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

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 LßT2 (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 LßT2 cells. Lhb begins to open in LßT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3-1 and LßT2, and Lhb in LßT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LßT2 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.

A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons.

Neurokinin B causes acute GnRH secretion and repression of GnRH transcription in GT1-7 GnRH neurons.

Genetic studies in human patients with idiopathic hypogonadotropic hypogonadism (IHH) identified mutations in the genes that encode neurokinin B (NKB) and the neurokinin 3 receptor (NK3R). However, determining the mechanism whereby NKB regulates gonadotropin secretion has been difficult because of conflicting results from in vivo studies investigating the luteinizing hormone (LH) response to senktide, a NK3R agonist. NK3R is expressed in a subset of GnRH neurons and in kisspeptin neurons that are known to regulate GnRH secretion. Thus, one potential source of inconsistency is that NKB could produce opposing direct and indirect effects on GnRH secretion. Here, we employ the GT1-7 cell model to elucidate the direct effects of NKB on GnRH neuron function. We find that GT1-7 cells express NK3R and respond to acute senktide treatment with c-Fos induction and increased GnRH secretion. In contrast, long-term senktide treatment decreased GnRH secretion. Next, we focus on the examination of the mechanism underlying the long-term decrease in secretion and determine that senktide treatment represses transcription of GnRH. We further show that this repression of GnRH transcription may involve enhanced c-Fos protein binding at novel activator protein-1 (AP-1) half-sites identified in enhancer 1 and the promoter, as well as chromatin remodeling at the promoter of the GnRH gene. These data indicate that NKB could directly regulate secretion from NK3R-expressing GnRH neurons. Furthermore, whether the response is inhibitory or stimulatory toward GnRH secretion could depend on the history or length of exposure to NKB because of a repressive effect on GnRH transcription.

Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism.

CONTEXT: Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader-Willi syndrome. AIM: To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH). PATIENTS AND METHODS: We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin. RESULTS: A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed. CONCLUSION: A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

Dynamic chromatin modifications control GnRH gene expression during neuronal differentiation and protein kinase C signal transduction.

GnRH, a neuropeptide produced by rare, specialized hypothalamic secretory neurons, is critical for reproduction. During development, GnRH gene expression increases as neurons migrate from the olfactory placode to the hypothalamus, with highest levels in the mature, postmitotic state. While neuronal differentiation is known to be controlled by chromatin modulations, the role of chromatin dynamics in GnRH gene regulation has not been studied. Here, we use mature and immature GnRH neuronal cell models to show that both neuron-specific and protein kinase C regulation of GnRH expression are mediated by chromatin structure and histone modifications. Only in GT1-7 mature GnRH neuronal cells did GnRH regulatory elements display high sensitivity to DNase and enrichment of active histone markers histone-H3 acetylation and H3 lysine 4 trimethylation (H3K4-Me3), as well as RNA polymerase II (RNAPII) binding and enhancer RNA transcription. In contrast, H3K9-Me2, a marker of inactive chromatin, was highest in nonneuronal cells, low in GT1-7 cells, and intermediate in immature GnRH neuronal cells. The chromatin of the GnRH gene was therefore active in mature GnRH neuronal cells, inactive in nonneuronal cells, but not fully inactive in immature GnRH neuronal cells. Activation of protein kinase C (PKC) potently represses GnRH expression. PKC activation caused closing of the chromatin and decreased RNAPII occupancy at the GnRH minimal promoter (-278/-97). At GnRH-Enhancer-1 (-2404/-2100), PKC activation decreased phosphorylated-RNAPII binding, enhancer RNA transcription, and H3 acetylation, and reciprocally increased H3K9-Me2. Chromatin modifications therefore participate in the dynamic regulation and specification of GnRH expression to differentiated hypothalamic neurons.

Enhancers of GnRH transcription embedded in an upstream gene use homeodomain proteins to specify hypothalamic expression.

