Otx2 Induction of the Gonadotropin-releasing Hormone Promoter Is Modulated by Direct Interactions with Grg Co-repressors
2009; Elsevier BV; Volume: 284; Issue: 25 Linguagem: Inglês
10.1074/jbc.m109.002485
ISSN1083-351X
AutoresRachel Larder, Pamela L. Mellon,
Tópico(s)Reproductive System and Pregnancy
ResumoHormonal communication between the hypothalamus, pituitary, and gonads orchestrates the development and regulation of mammalian reproductive function. In mice, gonadotropin-releasing hormone (GnRH) expression is limited to ∼1000 neurons that originate in the olfactory placode then migrate to specific positions scattered throughout the hypothalamus. Coordination of the hypothalamic-pituitary-gonadal axis is dependent upon correct migration of GnRH neurons into the hypothalamus followed by the appropriate synthesis and pulsatile secretion of GnRH. Defects in any one of these processes can cause infertility. Recently, substantial progress has been made in identifying transcription factors, and their cofactors, that regulate not only adult expression of GnRH, but also the maturation of GnRH neurons. Here, we show that expression of Otx2, a homeodomain protein required for the formation of the forebrain, is dramatically up-regulated during GnRH neuronal maturation and that overexpression of Otx2 increases GnRH promoter activity in GnRH neuronal cell lines. Furthermore, Otx2 transcriptional activity is modulated by Grg4, a member of the Groucho-related-gene (Grg) family. Using mutational analysis, we show that a WRPW peptide motif within the Otx2 protein is required for physical interaction between Otx2 and Grg4. Without this physical interaction, Grg4 cannot repress Otx2-dependent activation of GnRH gene transcription. Taken together, these data show that Otx2 is important for GnRH expression and that direct interaction between Otx2 and Grg co-repressors regulates GnRH gene expression in hypothalamic neurons. Hormonal communication between the hypothalamus, pituitary, and gonads orchestrates the development and regulation of mammalian reproductive function. In mice, gonadotropin-releasing hormone (GnRH) expression is limited to ∼1000 neurons that originate in the olfactory placode then migrate to specific positions scattered throughout the hypothalamus. Coordination of the hypothalamic-pituitary-gonadal axis is dependent upon correct migration of GnRH neurons into the hypothalamus followed by the appropriate synthesis and pulsatile secretion of GnRH. Defects in any one of these processes can cause infertility. Recently, substantial progress has been made in identifying transcription factors, and their cofactors, that regulate not only adult expression of GnRH, but also the maturation of GnRH neurons. Here, we show that expression of Otx2, a homeodomain protein required for the formation of the forebrain, is dramatically up-regulated during GnRH neuronal maturation and that overexpression of Otx2 increases GnRH promoter activity in GnRH neuronal cell lines. Furthermore, Otx2 transcriptional activity is modulated by Grg4, a member of the Groucho-related-gene (Grg) family. Using mutational analysis, we show that a WRPW peptide motif within the Otx2 protein is required for physical interaction between Otx2 and Grg4. Without this physical interaction, Grg4 cannot repress Otx2-dependent activation of GnRH gene transcription. Taken together, these data show that Otx2 is important for GnRH expression and that direct interaction between Otx2 and Grg co-repressors regulates GnRH gene expression in hypothalamic neurons. Normal reproductive function requires the precise orchestration and integration of hormonal regulation at all levels of the hypothalamic-pituitary-gonadal axis. Dysfunction at any of these levels leads to pathophysiologic disorders such as infertility, polycystic ovarian syndrome, and hypogonadotropic hypogonadism. Gonadotropin-releasing hormone (GnRH), 2The abbreviations used are: GnRHgonadotropin-releasing hormoneGrgGroucho-related-geneEMSAelectrophoretic mobility shift assayHDAChistone deacetylaseIVTin vitro translatedGAPDHglyceraldehyde-3-phosphate dehydrogenaseGFPgreen fluorescent proteinANOVAanalysis of varianceRTreverse transcriptionTKthymidine kinase. 