in sequence patterns.Our studies provide a quantitative map of ABRE activity at single-nucleotide resolution,which will facilitate the design of ABA-responsive promoters with desired activities in plants.展开更多
文摘自转录活性调节区测序(self-transcribing active regulatory region sequencing,STARR-seq)是一种可发现并同时验证全基因组增强子活性的高通量测序方法。其原理为:将待验证序列插入质粒载体并电转入细胞中,该序列在作为增强子提高靶基因转录的同时,其本身也作为靶基因被增强转录。通过对转录组进行测序,并对比未插入片段的测序结果,可获得增强子在基因组位置及活性的信息。在传统增强子研究方法中,通过对染色质开放区域和转录活性区域进行测序以预测增强子,但只能逐一验证预测结果,无法高通量验证增强子活性。STARR-seq技术解决了上述缺陷,可在对全基因组增强子高通量挖掘的同时,对其活性进行可靠的验证。自STARR-seq技术发明以来,已被广泛运用于不同物种与细胞中的增强子发现及活性验证研究。本文对传统增强子预测方法以及STARR-seq技术的基本原理、发展历史和具体运用进行了介绍,并对其发展前景进行展望,以期为后续增强子相关领域研究人员提供参考。
基金supported by the National Natural Science Foundation of China(32100502)the Ministry of Agriculture of China(2016ZX08009003-006)Science&Technology Department of Yunnan Province(202102AE090039).
文摘Background:The pig is an economically important livestock species and is a widely applied large animal model in medical research.Enhancers are critical regulatory elements that have fundamental functions in evolution,development and disease.Genome-wide quantification of functional enhancers in the pig is needed.Results:We performed self-transcribing active regulatory region sequencing(STARR-seq)in the porcine kidney epithelial PK15 and testicular ST cell lines,and reliably identified 2576 functional enhancers.Most of these enhancers were located in repetitive sequences and were enriched within silent and lowly expressed genes.Enhancers poorly overlapped with chromatin accessibility regions and were highly enriched in chromatin with the repressive histone modification H3K9me3,which is different from predicted pig enhancers detected using ChIP-seq for H3K27ac or/and H3K4me1 modified histones.This suggests that most pig enhancers identified with STARR-seq are endogenously repressed at the chromatin level and may function during cell type-specific development or at specific developmental stages.Additionally,the PPP3CA gene is associated with the loin muscle area trait and the QKI gene is associated with alkaline phosphatase activity that may be regulated by distal functional enhancers.Conclusions:In summary,we generated the first functional enhancer map in PK15 and ST cells for the pig genome and highlight its potential roles in pig breeding.
基金National Natural Science Foundation of China,Grant/Award Number:32270686。
文摘Abscisic acid(ABA)-responsive elements(ABREs)are the major cis-regulatory elements in ABA-induced gene expression.However,the impact of sequence variations on ABRE function is not yet well-understood.Here,we used synthetic STARR-seq to quantitatively assess the effects of singlenucleotide substitutions on ABRE activity.Our results revealed that the nucleotide substitutions in both the ACGT-core and ACGT-flank regions affected transcriptional strength.Alterations in the ACGT-core sequence had a more significant impact on ABRE activity than changes in the flanking region.Interestingly,we observed that the ACGT-flank variants with high activity exhibited a strong sequence preference in the downstream region,whereas the highly active core variants were diverse in sequence patterns.Our studies provide a quantitative map of ABRE activity at single-nucleotide resolution,which will facilitate the design of ABA-responsive promoters with desired activities in plants.
