Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress 2023 EHA Congress 2023 ASCO Annual Meeting
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on sleep in brain health

LIVE: Thursday 2nd May 2024, 18:00-19:30 (CEST)

Quality sleep is essential for health. But what happens to our brains when sleep patterns are disturbed? Join our experts to explore the interplay between sleep disruption and neurological diseases, and the questions that you need to be asking your patients to help you prevent the harmful effects of sleep deprivation.
ADVANCED SEARCH
Log in
Top
Reproductive Biology and Endocrinology
Published in: Reproductive Biology and Endocrinology 1/2017

Open Access 01-12-2017 | Research

Chromatin status and transcription factor binding to gonadotropin promoters in gonadotrope cell lines

Authors: Huimin Xie, Hanne M. Hoffmann, Anita K. Iyer, Melissa J. Brayman, Cindy Ngo, Mary Jean Sunshine, Pamela L. Mellon

Published in: Reproductive Biology and Endocrinology | Issue 1/2017

Login to get access

Abstract

Background

Proper expression of key reproductive hormones from gonadotrope cells of the pituitary is required for pubertal onset and reproduction. To further our understanding of the molecular events taking place during embryonic development, leading to expression of the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), we characterized chromatin structure changes, imparted mainly by histone modifications, in model gonadotrope cell lines.

Methods

We evaluated chromatin status and gene expression profiles by chromatin immunoprecipitation assays, DNase sensitivity assay, and RNA sequencing in three developmentally staged gonadotrope cell lines, αT1–1 (progenitor, expressing Cga), αT3–1 (immature, expressing Cga and Gnrhr), and LβT2 (mature, expressing Cga, Gnrhr, Lhb, and Fshb), to assess changes in chromatin status and transcription factor access of gonadotrope-specific genes.

Results

We found the common mRNA α-subunit of LH and FSH, called Cga, to have an open chromatin conformation in all three cell lines. In contrast, chromatin status of Gnrhr is open only in αT3–1 and LβT2 cells. Lhb begins to open in LβT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3–1 and LβT2, and Lhb in LβT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LβT2 cells. Finally, chromatin status correlates with the progressive access of LHX3 to Cga and Gnrhr, followed by PITX1 binding to the Lhb promoter.