2The abbreviations used are: GnRHgonadotropin-releasing hormoneGrgGroucho-related-geneEMSAelectrophoretic mobility shift assayHDAChistone deacetylaseIVTin vitro translatedGAPDHglyceraldehyde-3-phosphate dehydrogenaseGFPgreen fluorescent proteinANOVAanalysis of varianceRTreverse transcriptionTKthymidine kinase. the central regulator of the hypothalamic-pituitary-gonadal axis, is secreted in a pulsatile manner from a small, yet critical, population of neurons within the hypothalamus. The correct migration of GnRH neurons to the hypothalamus, from their origins within the olfactory placode, followed by the appropriate synthesis and pulsatile secretion of GnRH, is essential for effective coordination of the hypothalamic-pituitary-gonadal axis. Examination of the molecular mechanisms involved in GnRH neuron migration and transcription has benefited from the generation of immortalized GnRH neuronal cell lines (1Mellon P.L. Windle J.J. Goldsmith P.C. Padula C.A. Roberts J.L. Weiner R.I. Neuron. 1990; 5: 1-10Abstract Full Text PDF PubMed Scopus (893) Google Scholar, 2Radovick S. Wray S. Lee E. Nicols D.K. Nakayama Y. Weintraub B.D. Westphal H. Cutler G.B. Wondisford F.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3402-3406Crossref PubMed Scopus (193) Google Scholar). The GT1–7 cell line represents a fully differentiated neuron, which accurately processes the pro-GnRH precursor and secretes GnRH in a pulsatile pattern (3Wetsel W.C. Mellon P.L. Weiner R.I. Negro-Vilar A. 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Barbera J.P. Herman T.S. Connell S.O. Olson L. Ju B. Tollkuhn J. Baek S.H. Rose D.W. Rosenfeld M.G. Genes Dev. 2001; 15: 3193-3207Crossref PubMed Scopus (152) Google Scholar, 32Grbavec D. Stifani S. Biochem. Biophys. Res. Commun. 1996; 223: 701-705Crossref PubMed Scopus (131) Google Scholar, 33Zhu C.C. Dyer M.A. Uchikawa M. Kondoh H. Lagutin O.V. Oliver G. Development. 2002; 129: 2835-2849Crossref PubMed Google Scholar), the Q-domain is required for homo- and heterodimerization of Grg proteins (34Pinto M. Lobe C.G. J. Biol. Chem. 1996; 271: 33026-33031Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and the GP-domain is involved in the recruitment of histone deacetylases (HDACs) (35Chen G. Fernandez J. Mische S. Courey A.J. Genes Dev. 1999; 13: 2218-2230Crossref PubMed Scopus (352) Google Scholar). Grg1-S, Grg3b, and Grg5 lack the NLS, SP-domain, and WD40 repeats (24Leon C. Lobe C.G. Dev. Dyn. 1997; 208: 11-24Crossref PubMed Scopus (53) Google Scholar, 26Mallo M. Franco del Amo F. Gridley T. Mech. Dev. 1993; 42: 67-76Crossref PubMed Scopus (61) Google Scholar, 36Miyasaka H. Choudhury B.K. Hou E.W. Li S.S. Eur. J. Biochem. 1993; 216: 343-352Crossref PubMed Scopus (64) Google Scholar, 37Lepourcelet M. Shivdasani R.A. J. Biol. Chem. 2002; 277: 47732-47740Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The WD40 domain is conserved in Grg6, but the N terminus of the protein is truncated and has little similarity to the other Grgs. The short Grgs are proposed to function as dominant-negative forms of long Grgs; however, the exact mechanism by which they influence Grg-mediated repression remains to be resolved (38Swingler T.E. Bess K.L. Yao J. Stifani S. Jayaraman P.S. J. Biol. Chem. 2004; 279: 34938-34947Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 39Wang