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Changes in Gene Expression of Histone Modification Enzymes in Rat Maekawa et al. Journal of Ovarian Research (2016) 9:15 DOI 10.1186/s13048-016-0225-z RESEARCH Open Access Changes in gene expression of histone modification enzymes in rat granulosa cells undergoing luteinization during ovulation Ryo Maekawa, Lifa Lee, Maki Okada, Hiromi Asada, Masahiro Shinagawa, Isao Tamura, Shun Sato, Hiroshi Tamura and Norihiro Sugino* Abstract Background: The ovulatory LH surge rapidly alters the expression of steroidogenesis-related genes such as steroidogenic acute regulatory protein (StAR) in granulosa cells (GCs) undergoing luteinization. We recently reported that histone modifications contribute to these changes. Histone modifications are regulated by a variety of histone modification enzymes. This study investigated the changes in gene expression of histone modification enzymes in rat GCs undergoing luteinization after the induction of ovulation. The extracellular regulated kinase (ERK)-1/2 is a mediator in the intracellular signaling pathway stimulated by the ovulatory LH surge and regulates the expression of a number of genes in GCs. We further investigated whether ERK-1/2 is involved in the regulation of the histone modification at the StAR promoter region in GCs undergoing luteinization. Results: GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h) CG injection. The expressions of 84 genes regulating histone modifications or DNA methylation were measured using a PCR array. Five genes (HDAC4, HDAC10, EZH2, SETDB2, and CIITA) were identified as histone acetylation- or histone methylation-related genes, and were significantly altered after hCG injection. None of the genes were related to DNA methylation. mRNA levels of EZH2, SETDB2, HDAC4,andHDAC10 decreased and CIITA mRNA levels increased 4 or 12 h after hCG injection. GCs isolated after eCG injection were incubated with hCG for 4 h to induce luteinization. StAR mRNA levels were significantly increased by hCG accompanied by the increase in H3K4me3 of the StAR promoter region. StAR mRNA expression was inhibited by the ERK inhibitor with the significant decrease of H3K4me3. These results suggest that hCG increases StAR gene expression through the ERK-1/2-mediated signaling which is also associated with histone modification of the promoter region. Conclusions: Gene expressions of histone modification enzymes change in GCs undergoing luteinization after ovulation induction. This change may play important roles in regulating the expression of various genes during the early stage of luteinization, which may be critical for the subsequent corpus luteum formation. Keywords: Histone modification, Granulosa cells, Ovulation, Luteinization * Correspondence: [email protected] Department of Obstetrics and Gynecology, Yamaguchi University Graduate School of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan © 2016 Maekawa et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Maekawa et al. Journal of Ovarian Research (2016) 9:15 Page 2 of 9 Background on how and which histone modification enzymes The ovulatory LH surge alters the expression of various change in granulosa cells undergoing luteinization. genes, which are essential for ovulation, in granulosa The extracellular regulated kinase (ERK)-1/2 is a medi- cells undergoing luteinization [1]. Among the genes ator in the intracellular signaling pathway stimulated by involved in steroidogenesis, the expression of steroi- the ovulatory LH surge and regulates the expression of a dogenic acute regulatory (StAR) protein, a rate-limiting number of genes including StAR and cyp19a1 in granulosa enzyme for progesterone synthesis, is rapidly induced cells [27]. Recent studies have shown that LH surge- after the LH surge [2] while the expression of aromatase related signaling pathways regulate the expression of some (Cyp19a1), a key enzyme for estrogen synthesis, is rap- histone modification enzymes. CBP/p300, a histone acetyl- idly suppressed [3]. This functional change of steroido- transferase, acts downstream of the ERK-1/2 signal acti- genesis, in which there is a rapid shift from estrogen vated by the ovulatory LH surge in mouse granulosa cells synthesis to progesterone synthesis, plays a crucial role [26]. Two genes (NCOA7 and HP1BP3), which are related in follicle rupture and the following corpus luteum for- to histone modifications or chromatin remodeling, also mation [4, 5]. act downstream of the ERK-1/2 signal and are involved in In recent years, epigenetic mechanisms