Conclusion

Our data show the gonadotrope-specific genes Cga, Gnrhr, Lhb, and Fshb are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.
Literature
1.
Simmons DM, Voss JW, Ingraham HA, Holloway JM, Broide RS, Rosenfeld MG, et al. Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors. Genes Dev. 1990;4:695–711.CrossRefPubMed
2.
Scully KM, Rosenfeld MG. Pituitary development: regulatory codes in mammalian organogenesis. Science. 2002;295:2231–5.CrossRefPubMed
3.
Japon MA, Rubinstein M, Low MJ. In situ hybridization analysis of anterior pituitary hormone gene expression during fetal mouse development. J Histochem Cytochem. 1994;42:1117–25.CrossRefPubMed
4.
Hsieh J, Gage FH. Chromatin remodeling in neural development and plasticity. Curr Opin Cell Biol. 2005;17:664–71.CrossRefPubMed
5.
Laverriere JN, L'Hote D, Tabouy L, Schang AL, Querat B, Cohen-Tannoudji J. Epigenetic regulation of alternative promoters and enhancers in progenitor, immature, and mature gonadotrope cell lines. Mol Cell Endocrinol. 2016;434:250–65.CrossRefPubMed
6.
7.
Sims RJ 3rd, Nishioka K, Reinberg D. Histone lysine methylation: a signature for chromatin function. Trends Genet. 2003;19:629–39.CrossRefPubMed
8.
Gross DS, Garrard WT. Nuclease hypersensitive sites in chromatin. Annu Rev Biochem. 1988;57:159–97.CrossRefPubMed
9.
Shewchuk BM, Asa SL, Cooke NE, Liebhaber SA. Pit-1 binding sites at the somatotrope-specific DNase I hypersensitive sites I, II of the human growth hormone locus control region are essential for in vivo hGH-N gene activation. J Biol Chem. 1999;274:35725–33.CrossRefPubMed
10.
Venkatesh S, Workman JL. Histone exchange, chromatin structure and the regulation of transcription. Nat Rev Mol Cell Biol. 2015;16:178–89.CrossRefPubMed
11.
Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet. 2007;39:311–8.CrossRefPubMed
12.
Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH. Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc Natl Acad Sci U S A. 2004;101:16659–64.CrossRefPubMedPubMedCentral
13.
Siebzehnrubl FA, Buslei R, Eyupoglu IY, Seufert S, Hahnen E, Blumcke I. Histone deacetylase inhibitors increase neuronal differentiation in adult forebrain precursor cells. Exp Brain Res. 2007;176:672–8.CrossRefPubMed
14.
Windle JJ, Weiner RI, Mellon PL. Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol. 1990;4:597–603.CrossRefPubMed
15.
Alarid ET, Windle JJ, Whyte DB, Mellon PL. Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development. 1996;122:3319–29.
16.
Lew D, Brady H, Klausing K, Yaginuma K, Theill LE, Stauber C, et al. GHF-1 promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice. Genes Dev. 1993;7:683–93.CrossRefPubMed
17.
Pernasetti F, Spady TJ, Hall SB, Rosenberg SB, Givens ML, Anderson S, et al. Pituitary tumorigenesis targeted by the ovine follicle-stimulating hormone beta-subunit gene regulatory region in transgenic mice. Mol Cell Endocrinol. 2003;203:169–83.CrossRefPubMed
18.
Yusta B, Alarid ET, Gordon DF, Ridgway EC, Mellon PL. The thyrotropin β-subunit gene is repressed by thyroid hormone in a novel thryrotrope cell line, mouse TαT1 cells. Endocrinology. 1998;139:4476–82.
19.
Pernasetti F, Vasilyev VV, Rosenberg SB, Bailey JS, Huang H-J, Miller WL, et al. Cell-specific transcriptional regulation of FSHβ by activin and GnRH in the LβT2 pituitary gonadotrope cell model. Endocrinology. 2001;142:2284–95.CrossRefPubMed
20.
Graham KE, Nusser KD, Low MJ. LβT2 gonadotroph cells secrete follicle stimulating hormone (FSH) in response to activin A. J Endocrinol. 1999;162:R1–5.CrossRefPubMed
21.
Bailey JS, Rave-Harel N, Coss D, McGillivray SM, Mellon PL. Activin regulation of the follicle-stimulating hormone β-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1. Mol Endocrinol. 2004;18:1158–70.CrossRefPubMedPubMedCentral
22.
Barnhart KM, Mellon PL. The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone α-subunit gene in pituitary gonadotropes. Mol Endocrinol. 1994;8:878–85.PubMed