W. Wang Y.G. Reginato A.M. Glotzer D.J. Fukai N. Plotkina S. Karsenty G. Olsen B.R. Dev. Biol. 2004; 270: 364-381Crossref PubMed Scopus (58) Google Scholar).Herein, we have examined how Otx2 activation of GnRH gene transcription is regulated by the physical interaction of Otx2 with Grg4, a long Grg co-repressor. We show that Otx2 expression is restricted to GT1–7 cells and that Otx2 interacts in vivo with the GnRH promoter. Furthermore, we describe the identification and characterization of Grg4 as a repressor of Otx2-induced activation of GnRH transcription. We report that Otx2-induced activation is repressed by Grg4 through an HDAC-independent mechanism and demonstrate that this repression is mediated by physical interaction between the two proteins via a specific amino acid motif within the Otx2 protein.DISCUSSIONOtx2 is required for regionalization and patterning of the developing brain and plays an important role in establishing the identity and fate of progenitor neurons. The interaction of Otx2 with cofactors, together with a specific spatial and temporal expression pattern, controls the effects of Otx2 on the activity of a given promoter (20Danno H. Michiue T. Hitachi K. Yukita A. Ishiura S. Asashima M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 5408-5413Crossref PubMed Scopus (82) Google Scholar, 21Mochizuki T. Karavanov A.A. Curtiss P.E. Ault K.T. Sugimoto N. Watabe T. Shiokawa K. Jamrich M. Cho K.W. Dawid I.B. Taira M. Dev. Biol. 2000; 224: 470-485Crossref PubMed Scopus (59) Google Scholar, 22Nakano T. Murata T. Matsuo I. Aizawa S. Biochem. Biophys. Res. Commun. 2000; 267: 64-70Crossref PubMed Scopus (53) Google Scholar). Reports detailing the co-localization of Otx2 and GnRH during both development and adulthood, along with data identifying Otx2-target binding sites within the GnRH regulatory region, have established Otx2 as an important factor in the regulation of GnRH transcription (7Kelley C.G. Lavorgna G. Clark M.E. Boncinelli E. Mellon P.L. Mol. Endocrinol. 2000; 14: 1246-1256Crossref PubMed Scopus (64) Google Scholar, 60Mallamaci A. Di Blas E. Briata P. Boncinelli E. Corte G. Mech. Dev. 1996; 58: 165-178Crossref PubMed Scopus (81) Google Scholar). In this report, we have studied the regulation of GnRH expression by Otx2 in more detail and describe for the first time the identification and characterization of Grg proteins as co-repressors of Otx2-induced GnRH transcription.Groucho-related co-repressors (Grg1, -2, -3, and -4) are thought to repress promoter activity by down-regulating the induction of target genes by transcriptional activators, enhancing the effects of transcriptional repressors or converting transcriptional activators to repressors (27Gasperowicz M. Otto F. J. Cell. Biochem. 2005; 95: 670-687Crossref PubMed Scopus (92) Google Scholar). Our findings reveal that endogenous Grg4 and Otx2 proteins interact in GT1–7 cells and demonstrate that Grg4 represses Otx2-induced activation of both a GnRH reporter containing the rat enhancer and promoter regions and a reporter consisting of multiple copies of the Otx2 bicoid-like binding site. This suggests that Grg4 is not modulating GnRH transcription by down-regulating Otx2 expression, nor is it only augmenting the effects of known GnRH transcriptional repressors such as Msx1. Rather, these data indicate that Grg4 is either sequestering Otx2 away from the DNA, thus preventing activation of the promoter, or that interaction of Grg4 and Otx2 on the DNA blocks transcription from occurring. Because Grg4, like Otx2, has been shown to interact with the GnRH promoter chromatin in vivo (10Rave-Harel N. Miller N.L. Givens M.L. Mellon P.L. J. Biol. Chem. 2005; 280: 30975-30983Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), we hypothesize that, instead