The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced in the embryonic pituitary in response to delivery of the hypothalamic gonadotropin releasing hormone (GnRH). GnRH has a pivotal role in reestablishing gonadotropin levels at puberty in primates, and for many species with extended reproductive cycles, these are reinitiated in response to central nervous system-induced GnRH release. Thus, a clear role is evident for GnRH in overcoming repression of these genes. Although the mechanisms through which GnRH actively stimulates LH and FSH ␤-subunit (FSH␤) gene transcription have been described in some detail, there is currently no information on how GnRH overcomes repression in order to terminate reproductively inactive stages. We show here that GnRH overcomes histone deacetylase (HDAC)-mediated repression of the gonadotropin ␤-subunit genes in immature gonadotropes. The repressive factors associated with each of these genes comprise distinct sets of HDACs and corepressors which allow for differentially regulated derepression of these two genes, produced in the same cell by the same regulatory hormone. We find that GnRH activation of calcium/calmodulin-dependent protein kinase I (CaMKI) plays a crucial role in the derepression of the FSH␤ gene involving phosphorylation of several class IIa HDACs associated with both the FSH␤ and Nur77 genes, and we propose a model for the mechanisms involved. In contrast, derepression of the LH ␤-subunit gene is not CaMK dependent. This demonstration of HDACmediated repression of these genes could explain the temporal shut-down of reproductive function at certain periods of the life cycle, which can easily be reversed by the actions of the hypothalamic regulatory hormone.In the mouse embryo, the pituitary gonadotropes become fully differentiated between embryonic day (E)11.5 when the common ␣-subunit is first expressed, and E16.5 when the luteinizing hormone ␤-subunit (LH␤) gene is first detected, while expression of the follicle-stimulating hormone ␤-subunit (FSH␤) gene appears on the following day. The expression of these genes is facilitated by unique cell-specific groups of transcription factors which are expressed in response to local signals, and most are present by E9 to 13.5 (6, 23, 50). Expression of these transcription factors (e.g., SF-1, Egr-1, and Pitx-1 for LH and SF-1, AP-1, Lhx3, Ptx1, and Ptx2 for FSH) has been shown to be sufficient to induce activity of the transiently transfected LH␤ and FSH␤ promoters in reporter gene assays in heterologous cells (9,20,27,41,55,56,61,67). However, LH␤ and FSH␤ gene expression in the developing gonadotrope appears only following the migration of the gonadotropin releasing hormone (GnRH) neurons in the hypothalamus and GnRH delivery to the pituitary, which starts around E16. This indicates a role for GnRH in initiating gonadotropin gene expression, which might be distinct from its stimulating increases in gonadotropin gene expression in mature gonadotropes, in which the gonadotropin genes ...

The synthesis of the gonadotropin subunits is directed by pulsatile gonadotropin-releasing hormone (GnRH) from the hypothalamus, with the frequency of GnRH pulses governing the differential expression of the common α-subunit, luteinizing hormone β-subunit (LHβ) and follicle-stimulating hormone β-subunit (FSHβ). Three mitogen-activated protein kinases, (MAPKs), ERK1/2, JNK and p38, contribute uniquely and combinatorially to the expression of each of these subunit genes. In this study, using both experimental and computational methods, we found that dual specificity phosphatase regulation of the activity of the three MAPKs through negative feedback is required, and forms the basis for decoding the frequency of pulsatile GnRH. A fourth MAPK, ERK5, was shown also to be activated by GnRH. ERK5 was found to stimulate FSHβ promoter activity and to increase FSHβ mRNA levels, as well as enhancing its preference for low GnRH pulse frequencies. The latter is achieved through boosting the ultrasensitive behavior of FSHβ gene expression by increasing the number of MAPK dependencies, and through modulating the feedforward effects of JNK activation on the GnRH receptor (GnRH-R). Our findings contribute to understanding the role of changing GnRH pulse-frequency in controlling transcription of the pituitary gonadotropins, which comprises a crucial aspect in regulating reproduction. Pulsatile stimuli and oscillating signals are integral to many biological processes, and elucidation of the mechanisms through which the pulsatility is decoded explains how the same stimulant can lead to various outcomes in a single cell.

