Estrogen Stimulates Histone Citrullination to Regulate Gene Expression in Somatolactotrope Cells

Presentation Number: OR17-4
Date of Presentation: April 2nd, 2017

Stanley B DeVore*1, Aaron Muth2, Paul R Thompson2, Anitha Sundararajan3, Faye D Schilkey3 and Brian D Cherrington1
1University of Wyoming, Laramie, WY, 2University of Massachusetts Medical School, Worcester, MA, 3National Center for Genome Resources (NCGR) and NM-INBRE, Santa Fe, NM

Abstract

Estrogen strongly stimulates gene expression in anterior pituitary lactotrope cells; however, the epigenetic mechanisms induced by estrogen are not well-defined. One candidate mechanism initiated by estrogen is the citrullination of histone tail arginine residues by the peptidylarginine deiminase (PAD) family of enzymes. Histone citrullination decondenses chromatin and promotes the recruitment of transcriptional machinery to modulate gene expression. Previous studies localized PAD expression in rodent lactotrope cells, although function was not investigated. This finding prompted us to hypothesize that PADs regulate gene expression in lactotrope cells. To test this, we used confocal microscopy to show that PAD2 and 4 expression in mouse lactotrope cells changes across the estrous cycle. Given the coincident expression of PADs with stages of high estrogen, we investigated if estrogen stimulates PAD expression. To assess this, we used the GH3 cell line, which was derived from a rat somatolactotrope tumor. GH3 cells were treated with vehicle or 10 nM 17-β estradiol (E2), and western blot analysis indicated that PAD2 and 4 protein levels increase significantly following 24 and 48 hours of treatment (P<0.05). We next examined if E2 stimulates PAD-catalyzed histone citrullination in GH3 cells. Following 1 hr of treatment with vehicle or 10 nM E2, GH3 histones were purified and probed by western blot with an antibody that specifically recognizes citrullinated arginine residues 2, 8, and 17 on the histone H3 tail (H3Cit2,8,17). Results showed that E2 significantly increases H3Cit2,8,17 levels compared to vehicle (P<0.001). To confirm specificity, GH3 cells were pre-treated for 2 hr with 1.25 μM PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA) before addition of vehicle or E2. Western blot analysis revealed that BB-ClA significantly decreases both basal and E2-stimulated H3Cit2,8,17 levels. Given this finding, we next used RNA sequencing as a non-biased approach to identify genes regulated by histone citrullination in GH3 cells. Target genes identified include c-Myc and prolactin, whose mRNA levels at 1 and 12 hr, respectively, decrease in the presence of BB-ClA (P<0.001). Since GH3 cells were derived from tumors, at issue is the role of histone citrullination in normal lactotrope function versus somatolactotrope tumors. To begin to address this question, we examined human somatolactotrope tumors by IHC and detected PAD2, PAD4, and H3Cit2,8,17. Collectively, our results suggest that estrogen stimulates the expression of PADs, which in turn regulate estrogen-dependent gene expression via histone citrullination. Although likely important in normal lactotrope function, dysregulation of PAD expression or activity may represent a potential mechanism regulating gene expression in somatolactotrope tumors.

 

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