23.
Coss D, Thackray VG, Deng CX, Mellon PL. Activin regulates luteinizing hormone beta-subunit gene expression through smad-binding and homeobox elements. Mol Endocrinol. 2005;19:2610–23.CrossRefPubMedPubMedCentral
24.
Corpuz PS, Lindaman LL, Mellon PL, Coss D. FoxL2 is required for activin induction of the mouse and human follicle-stimulating hormone β-subunit genes. Mol Endocrinol. 2010;24:1037–51.CrossRefPubMedPubMedCentral
25.
McGillivray SM, Bailey JS, Ramezani R, Kirkwood BJ, Mellon PL. Mouse GnRH receptor gene expression is mediated by the LHX3 homeodomain protein. Endocrinology. 2005;146:2180–5.CrossRefPubMedPubMedCentral
26.
Xie H, Hoffmann HM, Meadows JD, Mayo SL, Trang C, Leming SS, et al. Homeodomain proteins SIX3 and SIX6 regulate gonadotrope-specific genes during pituitary development. Mol Endocrinol. 2015;29:842–55.CrossRefPubMedPubMedCentral
27.
Steger DJ, Hecht JH, Mellon PL. GATA-binding proteins regulate the human gonadotropin α-subunit gene in placenta and pituitary gland. Mol Cell Biol. 1994;14:5592–602.CrossRefPubMedPubMedCentral
28.
Rosenberg SB, Mellon PL. An Otx-related homeodomain protein binds an LHβ promoter element important for activation during gonadotrope maturation. Mol Endocrinol. 2002;16:1280–98.PubMedPubMedCentral
29.
Harris AN, Mellon PL. The basic helix-loop-helix leucine zipper transcription factor USF is a key regulator of SF-1 gene expression in pituitary gonadotrope and steroidogenic cells. Mol Endocrinol. 1998;12:714–27.CrossRefPubMed
30.
Breen KM, Thackray VG, Coss D, Mellon PL. Runt-related transcription factors impair activin induction of the follicle-stimulating hormone beta-subunit gene. Endocrinology. 2010;151:2669–80.CrossRefPubMedPubMedCentral
31.
Schang AL, Granger A, Querat B, Bleux C, Cohen-Tannoudji J, Laverriere JN. GATA2-induced silencing and LIM-homeodomain protein-induced activation are mediated by a bi-functional response element in the rat GnRH receptor gene. Mol Endocrinol. 2013;27:74–91.CrossRefPubMed
32.
Zubair M, Ishihara S, Oka S, Okumura K, Morohashi K. Two-step regulation of Ad4BP/SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP/SF-1. Mol Cell Biol. 2006;26:4111–21.CrossRefPubMedPubMedCentral
33.
Pick L, Schier A, Affolter M, Schmidt-Glenewinkel T, Gehring WJ. Analysis of the ftz upstream element: germ layer-specific enhancers are independently autoregulated. Genes Dev. 1990;4:1224–39.CrossRefPubMed
34.
Shima Y, Zubair M, Komatsu T, Oka S, Yokoyama C, Tachibana T, et al. Pituitary homeobox 2 regulates adrenal4 binding protein/steroidogenic factor-1 gene transcription in the pituitary gonadotrope through interaction with the intronic enhancer. Mol Endocrinol. 2008;22:1633–46.CrossRefPubMedPubMedCentral
35.
Halvorson LM, Kaiser UB, Chin WW. Stimulation of luteinizing hormone beta gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1. J Biol Chem. 1996;271:6645–50.CrossRefPubMed
36.
Keri RA, Nilson JH. A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone beta subunit promoter in gonadotropes of transgenic mice. J Biol Chem. 1996;271:10782–5.CrossRefPubMed
37.
Iyer AK, Brayman MJ, Mellon PL. Dynamic chromatin modifications control GnRH gene expression during neuronal differentiation and protein kinase C signal transduction. Mol Endocrinol. 2011;25:460–73.CrossRefPubMedPubMedCentral
38.
Iyer AK, Miller NL, Yip K, Tran BH, Mellon PL. Enhancers of GnRH transcription embedded in an upstream gene use homeodomain proteins to specify hypothalamic expression. Mol Endocrinol. 2010;24:1949–64.CrossRefPubMedPubMedCentral
39.
Haring M, Offermann S, Danker T, Horst I, Peterhansel C, Stam M. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods. 2007;3:11.CrossRefPubMedPubMedCentral
40.
Zhang H, Bailey JS, Coss D, Lin B, Tsutsumi R, Lawson MA, et al. Activin modulates the transcriptional response of LbetaT2 cells to gonadotropin-releasing hormone and alters cellular proliferation. Mol Endocrinol. 2006;20:2909–30.CrossRefPubMedPubMedCentral