of sequestering Otx2 away from the DNA, Grg4 is recruited to DNA-bound Otx2 resulting in an attenuation of GnRH transcription. Long term repression by Grgs is thought to involve the recruitment of HDACs, via the GP-domain of Grg protein, which is highly conserved between all the long Grgs (35Chen G. Fernandez J. Mische S. Courey A.J. Genes Dev. 1999; 13: 2218-2230Crossref PubMed Scopus (352) Google Scholar). However, in this report, we demonstrate that a mutant Grg4 protein, in which the GP-domain has been deleted, remains capable of repressing Otx2-induced transcription as efficiently as wild-type Grg4. This suggests that, as in the case of Grg4-mediated repression of Pax2 and Oct-2 activation (61Cai Y. Brophy P.D. Levitan I. Stifani S. Dressler G.R. EMBO J. 2003; 22: 5522-5529Crossref PubMed Scopus (68) Google Scholar, 62Malin S. Linderson Y. Almqvist J. Ernberg I. Tallone T. Pettersson S. Nucleic Acids Res. 2005; 33: 4618-4625Crossref PubMed Scopus (15) Google Scholar), repression occurs via a HDAC-independent mechanism. In the case of Pax2, phosphorylation of Pax2 by c-Jun N-terminal kinase is blocked by association with Grg4, decreasing the transcriptional activity of Pax2. Analogously, Grg4 may inhibit Otx2 activity by preventing its post-translational modification. However, further investigation is required to determine whether protein modifications, such as phosphorylation, control the activity of Otx2.Interestingly, although Grg4 was able to repress Otx2-induced activation of the GnRHe/p and the −152-bp multimer, it was unable to exert any repressive effects on Otx2 activation of the −198-bp multimer. Bioinformatic analysis reveals that both sites are conserved in various mammals. However, only the binding site at −152 bp is a 100% match to a consensus Otx2-binding site sequence. Otx2 has been shown to bind the core elements contained within both of the sites, but has a greater affinity for the TAATCCC element found at −152 bp (63Briata P. Ilengo C. Bobola N. Corte G. FEBS Lett. 1999; 445: 160-164Crossref PubMed Scopus (37) Google Scholar), and our in vitro experiments confirmed this. Although we were able to demonstrate that overexpression of Otx2 significantly activates both multimers, and in vitro translated Otx2 protein binds to both sites, we did not see the formation of an Otx2 protein complex on the −198-bp probe when using GT1–7 cell nuclear extracts, suggesting that, although the site is capable of binding Otx2, and being activated by it, it preferentially binds other nuclear proteins in EMSAs. Although chromatin immunoprecipitation experiments demonstrated that Otx2 interacts with the GnRH promoter in GT1–7 cells, the proximity of the two sites prevented us from determining whether Otx2 is capable of binding to both sites in vivo and, if so, whether the −152-bp site was preferentially bound. We hypothesize that the differential effects of Grg4 on the two multimers may be explained by the affinity with which Otx2 binds to the DNA, as is observed with the interaction of Pax2 and Groucho (61Cai Y. Brophy P.D. Levitan I. Stifani S. Dressler G.R. EMBO J. 2003; 22: 5522-5529Crossref PubMed Scopus (68) Google Scholar). Weak binding of Otx2 to the DNA may affect the stability of the Otx2-Grg4 interaction and thus the ability of Grg4 to exert its repressive effects.Previous studies have shown that Hairy-related proteins interact with Grg family members and that deletion of the Hairy protein WRPW motif is sufficient to prevent this physical interaction (57Paroush Z. Finley R.L. Kidd T. Wainwright S.M. Ingham P.W. Brent R. Ish-Horowicz D. Cell. 1994; 79: 805-815Abstract Full Text PDF PubMed Scopus (493) Google Scholar). Otx2 contains a sequence homologous to the WRPW motif, and we have shown that mutation of this