scholarly journals GATA2-Induced Silencing and LIM-Homeodomain Protein-Induced Activation Are Mediated by a Bi-Functional Response Element in the Rat GnRH Receptor Gene

2013 ◽  
Vol 27 (1) ◽  
pp. 74-91 ◽  
Author(s):  
Anne-Laure Schang ◽  
Anne Granger ◽  
Bruno Quérat ◽  
Christian Bleux ◽  
Joëlle Cohen-Tannoudji ◽  
...  
Keyword(s):  
Cell Fate ◽  
Promoter Activity ◽  
Receptor Gene ◽  
Gnrh Receptor ◽  
Expression Vectors ◽  
Homeodomain Protein ◽  
Homeodomain Proteins ◽  
Enhancer Activity ◽  
Response Element ◽  
Lim Homeodomain

GATA2 transcription factor and LIM homeodomain proteins Islet1 (ISL1) and LIM homeobox 3 (LHX3) are suspected to be involved in gonadotrope cell fate and maintenance. The GnRH receptor gene (Gnrhr), crucial for gonadotrope function, is expressed in the pituitary gland from embryonic day 13.5 onward, well before LH and FSH β-subunits. This expression pattern together with the presence of WGATAR and TAAT motifs in Gnrhr promoter sequences suggests the involvement of early transcription factors in promoter activation. In this study, using a well-characterized transgenic mouse model, GATA2 was found colocalized with Gnrhr promoter activity in the pituitary. Transient transfection of Gnrhr promoter luciferase fusion constructs together with either GATA2 expression vectors or small interfering RNA in gonadotrope cell lines indicated that GATA2, which typically acts as a trans-activator, unexpectedly repressed Gnrhr promoter activity. Using DNA chromatography affinity and EMSA, we demonstrated that GATA2 operates via a response element containing a peculiar palindromic GATA motif that overlaps a critical TAAT motif involved in LHX3/ISL1 trans-activation. Indeed, despite the inhibitory action of GATA2, this element displayed a clear-cut enhancer activity in gonadotrope cells. Chromatin immunoprecipitation assays indicated that GATA2, LHX3, and ISL1 interact with a Gnrhr promoter fragment encompassing this element. The trans-repressive action of GATA2 on Gnrhr promoter activity is likely balanced or even hindered by trans-activating effects of LIM homeodomain proteins via this novel bifunctional LIM/GATA response element. Such a hierarchical interplay may contribute to finely adjust Gnrhr gene expression in gonadotrope cell lineage during pituitary development as well as in the adult animal.

The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 881-896 ◽  
Author(s):  
I. Valarche ◽  
J.P. Tissier-Seta ◽  
M.R. Hirsch ◽  
S. Martinez ◽  
C. Goridis ◽  
...  
Keyword(s):  
Nervous System ◽  
Cell Adhesion ◽  
Peripheral Nervous System ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Homeodomain Protein ◽  
Sensory Ganglia ◽  
Homeodomain Proteins ◽  
Regulatory Cascade

Transcriptional regulation of the gene encoding the cell adhesion receptor NCAM (neural cell adhesion molecule), a putative effector molecule of a variety of morphogenetic events, is likely to involve important regulators of morphogenesis. Here we identify two mouse homeodomain proteins that bind to an upstream regulatory element in the Ncam promoter: Cux, related to Drosophila cut and human CDP, and Phox2, a novel protein with a homeodomain related to that of the Drosophila paired gene. In transient transfection experiments, Cux was found to be a strong inhibitor of Ncam promoter activity, and this inhibition could be relieved by simultaneously overexpressing Phox2. These results suggest that the Ncam gene might be a direct target of homeodomain proteins and provide a striking example of regulatory cross-talk between homeodomain proteins of different classes. Whereas the expression pattern of Cux/CDP includes many NCAM-negative sites, Phox2 expression was restricted to cells also expressing Ncam or their progenitors. The localisation data thus strongly reinforce the notion that Phox2 plays a role in transcriptional activation of Ncam in Phox2-positive cell types. In the peripheral nervous system, Phox2 was strongly expressed in all ganglia of the autonomic nervous system and more weakly in some cranial sensory ganglia, but not in the sensory ganglia of the trunk. Phox2 transcripts were detected in the primordia of sympathetic ganglia as soon as they form. Phox2 expression in the brain was confined to spatially restricted domains in the hindbrain, which correspond to the noradrenergic and adrenergic nuclei once they are identifiable. All Phox2-expressing components of the peripheral nervous system are at least transiently adrenergic or noradrenergic. In the developing brain, Phox2 was expressed at all known locations of (nor)adrenergic neurones and of their precursors. These results suggest that Phox2, in addition to regulating the NCAM gene, may be part of the regulatory cascade that controls the differentiation of neurons towards this neurotransmitter phenotype.