41.
42.
McArthur M, Gerum S, Stamatoyannopoulos G. Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse beta-globin LCR. J Mol Biol. 2001;313:27–34.CrossRefPubMedPubMedCentral
43.
Perissi V, Jepsen K, Glass CK, Rosenfeld MG. Deconstructing repression: evolving models of co-repressor action. Nat Rev Genet. 2010;11:109–23.CrossRefPubMed
44.
45.
Barrera LO, Ren B. The transcriptional regulatory code of eukaryotic cells--insights from genome-wide analysis of chromatin organization and transcription factor binding. Curr Opin Cell Biol. 2006;18:291–8.CrossRefPubMed
46.
Roybal LL, Hambarchyan A, Meadows JD, Barakat NH, Pepa PA, Breen KM, et al. Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHbeta. Mol Endocrinol. 2014;28:1640–55.CrossRefPubMedPubMedCentral
47.
McGillivray SM, Thackray VG, Coss D, Mellon PL. Activin and glucocorticoids synergistically activate follicle-stimulating hormone β-subunit gene expression in the immortalized LβT2 gonadotrope cell line. Endocrinology. 2007;148:762–73.CrossRefPubMed
48.
Zhang Y, Fatima N, Dufau ML. Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription. Mol Cell Biol. 2005;25:7929–39.CrossRefPubMedPubMedCentral
49.
Ooi GT, Tawadros N, Escalona RM. Pituitary cell lines and their endocrine applications. Mol Cell Endocrinol. 2004;228:1–21.CrossRefPubMed
50.
Sanz E, Yang L, Su T, Morris DR, McKnight GS, Amieux PS. Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci U S A. 2009;106:13939–44.CrossRefPubMedPubMedCentral
51.
Hoffmann HM, Tamrazian A, Xie H, Perez-Millan MI, Kauffman AS, Mellon PL. Heterozygous deletion of ventral anterior homeobox (Vax1) causes subfertility in mice. Endocrinology. 2014;155:4043–53.CrossRefPubMedPubMedCentral
52.
Zhao Y, Zhu M, Yu Y, Qiu L, Zhang Y, He L, et al. Brain REST/NRSF Is Not Only a Silent Repressor but Also an Active Protector. Mol Neurobiol. 2017;54:541–50.CrossRefPubMed
53.
Ichiyanagi K. Epigenetic regulation of transcription and possible functions of mammalian short interspersed elements, SINEs. Genes Genet Syst. 2013;88:19–29.CrossRefPubMed
54.
Magotra A, Naskar S, Das B, Ahmad T. A comparative study of SINE insertion together with a mutation in the first intron of follicle stimulating hormone beta gene in indigenous pigs of India. Mol Biol Rep. 2015;42:465–70.CrossRefPubMed
55.
Coss D, Hand CM, Yaphockun KK, Ely HA, Mellon PL. p38 mitogen-activated protein kinase is critical for synergistic induction of the FSH(beta) gene by gonadotropin-releasing hormone and activin through augmentation of c-Fos induction and Smad phosphorylation. Mol Endocrinol. 2007;21:3071–86.CrossRefPubMedPubMedCentral
56.
Lim S, Luo M, Koh M, Yang M, bin Abdul Kadir MN, Tan JH, et al. Distinct mechanisms involving diverse histone deacetylases repress expression of the two gonadotropin beta-subunit genes in immature gonadotropes, and their actions are overcome by gonadotropin-releasing hormone. Mol Cell Biol. 2007;27:4105–20.CrossRefPubMedPubMedCentral
57.
Burger LL, Haisenleder DJ, Aylor KW, Marshall JC. Regulation of intracellular signaling cascades by GNRH pulse frequency in the rat pituitary: roles for CaMK II, ERK, and JNK activation. Biol Reprod. 2008;79:947–53.CrossRefPubMedPubMedCentral
58.
Haisenleder DJ, Burger LL, Walsh HE, Stevens J, Aylor KW, Shupnik MA, et al. Pulsatile gonadotropin-releasing hormone stimulation of gonadotropin subunit transcription in rat pituitaries: evidence for the involvement of Jun N-terminal kinase but not p38. Endocrinology. 2008;149:139–45.CrossRefPubMed
59.
Dalkin AC, Haisenleder DJ, Ortolano GA, Ellis TR, Marshall JC. The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression. Endocrinology. 1989;125:917–24.CrossRefPubMed
60.
