Regulatory Architecture of the LβT2 Gonadotrope Cell Underlying the Response to Gonadotropin-Releasing Hormone

Abstract

The LβT2 mouse pituitary cell line has many characteristics of a mature gonadotrope and is a widely used model system for studying the 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. In addition, we examine cell-to-cell variability in the transcriptional response to GnRH using Gel bead-in-Emulsion Drop-seq technology. Analysis of a bulk RNA-seq data set obtained 45 min 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 [assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq)] data sets generated from GnRH-treated LβT2 cells identified more than 58,000 open chromatin regions, some containing notches consistent with bound transcription factor footprints. The 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 bead-in-Emulsion Drop-seq transcriptome data, with an average of >4,000 expressed genes/cell, from 1,992 vehicle- and 1,889 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.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity

“…We assessed the use of this protocol for the analysis of cell-to-cell variability in individual cycling cells using gel-in-emulsion (GEM) Drop-seq ( 24 ). We recently used GEM Drop-seq in fresh samples of LβT2 cells to determine the SC cell cycle stage assignment and the interaction of cell cycle with the effects of GnRH ( 14 ). We reproduced this experiment using cells preserved with RNA-Best.…”

“…The recent development of SC transcriptomics has enabled researchers to explore cell-to-cell variability and grasp the complex dynamics of gene regulation. Our recent SC transcriptome analysis of gonadotrope cells exposed to static GnRH stimulation highlighted cellular heterogeneity in the response to GnRH and the absence of an effect of cell cycle on this response ( 14 ). However, SC sequencing approaches are limited in measurement accuracy due to statistical limitations on measuring rare transcripts.…”

Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines

“…In addition, our reexamination of previous genome-wide transcriptome data in L β T2 cells [32] showed the absence of chromosome Y‒expressed genes compared with male pituitaries. Further, our reanalysis of previous genome-wide chromatin accessibility data [30] provided evidence for the absence of the Y chromosome and the presence of two X chromosomes in comparison with autosome coverage. Therefore, three independent analyses converged toward a female origin for L β T2 cells.…”

“…Quantitative real-time PCR experiments were performed as previously described [30]. Following total RNA isolation, 1 μg of RNA was reverse-transcribed with the Affinity Script reverse-transcriptase (Agilent).…”

GLUT1-mediated glycolysis supports GnRH-induced secretion of luteinizing hormone from female gonadotropes

“…To resolve the contribution of glucose availability versus glucose uptake, we leveraged the intrinsic heterogeneity of the LβT2 cell line 36 . Based on data showing that LH pulses are correlated with glycolytic bursts (Fig.…”