Differential expression of a transcription regulatory factor, the LIM domain only 4 protein Lmo4, in muscle sensory neurons

Development ◽  
2002 ◽  
Vol 129 (21) ◽  
pp. 4879-4889
Author(s):  
Hsiao-Huei Chen ◽  
Joseph W. Yip ◽  
Alexandre F. R. Stewart ◽  
Eric Frank
Keyword(s):  
Differential Expression ◽  
Sensory Neurons ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
Expression Patterns ◽  
Cdna Libraries ◽  
Homeodomain Protein ◽  
Lim Domain ◽  
Homeodomain Proteins ◽  
Lim Homeodomain

In the stretch-reflex system, proprioceptive sensory neurons make selective synaptic connections with different subsets of motoneurons, according to the peripheral muscles they supply. To examine the molecular mechanisms that may influence the selection of these synaptic targets, we constructed single-cell cDNA libraries from sensory neurons that innervate antagonist muscles. Differential screening of these libraries identified a transcription regulatory co-factor of the LIM homeodomain proteins, the LIM domain only 4 protein Lmo4, expressed in most adductor but few sartorius sensory neurons. Differential patterns of Lmo4 expression were also seen in sensory neurons supplying three other muscles. A subset of motoneurons also expresses Lmo4 but the pattern of expression is not specific for motor pools. Differential expression of Lmo4 occurs early, as neurons develop their characteristic LIM homeodomain protein expression patterns. Moreover, ablation of limb buds does not block Lmo4 expression, suggesting that an intrinsic program controls the early differential expression of Lmo4. LIM homeodomain proteins are known to regulate several aspects of sensory and motor neuronal development. Our results suggest that Lmo4 may participate in this differentiation by regulating the transcriptional activity of LIM homeodomain proteins.

Identification and Analysis of Two Novel Sites of Rat GnRH Receptor Gene Promoter Activity: The Pineal Gland and Retina

2012 ◽  
Vol 97 (2) ◽  
pp. 115-131 ◽  
Author(s):  
Anne-Laure Schang ◽  
Christian Bleux ◽  
Marie-Claude Chenut ◽  
Valérie Ngô-Muller ◽  
Bruno Quérat ◽  
...  
Keyword(s):  
Pineal Gland ◽  
Promoter Activity ◽  
Receptor Gene ◽  
Gene Promoter ◽  
Gnrh Receptor

scholarly journals Insulin Stimulates the Expression of Carbohydrate Response Element Binding Protein (ChREBP) by Attenuating the Repressive Effect of Pit-1, Oct-1/Oct-2, and Unc-86 Homeodomain Protein Octamer Transcription Factor-1

Endocrinology ◽  
2009 ◽  
Vol 150 (8) ◽  
pp. 3483-3492 ◽  
Author(s):  
Adam S. Sirek ◽  
Ling Liu ◽  
Mark Naples ◽  
Khosrow Adeli ◽  
Dominic S. Ng ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Expression ◽  
Binding Site ◽  
Promoter Activity ◽  
Homeodomain Protein ◽  
Response Element ◽  
Repressive Effect ◽  
Element Binding Protein ◽  
Mrna And Protein Expression ◽  
Transcription Factor 1

The carbohydrate response element binding protein (ChREBP) has been recognized as a key controller of hepatic lipogenesis. Whereas the function of ChREBP has been extensively investigated, mechanisms underlying its transcription remain largely unknown, although ChREBP production is elevated in a hyperinsulinemic mouse model. We located a conserved Pit-1, Oct-1/Oct-2, and Unc-86 (POU) protein binding site (ATGCTAAT) within the proximal promoter region of human ChREBP. This site interacts with the POU homeodomain protein octamer transcription factor-1 (Oct-1), as detected by gel shift and chromatin immunoprecipitation assays. Oct-1 cotransfection in the human HepG2 cell line repressed ChREBP promoter activity approximately 50–75% (P < 0.01 to P < 0.001), and this repression was dependent on the existence of the POU binding site. Furthermore, overexpression of Oct-1 repressed endogenous ChREBP mRNA and protein expression, whereas knockdown of Oct-1 expression, using a lentivirus-based small hairpin RNA approach, led to increased ChREBP mRNA and protein expression. In contrast, HepG2 cells treated with 10 or 100 nm insulin for 4 or 8 h resulted in an approximately 2-fold increase of ChREBP promoter activity (P < 0.05 to P < 0.01). Insulin (10 nm) also stimulated endogenous ChREBP expression in HepG2 and primary hamster hepatocytes. More importantly, we found that the stimulatory effect of insulin on ChREBP promoter activity was dependent on the presence of the POU binding site, and insulin treatment reduced Oct-1 expression levels. Our observations therefore identify Oct-1 as a transcriptional repressor of ChREBP and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.