domain relieves Grg4-mediated repression of Otx2-induced GnRH transcription by preventing physical interaction of the two proteins. A recent report shows that the WD40 repeats of Grg4 are required for physical interaction of Grg4 and Otx2 (53Heimbucher T. Murko C. Bajoghli B. Aghaallaei N. Huber A. Stebegg R. Eberhard D. Fink M. Simeone A. Czerny T. Mol. Cell. Biol. 2007; 27: 340-351Crossref PubMed Scopus (38) Google Scholar). Therefore, it is likely that deletion or mutation of the Grg4 WD40-domain would also relieve Grg4-mediated repression of the GnRH promoter. Recently, Buscarlet et al. (56Buscarlet M. Perin A. Laing A. Brickman J.M. Stifani S. J. Biol. Chem. 2008; 283: 24881-24888Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) reported that the anti-neurogenic properties of long Grg proteins are reliant upon the interaction of the Grg protein WD40-domain, with WRPW motif-containing proteins. Overexpression of long Grg proteins results in the inhibition of neuronal development in vivo and decreases neuronal differentiation of progenitor cells (64Yao J. Liu Y. Lo R. Tretjakoff I. Peterson A. Stifani S. Mech. Dev. 2000; 93: 105-115Crossref PubMed Scopus (46) Google Scholar, 65Nuthall H.N. Joachim K. Stifani S. Mol. Cell. Biol. 2004; 24: 8395-8407Crossref PubMed Scopus (55) Google Scholar). Interestingly, Otx2 has been reported to control the identity and fate of neuronal precursors by suppressing their differentiation (66Puelles E. Acampora D. Gogoi R. Tuorto F. Papalia A. Guillemot F. Ang S.L. Simeone A. J. Neurosci. 2006; 26: 5955-5964Crossref PubMed Scopus (46) Google Scholar). However, it has not been established whether this effect is mediated by a co-repressor, or is a consequence of Otx2 acting alone.The anti-neurogenic properties of both long Grg proteins and Otx2 are of particular interest given that GnRH-producing neurons have a unique cellular origin within the olfactory placode. During embryogenesis, they migrate from the olfactory placode, across the cribriform plate, and into the basal forebrain to arrive at their final destination within the hypothalamus (67Schwanzel-Fukuda M. Pfaff D.W. Nature. 1989; 338: 161-164Crossref PubMed Scopus (935) Google Scholar, 68Schwanzel-Fukuda M. Jorgenson K.L. Bergen H.T. Weesner G.D. Pfaff D.W. Endocr. Rev. 1992; 13: 623-634Crossref PubMed Scopus (94) Google Scholar). The factors that specify the onset of GnRH expression and the initiation of these neurons on their migratory pathway have yet to be determined, however, the birth of these cells may be mediated by the spatiotemporal expression of transcription factors. Given that Otx2 expression is observed within the olfactory placode at the time of GnRH neuronal birth, and co-localizes with GnRH during development and into adulthood, it would be interesting to determine the role of Otx2 in GnRH neuronal development and GnRH transcription. However, Otx2-null mice lack the entire forebrain and olfactory placode, preventing analysis of GnRH neurons. In contrast, heterozygotes display highly variable phenotypes with defects in craniofacial, ocular, and olfactory placode development (12Acampora D. Mazan S. Lallemand Y. Avantaggiato V. Maury M. Simeone A. BrÛlet P. Development. 1995; 121: 3279-3290Crossref PubMed Google Scholar, 18Ang S.L. Jin O. Rhinn M. Daigle N. Stevenson L. Rossant J. Development. 