Haisenleder DJ, Katt JA, Ortolano GA, el Gewely MR, Duncan JA, Dee C, et al. Influence of gonadotropin-releasing hormone pulse amplitude, frequency, and treatment duration on the regulation of luteinizing hormone (LH) subunit messenger ribonucleic acids and LH secretion. Mol Endocrinol. 1988;2:338–43.CrossRefPubMed
61.
Papavasiliou SS, Zmeili S, Khoury S, Landefeld TD, Chin WW, Marshall JC. Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone alpha and beta subunits in male rats. Proc Natl Acad Sci U S A. 1986;83:4026–9.CrossRefPubMedPubMedCentral
62.
Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013;10:1213–8.CrossRefPubMedPubMedCentral
63.
Mullen RD, Colvin SC, Hunter CS, Savage JJ, Walvoord EC, Bhangoo AP, et al. Roles of the LHX3 and LHX4 LIM-homeodomain factors in pituitary development. Mol Cell Endocrinol. 2007;2:190–5.
64.
Tremblay JJ, Lanctôt C, Drouin J. The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol. 1998;12:428–41.CrossRefPubMed
65.
Sheng HZ, Zhadanov AB, Mosinger BJ, Fujii T, Bertuzzi S, Grinberg A, et al. Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science. 1996;272:1004–7.CrossRefPubMed
66.
Charles MA, Mortensen AH, Potok MA, Camper SA. Pitx2 deletion in pituitary gonadotropes is compatible with gonadal development, puberty, and fertility. Genesis. 2008;46:507–14.CrossRefPubMedPubMedCentral
67.
Tremblay JJ, Drouin J. Egr-1 is a downstream effector of GnRH and synergizes by direct interaction with Ptx1 and SF-1 to enhance luteinizing hormone β gene transcription. Mol Cell Biol. 1999;19:2567–76.CrossRefPubMedPubMedCentral
68.
Fortin J, Lamba P, Wang Y, Bernard DJ. Conservation of mechanisms mediating gonadotrophin-releasing hormone 1 stimulation of human luteinizing hormone beta subunit transcription. Mol Hum Reprod. 2009;15:77–87.CrossRefPubMed
69.
Cederberg RA, Smith JE, McDonald EA, Lee C, Perkins AR, White BR. Activity of the porcine gonadotropin-releasing hormone receptor gene promoter is partially conferred by a distal gonadotrope specific element (GSE) within an upstream enhancing region, two proximal GSEs and a retinoid X receptor binding site. Reprod Biol Endocrinol. 2015;13:45.
70.
Jacobs SB, Coss D, McGillivray SM, Mellon PL. Nuclear factor Y and steroidogenic factor 1 physically and functionally interact to contribute to cell-specific expression of the mouse Follicle-stimulating hormone-beta gene. Mol Endocrinol. 2003;17:1470–83.CrossRefPubMedPubMedCentral
71.
West BE, Parker GE, Savage JJ, Kiratipranon P, Toomey KS, Beach LR, et al. Regulation of the follicle-stimulating hormone beta gene by the LHX3 LIM-homeodomain transcription factor. Endocrinology. 2004;145:4866–79.CrossRefPubMed
Metadata
Title
Chromatin status and transcription factor binding to gonadotropin promoters in gonadotrope cell lines
Authors
Huimin Xie
Hanne M. Hoffmann
Anita K. Iyer
Melissa J. Brayman
Cindy Ngo
Mary Jean Sunshine
Pamela L. Mellon
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Reproductive Biology and Endocrinology / Issue 1/2017
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/s12958-017-0304-z

Other articles of this Issue 1/2017

Reproductive Biology and Endocrinology 1/2017 Go to the issue

Research

Determining optimal gestational weight gain in the Korean population: a retrospective cohort study

Commentary

Comment on "Oestrogen-induced angiogenesis and implantation contribute to the development of parasitic myomas after laparoscopic morcellation"

Research

Arcyriaflavin a, a cyclin D1–cyclin-dependent kinase4 inhibitor, induces apoptosis and inhibits proliferation of human endometriotic stromal cells: a potential therapeutic agent in endometriosis

Research

An inverse association between serum soluble receptor of advanced glycation end products and hyperandrogenism and potential implication in polycystic ovary syndrome patients

Review

5α-reductase activity in women with polycystic ovary syndrome: a systematic review and meta-analysis

Research

Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model