scholarly journals The Tripartite Basal Enhancer of the Gonadotropin-Releasing Hormone (GnRH) Receptor Gene Promoter Regulates Cell-Specific Expression Through a Novel GnRH Receptor Activating Sequence

1997 ◽  
Vol 11 (12) ◽  
pp. 1814-1821 ◽  
Author(s):  
Dawn L. Duval ◽  
Scott E. Nelson ◽  
Colin M. Clay
Keyword(s):  
Transcriptional Activation ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Receptor Gene ◽  
Gnrh Receptor ◽  
Luciferase Expression ◽  
Functional Requirements ◽  
Specific Expression ◽  
Core Motif

Abstract The molecular mechanisms regulating restricted expression of GnRH receptor and gonadotropin subunit genes to gonadotrope cells have been the focus of intense interest. Using deletion and mutational analysis we have identified a tripartite enhancer that regulates cell-specific expression of the GnRH receptor gene in the gonadotrope-derived αT3–1 cell line. Individual elements of this enhancer include binding sites for steroidogenic factor-1; activator protein 1 (AP-1); and a novel element referred to as the GnRH receptor activating sequence (GRAS). Mutation of each element alone results in loss of approximately 60% of promoter activity. Combinatorial mutations of any two elements decreases promoter activity by approximately 80%. Finally, mutation of all three elements reduces promoter activity to a level not different from promoterless vector. Using 2-bp mutations, we have defined the functional requirements for transcriptional activation by GRAS. The core motif of GRAS is at −391 to −380 bp relative to the start site of translation and has the sequence 5′-CTAGTCACAACA-3′. Three copies of GRAS or GRAS with a 2-bp mutation (μGRAS) were cloned into a luciferase expression vector immediately upstream of the thymidine kinase minimal promoter (TK) and tested for expression in αT3–1 cells. When compared with TK promoter alone, activity of 3xGRAS-TKLUC was increased by more than 5-fold while activity of 3xμGRAS-TKLUC was unchanged. When 3xGRAS-TKLUC was transfected into a variety of nongo-nadotrope cell lines, it did not increase activity of the TK promoter. We propose that basal activity of the GnRH receptor gene is regulated by a tripartite enhancer, and the key component of this enhancer is an element, GRAS, that activates transcription in a cell-specific fashion.

Sp1/Sp3 binding is associated with cell-specific expression of the glucose-dependent insulinotropic polypeptide receptor gene

2006 ◽  
Vol 290 (6) ◽  
pp. E1287-E1295 ◽  
Author(s):  
Michael O. Boylan ◽  
Lisa I. Jepeal ◽  
M. Michael Wolfe
Keyword(s):  
Promoter Activity ◽  
Receptor Gene ◽  
Expression Vectors ◽  
Target Cells ◽  
Specific Expression ◽  
Binding Motifs ◽  
Insulinoma Cell ◽  
Specific Receptors ◽  
Rat Insulinoma Cell Line ◽  
Flanking Regions

The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor ( GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between −1 and −100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5′-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between −85 and −40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions −77, −60, and −50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at −50 and −60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at −70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between −100 and −40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.

Molecular structure of the human gonadotropin-releasing hormone receptor gene

1997 ◽  
pp. 183-192 ◽  
Author(s):  
SS Kakar
Keyword(s):  
Cell Line ◽  
Receptor Gene ◽  
Tumor Cell Line ◽  
Gnrh Receptor ◽  
Response Element ◽  
Reading Frame ◽  
Gnrh Receptors ◽  
Untranslated Sequence ◽  
Flanking Region ◽  
Transcriptional Start Sites