1996; 122: 243-252PubMed Google Scholar, 19Matsuo I. Kuratani S. Kimura C. Takeda N. Aizawa S. Genes Dev. 1995; 9: 2646-2658Crossref PubMed Scopus (585) Google Scholar). The varying degree of penetrance observed in these mice suggests that correct dosage of Otx2 is critical for normal development to occur, and this hypothesis is supported by data from human patients where a heterozygous loss-of-function mutation can have highly pleiotropic effects within an affected family. Furthermore, both duplication and deletion of the OTX2-containing region of human chromosome 14q22 lead to developmental abnormalities (69Ou Z. Martin D.M. Bedoyan J.K. Cooper M.L. Chinault A.C. Stankiewicz P. Cheung S.W. Am. J. Med. Genet. A. 2008; 146A: 2480-2489Crossref PubMed Scopus (33) Google Scholar, 70Wyatt A. Bakrania P. Bunyan D.J. Osborne R.J. Crolla J.A. Salt A. Ayuso C. Newbury-Ecob R. Abou-Rayyah Y. Collin J.R. Robinson D. Ragge N. Hum. Mutat. 2008; 29: E278-E283Crossref PubMed Scopus (82) Google Scholar), whereas specific mutations can cause a number of deficiencies including hypopituitarism, which includes low gonadotropins (71Diaczok D. Romero C. Zunich J. Marshall I. Radovick S. J. Clin. Endocrinol. Metab. 2008; 93: 4351-4359Crossref PubMed Scopus (98) Google Scholar, 72Ragge N.K. Brown A.G. Poloschek C.M. Lorenz B. Henderson R.A. Clarke M.P. Russell-Eggitt I. Fielder A. Gerrelli D. Martinez-Barbera J.P. Ruddle P. Hurst J. Collin J.R. Salt A. Cooper S.T. Thompson P.J. Sisodiya S.M. Williamson K.A. Fitzpatrick D.R. van Heyningen V. Hanson I.M. Am. J. Hum. Genet. 2005; 76: 1008-1022Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Ongoing studies are utilizing a GnRH-neuron-specific conditional Otx2 knockout approach to determine the role of Otx2 in GnRH neuronal development and regulation of GnRH transcription in the adult. Because expression of Grg family members also co-localizes with GnRH during development (10Rave-Harel N. Miller N.L. Givens M.L. Mellon P.L. J. Biol. Chem. 2005; 280: 30975-30983Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), it would also be interesting to study the influence of Otx2 and Grg protein interactions on the differentiation and migration of GnRH neurons. Unfortunately, the ability to study Grg protein function during development is also complicated due to the presence of multiple family members that are expressed in overlapping domains, making loss-of-function analyses difficult because of redundancy issues. Because long Grg proteins and Otx2 interact via the Grg WD40-domain, this may explain why no difference in GnRH promoter activity was observed when co-transfecting Otx2 with Grg5, which lacks the GP-domain, SP-domain, and WD40 repeats. It also suggests that other long Grg family members could negatively regulate Otx2 activation of the GnRH promoter. Indeed, analysis of mRNA expression reveals that all of the Grg family members, apart from Grg2, are expressed in both GnRH neuronal cell lines, indicating that they may be interchangeable in repression of Otx2.In conclusion, we have explored novel interactions between the homeodomain-containing protein Otx2 and members of the Groucho-related gene family of proteins. We have demonstrated that Grg4 physically interacts with Otx2 resulting in repression of GnRH promoter activity via an HDAC-independent mechanism. We hypothesize that modulation of the interactions of Otx2 with co-repressors and/or co-activators may contribute to the dynamic control of GnRH gene expression that is necessary during development and for reproduction. This modulation of Otx2 activity may be especially critical during puberty and in the control of the estrous cycle. Normal reproductive function requires the precise orchestration and integration of hormonal regulation at all levels of the hypothalamic-pituitary-gonadal axis. Dysfunction at any of these levels leads to pathophysiologic disorders such as infertility, polycystic ovarian syndrome, and hypogonadotropic hypogonadism. Gonadotropin-releasing hormone (GnRH), 2The abbreviations used are: GnRHgonadotropin-releasing hormoneGrgGroucho-related-geneEMSAelectrophoretic mobility shift assayHDA
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