Project description: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.

Project description:Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus. GnRH regulates follicle-stimulating hormone b-subunit (FSHb) gene expression in pituitary gonadotropes in a frequency-sensitive manner that is central to reproductive physiology. The mechanisms underlying the preferential and paradoxical induction of FSHb by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a novel autocrine inducer of FSHb gene expression. GDF9 gene expression was preferentially suppressed by high frequency GnRH pulses due to reduced transcription. Exogenous GDF9 induced FSHb mRNA expression and knockdown or immunoneutralization of GDF9 reduced FSHb gene expression. Treatment with GDF9 stimulated Smad2/3 phosphorylation. The activin receptor-like kinase (ALK) receptor inhibitor SB-505124 antagonized GDF9-induced Smad2/3 phosphorylation and FSHb mRNA induction. Smad2 and Smad3 knockdown studies indicated that the induction of FSHb by GDF9 involves both Smad2 and Smad3. GDF9 and GnRH synergistically induced FSHb mRNA expression and high frequency GnRH pulses suppressed GDF9. We hypothesized that GDF9 contributes to a regulatory loop that tunes the GnRH frequency-response characteristics of the FSHb gene. To test this, we determined the effects of GDF9 knockdown on FSHb induction at different GnRH pulse frequencies using a parallel perifusion system. Reduction of GDF9 shifted the characteristic pattern of GnRH pulse frequency sensitivity. These results identify GDF9 as contributing to an incoherent feed-forward loop, comprised of both intracellular and secreted elementscomponents, that regulates FSHb expression in response to activation of the cell surface GnRH receptor. LbT2 microarray datasets as well as RNA-Seq data (GSE42120) were interrogated to determine the levels of expression of ALK4/5/7. LM-NM-2T2 cells were transfected with either scrambled siRNA or Gas siRNA for 48 h. RNA samples were snap-frozen in dry ice prior to whole-genome expression profiling analysis using MouseWG-6 v2.0 Expression BeadChip (Illumina, San Diego, CA). A total of 6 concentrated conditioned media samples were independently prepared: 3 replicates from control siRNA-treated cells, and 3 replicates from Gas siRNA-treated cells. This submission represents the microarray component of study.

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Regulation of murine follicle-stimulating hormone β subunit transcription by newly identified enhancers [ChIP-seq; LβT2 cells]

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