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation.

Most receptor tyrosine kinases and G protein-coupled receptors (GPCRs) operate via a limited number of MAPK cascades but still exert diverse functions, and therefore signal specificity remains an enigma. Also, most GPCR ligands utilize families of receptors for mediation of diverse biological actions; however, the mammalian type I GnRH receptor (GnRHR) seems to be the sole receptor mediating GnRH-induced gonadotropin synthesis and release. Signaling complexes associated with GPCRs may thus provide the means for signal specificity. Here we describe a signaling complex associated with the GnRHR, which is a unique GPCR lacking a C-terminal tail. Unlike other GPCRs, this signaling complex is preformed, and exposure of L beta T2 gonadotropes to GnRH induces its dynamic rearrangement. The signaling complex includes c-Src, protein kinase C delta, -epsilon, and -alpha, Ras, MAPK kinase 1/2, ERK1/2, tubulin, focal adhesion kinase (FAK), paxillin, vinculin, caveolin-1, kinase suppressor of Ras-1, and the GnRHR. Exposure to GnRH (5 min) causes MAPK kinase 1/2, ERK1/2, tubulin, vinculin, and the GnRHR to detach from c-Src, but they reassociate within 30 min. On the other hand, FAK, paxillin, the protein kinase Cs, and caveolin-1 stay bound to c-Src, whereas kinase suppressor of Ras-1 appears in the complex only 30 min after GnRH stimulation. GnRH was found to activate ERK1/2 in the complex in a c-Src-dependent manner, and the activated ERK1/2 subsequently phosphorylates FAK and paxillin. In parallel, caveolin-1, FAK, vinculin, and paxillin are phosphorylated on Tyr residues apparently by GnRH-activated c-Src. Receptor tyrosine kinases and GPCRs translocate ERK1/2 to the nucleus to phosphorylate and activate transcription factors. We therefore propose that the role of the multiprotein signaling complex is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions.

Gonadotrophin‐releasing hormone (GnRH) regulates reproduction via binding a G‐protein coupled receptor on the surface of the gonadotroph, through which it transmits signals, mostly via the mitogen‐activated protein (MAPK) cascade, to increase synthesis of the gonadotrophin hormones: luteinising hormone (LH) and follicle‐stimulating hormone (FSH). Activation of the MAPK cascade requires an elevation in cytosolic Ca2+ levels, which is a result of both calcium influx and mobilisation from intracellular stores. However, Ca2+ also transmits signals via an MAPK‐independent pathway, through binding calmodulin (CaM), which is then able to bind a number of proteins to impart diverse downstream effects. Although the ability of GnRH to activate CaM was recognised over 20 years ago, only recently have some of the downstream effects been elucidated. GnRH was shown to activate the CaM‐dependent phosphatase, calcineurin, which targets gonadotrophin gene expression both directly and indirectly via transcription factors such as nuclear factor of activated T‐cells and Nur77, the Transducer of Regulated CREB (TORC) co‐activators and also the prolyl isomerase, Pin1. Gonadotrophin gene expression is also regulated by GnRH‐induced CaM‐dependent kinases (CaMKs); CaMKI is able to derepress the histone deacetylase‐inhibition of β‐subunit gene expression, whereas CaMKII appears to be essential for the GnRH‐activation of all three subunit genes. Asides from activating gonadotrophin gene expression, GnRH also exerts additional effects on gonadotroph function, some of which clearly occur via CaM, including the proliferation of immature gonadotrophs, which is dependent on calcineurin. In this review, we summarise these pathways, and discuss the additional functions that have been proposed for CaM with respect to modifying GnRH‐induced signalling pathways via the regulation of the small GTP‐binding protein, Gem, and/or the regulator of G‐protein signalling protein 2.