Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone [ATAC-Seq]

The LβT2 mouse pituitary cell line has many characteristics of a mature gonadotrope and is a widely used model system for studying developmental processes and the response to gonadotropin-releasing hormone (GnRH). The global epigenetic landscape, which contributes to cell-specific gene regulatory mechanisms, and the single-cell transcriptome response variation of LβT2 cells have not been previously investigated. Here, we integrate the transcriptome and genome-wide chromatin accessibility state of LβT2 cells during GnRH stimulation. Additionally, we examine cell-to-cell variability in the transcriptional response to GnRH using gel-in-emulsion Drop-seq technology. Analysis of a bulk RNA-seq dataset obtained 45 minutes after exposure to either GnRH or vehicle identified 112 transcripts that were regulated > 4-fold by GnRH (FDR < 0.05). The top regulated transcripts constitute, as determined by Bayesian massive public data integration analysis, a human pituitary-relevant coordinated gene program. Chromatin accessibility (ATAC-seq) datasets generated from GnRH-treated LβT2 cells identified more than 58,000 open chromatin regions, some containing notches consistent with bound transcription factor footprints. Study of the most prominent open regions showed that 75% were in transcriptionally active promoters or introns, supporting their involvement in active transcription. Lhb, Cga, and Egr1 showed significantly open chromatin over their promoters. While Fshb was closed over its promoter, several discrete significantly open regions were found at -40 to -90 kb, which may represent novel upstream enhancers. Chromatin accessibility determined by ATAC-seq was associated with high levels of gene expression determined by RNA-seq. We obtained high quality single-cell gel-in-emulsion Drop-seq transcriptome data, with an average of >4000 expressed genes/cell, from 1992 vehicle- and 1889 GnRH-treated cells. While the individual cell expression patterns showed high cell-to-cell variation, representing both biological and measurement variation, the average expression patterns correlated well with bulk RNA-seq data. Computational assignment of each cell to its precise cell cycle phase showed that the response to GnRH was unaffected by cell cycle. To our knowledge, this study represents the first genome-wide epigenetic and single-cell transcriptomic characterization of this important gonadotrope model. The data have been deposited publicly and should provide a resource for hypothesis generation and further study. ATAC-seq was performed on two replicate samples of LβT2 cells treated with 2 nM GnRH pulses every 2 h for a duration of 6 h and 45 min (4 pulses in total; cells harvested 45 min after last pulse) in a high throughput GnRH pulse system.

LβT2小鼠垂体细胞系具备成熟促性腺激素细胞的诸多特征，是研究发育进程及促性腺激素释放激素（gonadotropin-releasing hormone, GnRH）应答反应的经典模型体系。此前尚未有研究针对LβT2细胞的全基因组表观遗传调控景观（该景观是细胞特异性基因调控机制的基础）以及单细胞转录组应答异质性展开探索。本研究整合了GnRH刺激下LβT2细胞的转录组与全基因组染色质开放状态数据；同时利用凝胶微滴Drop-seq（gel-in-emulsion Drop-seq）技术，解析了细胞间GnRH转录应答的异质性。对经GnRH或溶剂对照处理45分钟后获取的批量RNA测序（bulk RNA-seq）数据集进行分析，共鉴定出112个受GnRH调控幅度超过4倍的转录本（错误发现率（FDR）<0.05）。经贝叶斯大规模公共数据整合分析证实，显著调控的核心转录本共同构成了与人垂体相关的协同基因调控程序。对GnRH处理后的LβT2细胞生成的转座酶可及性染色质测序（ATAC-seq）数据集进行分析，共鉴定出超过58000个染色质开放区域，其中部分区域存在与结合型转录因子足迹一致的缺口特征。对最显著的染色质开放区域的分析显示，其中75%位于转录活跃的启动子或内含子区域，佐证了其在活跃转录过程中的调控作用。Lhb、Cga及Egr1基因的启动子区域呈现显著开放的染色质状态；而Fshb基因的启动子区域处于闭合状态，但在其上游-40kb至-90kb区域内存在数个离散的显著染色质开放区域，这可能代表全新的上游增强子。转座酶可及性染色质测序（ATAC-seq）检测到的染色质开放状态与RNA-seq测得的高基因表达水平呈显著关联。本研究从1992个溶剂对照处理细胞及1889个GnRH处理细胞中获取了高质量的凝胶微滴Drop-seq单细胞转录组数据，平均每个细胞可检测到超过4000个表达基因。尽管单个细胞的表达模式存在较高的细胞间异质性（涵盖生物学变异与检测技术变异），但群体平均表达模式与批量RNA测序（bulk RNA-seq）数据具有良好的相关性。通过计算方法将每个细胞精准分配至相应的细胞周期时相后发现，GnRH的应答反应不受细胞周期状态的影响。据我们所知，本研究是首次针对这一重要促性腺激素细胞模型开展全基因组表观遗传与单细胞转录组学表征的研究。本研究产生的数据已公开存储，可为后续假说构建与相关研究提供宝贵的资源。本研究在高通量GnRH脉冲处理体系中，对两组重复的LβT2细胞样本进行了转座酶可及性染色质测序（ATAC-seq）检测：该样本以每2小时一次的频率施加2nM GnRH脉冲，持续处理6小时45分钟（共计4次脉冲，于末次脉冲后45分钟收集细胞）。