such as DNA luteinization of granulosa cells after ovulation induction methylation and histone modifications have been shown [27]. These findings led us to investigate that ERK-1/2 is to be involved in the regulation of gene expression [6–9]. involved in regulating the gene expression through histone DNA methylation occurs at cytosines within CpG dinucle- modifications in granulosa cells undergoing luteinization otides through DNA methyltransferases (DNMT), which after the ovulatory LH surge. can silence gene expression by altering chromatin struc- In this study, we first investigated the changes in gene ture and preventing the binding of transcription factors expression of histone modification enzymes in rat granu- [10–13]. Histone modifications also affect chromatin losa cells undergoing luteinization after ovulation induc- structure, which is critical for the interaction of transcrip- tion. Furthermore, we investigated whether ERK-1/2 is tional factors with response elements in the promoters involved in the regulation of the histone modification at [10, 14–16]. Histone modifications such as acetylation of the StAR promoter region and its mRNA expression in histone H3 and histone H4 or trimethylation of the site of granulosa cells undergoing luteinization. lysine 4 on histone H3 (H3K4me3) activate transcription by loosening the chromatin structure and allowing the Methods recruitment of transcriptional factors to their response The present study was reviewed and approved by the elements [17–19]. On the other hand, histone modifica- Committee for the Ethics on Animal Experiment in tions such as trimethylation of the site of lysines 9 and 27 Yamaguchi University Graduate School of Medicine. on histone H3 (H3K9me and H3K27me3) inactivate tran- scription by condensing the chromatin [20, 21]. Animal models Histone modifications have been reported to regulate Female Sprague–Dawley rats (Japan SLC, Hamamatsu, the gene expression of StAR, steroidogenesis-related Japan) in 21–24 days old were injected subcutaneously enzymes, and LH receptor in the ovary [22–24]. We with 15 IU of equine chorionic gonadotropin (eCG) recently reported that histone modifications and chro- (Sigma, St. Louis, MO, USA) to stimulate follicular matin remodeling contribute to the rapid induction of growth. After 48 h, the rats were injected subcutane- the StAR gene and the rapid suppression of the Cyp19a1 ously with 15 IU of human chorionic gonadotropin gene in granulosa cells undergoing luteinization dur- (hCG) (Sigma) to induce ovulation and luteinization. ing ovulation in rats [25]. Levels of histone H4 acetyl- The ovaries were dissected before hCG (0 h), 4 and 12 h ation and H3K4me3 increased whereas H3K9me3 and after hCG injection. Then, follicles were punctured using H3K27me3 decreased in the StAR promoter after ovu- 27 G needles, and granulosa cells were isolated. The lation induction [25]. On the other hand, the levels of cells were centrifuged at 800 x g, pelleted, and washed histone H3/H4 acetylation and H3K4me3 decreased, in cold PBS twice. Total RNA was isolated from the cells and the level of H3K27me3 increased in the cyp19a1 with Isogen (Wako Pure Chemical Industries Ltd., Osaka) promoter after ovulation induction [25]. Histone and purified by RNeasy Mini (QIAGEN, Chatsworth, CA). modifications are regulated by a variety of histone Three to five rats in each time point were used for RT2 modification enzymes. Recently, CREB-binding protein profiler PCR array or real-time reverse transcription (CBP)/p300 was shown to regulate the histone acetyl- (RT)-PCR described below. ation in the promoters of the LH-induced target genes as a histone acetyltransferase in mouse granu- Collection and culture of granulosa cells losa cells undergoing luteinization after the ovulatory Culture of granulosa cells was performed as reported LH surge [26]. However, little information is available previously [28]. Immature (3 weeks) ICR mice (Japan Maekawa et al. Journal of Ovarian Research (2016) 9:15 Page 3 of 9 SLC Inc.) received a subcutaneous injection of 20 units bifurcated 2 (SETDB2), histone deacetylase 4 (HDAC4), of eCG to stimulate the development of multiple HDAC10, class II, major histocompatibility complex, follicles. Mice were laparotomized under deep ether transactivator (CIITA), nuclear receptor coactivator 7 anesthesia 48 h after the eCG injection; the ovaries were (NCOA7), heterochromatin protein 1-binding protein 3 quickly removed for the following experiments, and the (HP1BP3),
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