GnRH, the central regulator of reproductive function, is produced by only approximately 800 highly specialized hypothalamic neurons. Previous studies identified a minimal promoter [GnRH minimal promoter (GnRH-P)] (-173/+1) and a neuron-specific enhancer [GnRH-enhancer (E)1] (-1863/-1571) as regulatory regions in the rat gene that confer this stringent specificity of GnRH expression to differentiated GnRH neurons. In transgenic mice, these two elements target only GnRH neurons but fail to drive expression in the entire population, suggesting the existence of additional regulatory regions. Here, we define two novel, highly conserved, upstream enhancers in the GnRH gene termed GnRH-E2 (-3135/-2631) and GnRH-E3 (-4199/-3895) that increase neuron-specific GnRH expression through interactions with GnRH-E1 and GnRH-P. GnRH-E2 and GnRH-E3 regulate GnRH expression through similar mechanisms via Oct-1, Msx1, and Dlx2, which bind both GnRH-E2 and the GnRH-E3 critical region at -3952/-3895. Overexpression of Dlx2 increases transcription through GnRH-E2 and GnRH-E3. Remarkably, these novel elements are contained within the 3' untranslated region of the neighboring upstream gene, yet are marked endogenously by histone modification signatures consistent with those of enhancers. Thus, GnRH-E2 and GnRH-E3 are novel regulatory elements that, together with GnRH-E1 and GnRH-P, confer the specificity of GnRH expression to differentiated and mature GnRH neurons.

EWS/FLI and its downstream target NR0B1 interact directly to modulate transcription and oncogenesis in Ewing's sarcoma.

Most Ewing's sarcomas harbor chromosomal translocations that encode fusions between EWS and ETS family members. The most common fusion, EWS/FLI, consists of an EWSR1-derived strong transcriptional activation domain fused, in-frame, to the DNA-binding domain-containing portion of FLI1. EWS/FLI functions as an aberrant transcription factor to regulate genes that mediate the oncogenic phenotype of Ewing's sarcoma. One of these regulated genes, NR0B1, encodes a corepressor protein, and likely plays a transcriptional role in tumorigenesis. However, the genes that NR0B1 regulates and the transcription factors it interacts with in Ewing's sarcoma are largely unknown. We used transcriptional profiling and chromatin immunoprecipitation to identify genes that are regulated by NR0B1, and compared these data to similar data for EWS/FLI. Although the transcriptional profile overlapped as expected, we also found that the genome-wide localization of NR0B1 and EWS/FLI overlapped as well, suggesting that they regulate some genes coordinately. Further analysis revealed that NR0B1 and EWS/FLI physically interact. This protein-protein interaction is likely to be relevant for the development of Ewing's sarcoma because mutations in NR0B1 that disrupt the interaction have transcriptional consequences and also abrogate oncogenic transformation. Taken together, these data suggest that EWS/FLI and NR0B1 physically interact, coordinately modulate gene expression, and mediate the transformed phenotype of Ewing's sarcoma.

LXXLL motifs and AF-2 domain mediate SHP (NR0B2) homodimerization and DAX1 (NR0B1)-DAX1A heterodimerization.

Small heterodimer partner (SHP; NR0B2) is an unusual orphan member of the nuclear receptor superfamily that functions as a corepressor of other nuclear receptors through heterodimeric interactions. Mutations in SHP are associated with mild obesity and insulin resistance. The protein domain structure of SHP is similar to Dosage-sensitive sex reversal adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1 (DAX1; NR0B1). Mutations in DAX1 cause AHC with associated hypogonadotropic hypogonadism. DAX1A is an alternatively spliced isoform of DAX1 that lacks the last 80 amino acids of the DAX1 C-terminal repressor domain and is replaced by a novel 10-amino acid motif. We have previously shown homodimerization of SHP and DAX1 individually, heterodimerization of DAX1 with SHP, and heterodimerization of DAX1 with DAX1A. In these studies, we investigated the domains and residues of SHP involved in SHP homodimerization and DAX1-SHP heterodimerization and also further characterized DAX1-DAX1 homodimerization and DAX1-DAX1A heterodimerization. We showed involvement of the SHP LXXLL motifs and AF-2 domain in SHP homodimerization and DAX1-SHP heterodimerization. We demonstrated redundancy of the LXXLL motifs in DAX1 homodimerization. While DAX1A subcellular localization is mostly cytoplasmic, DAX1-DAX1A heterodimers existed in the nucleus, suggesting differential functions for DAX1A in each compartment. We showed that the AF-2 domain of DAX1 is involved in DAX1-DAX1A heterodimerization. These results indicate that NR0B family members use similar mechanisms for homodimerization as well as heterodimerization. These resemble coactivator-receptor interactions that may have potential functional consequences for molecular mechanisms of the NR0B family.

Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1) and small heterodimer partner (SHP) (NR0B2) form homodimers individually, as well as DAX1-SHP heterodimers.

Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1) (NR0B1), and small heterodimer partner (SHP) (NR0B2) are atypical nuclear receptor superfamily members that function primarily as corepressors through heterodimeric interactions with other nuclear receptors. Mutations in DAX1 cause adrenal hypoplasia congenita, and mutations in SHP lead to mild obesity and insulin resistance, but the mechanisms are unclear. We investigated the existence and subcellular localization of DAX1 and SHP homodimers and the dynamics of homodimerization. We demonstrated DAX1 homodimerization in the nucleus and cytoplasm, and dissociation of DAX1 homodimers upon heterodimerization with steroidogenic factor 1 (SF1) or ligand-activated estrogen receptor-alpha (ERalpha). DAX1 homodimerization involved an interaction between its amino and carboxy termini involving its LXXLL motifs and activation function (AF)-2 domain. We observed SHP homodimerization in the nucleus of mammalian cells and showed dissociation of SHP homodimers upon heterodimerization with ligand-activated ERalpha. We observed DAX1-SHP heterodimerization in the nucleus of mammalian cells and demonstrated the involvement of the LXXLL motifs and AF-2 domain of DAX1 in this interaction. We further demonstrate heterodimerization of DAX1 with its alternatively spliced isoform, DAX1A. This is the first evidence of homodimerization of individual members of the unusual NR0B nuclear receptor family and heterodimerization between its members. Our results suggest that DAX1 forms antiparallel homodimers through the LXXLL motifs and AF-2 domain. These homodimers may function as holding reservoirs in the absence of heterodimeric partners. The formation of DAX1 and SHP homodimers and DAX1-SHP and DAX1-DAX1A heterodimers suggests the possibility of novel functions independent of their coregulator roles, suggesting additional complexity in the molecular mechanisms of DAX1 and SHP action.

Molecular mechanisms of DAX1 action.

DAX1 (dosage sensitive sex reversal (DSS), adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1) encoded by the gene NR0B1, is an unusual orphan nuclear receptor that when mutated causes AHC with associated hypogonadotropic hypogonadism (HH), and when duplicated causes DSS. DAX1 expression has been shown in all regions of the hypothalamic-pituitary-adrenal-gonadal (HPAG) axis during development and in adult tissues, suggesting a critical role for DAX1 in the normal development and function of this axis. Steroidogenic factor 1 (SF1, NR5A1) knockout mice show similar developmental defects as AHC and HH patients, but paradoxically, DAX1 is a negative coregulator of SF1 transactivation. The function of DAX1 as an antagonist of SF1 in gonadal development is consistent with the fact that in humans, duplication of the region of the X chromosome containing DAX1 causes a similar phenotype as mutations in SF1. However, how disruption of DAX1 leads to adrenal, hypothalamic, and pituitary developmental defects similar to SF1 disruption remains to be clarified. The exact mechanism of DAX1 action in each of these tissues during adulthood and critical stages of development are not fully understood. Recent evidence suggests a broader functional role for DAX1 as a negative coregulator of estrogen receptor (ER, NR3A1-2), liver receptor homologue-1 (LRH-1, NR5A2), androgen receptor (AR, NR3C4), and progesterone receptor (PR, NR3C3), each by distinct repression mechanisms. DAX1 may have pleiotropic roles in addition to its function as a negative regulator of steroidogenesis during the development and adult function of the HPAG axis.