GnRH receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent tumor tissues. Recently, cDNAs for the GnRH receptors of several species including the human have been cloned. To determine the structure of the gene encoding the human GnRH receptor, we isolated the receptor gene clones from the human genomic libraries. Comparison of the genomic and cDNA sequences revealed that the human GnRH receptor gene is composed of three exons and two introns and spans over 20 kb in size. Exon 1 encodes the 5' untranslated sequence and nucleotide +1 to +522 in the open reading frame, exon 2 encodes nucleotide +523 to +742 and exon 3 encodes nucleotide +743 to +987 in the open reading frame as well as the 3' untranslated sequence. Southern blot analysis of genomic DNA and localization of the GnRH receptor gene to a single site on human chromosome 4 (4q13) indicate the presence of a single copy of the gene in the human genome. Several regulatory sequences for various hormones and other regulatory factors were identified, including PEA-3, AP-1, AP-2, and Pit-1 sites. In addition, glucocorticoid/progesterone response element thyroid hormone response element, and cAMP response element sequences were identified. Reverse transcriptase-primer extension and 5' RACE analysis of the human pituitary RNA demonstrated the presence of multiple transcriptional start sites upstream of the translational start site. Analysis of the 5' flanking region of the gene also revealed the presence of multiple TATA and CAAT sequences. The finding of multiple transcriptional start sites raises the possibility of tissue-specific regulation and the existence of variable size transcripts. Chimeras containing 1.26 kb (-534 to 728) of the 5' flanking region of the receptor gene and the luciferase (Luc) gene expressed a significant luciferase activity when transfected into a human endometrial tumor cell line (HEC-1A) and a breast tumor cell line (MCF-7) but not in a mouse pituitary gonadotrope cell line (alpha T3-1), suggesting the existence of multiple promoter elements in the gene. These findings indicate a multiplicity of regulation of expression of the GnRH receptor and provide the substrate for detailed investigation in the reproductive system.

scholarly journals The LIM-Homeodomain Proteins Isl-1 and Lhx3 Act with Steroidogenic Factor 1 to Enhance Gonadotrope-Specific Activity of the Gonadotropin-Releasing Hormone Receptor Gene Promoter

2006 ◽  
Vol 20 (9) ◽  
pp. 2093-2108 ◽  
Author(s):  
Anne Granger ◽  
Christian Bleux ◽  
Marie-Laure Kottler ◽  
Simon J. Rhodes ◽  
Raymond Counis ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Specific Activity ◽  
Receptor Gene ◽  
Marker Gene ◽  
Gene Promoter ◽  
Dominant Negative ◽  
R Gene ◽  
Specific Expression ◽  
Steroidogenic Factor 1 ◽  
Lim Homeodomain

Abstract The GnRH receptor (GnRH-R) plays a central role in mammalian reproductive function throughout adulthood. It also appears as an early marker gene of the presumptive gonadotrope lineage in developing pituitary. Here, using transient transfections combined with DNA/protein interaction assays, we have delineated cis-acting elements within the rat GnRH-R gene promoter that represent targets for the LIM-homeodomain (LIM-HD) proteins, Isl-1 and Lhx3. These factors, critical in early pituitary development, are thus also crucial for gonadotrope-specific expression of the GnRH-R gene. In heterologous cells, the expression of Isl-1 and Lhx3, together with steroidogenic factor 1 (SF-1), culminates in the activation of both the rat as well as human GnRH-R promoter, suggesting that this combination is evolutionarily conserved among mammals. The specificity of these LIM-HD factors is attested by the inefficiency of related proteins, including Lhx5 and Lhx9, to activate the GnRH-R gene promoter, as well as by the repressive capacity of a dominant-negative derivative of Lhx3. Accordingly, targeted deletion of the LIM response element decreases promoter activity. In addition, experiments with Gal4-SF-1 fusion proteins suggest that LIM-HD protein activity in gonadotrope cells is dependent upon SF-1 binding. Finally, using a transgenic model that allows monitoring of in vivo promoter activity, we show that the overlapping expression of Isl-1 and Lhx3 in the developing pituitary correlates with promoter activity. Collectively, these data suggest the occurrence of a specific LIM-HD pituitary code and designate the GnRH-R gene as the first identified transcriptional target of Isl-1 in the anterior pituitary.

scholarly journals Mouse GnRH Receptor Gene Expression Is Mediated by the LHX3 Homeodomain Protein

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2180-2185 ◽  
Author(s):  
Shauna M. McGillivray ◽  
Janice S. Bailey ◽  
Rosha Ramezani ◽  
Brian J. Kirkwood ◽  
Pamela L. Mellon
Keyword(s):  
Hormonal Regulation ◽  
Transcriptional Start Site ◽  
Receptor Gene ◽  
Regulatory Elements ◽  
Gnrh Receptor ◽  
Homeodomain Protein ◽  
Pituitary Development ◽  
Basal Expression

Abstract Appropriate expression of GnRH receptor (GnRHR) is necessary for the correct regulation of the gonadotropins, LH and FSH, by GnRH. GnRHR is primarily expressed in the gonadotrope cells of the anterior pituitary, and a number of regulatory elements important for both basal and hormonal regulation of the gene have been identified. Using the gonadotrope-derived cell line, αT3-1, that endogenously expresses GnRHR, we have identified an ATTA element located at −298 relative to the transcriptional start site that is essential for basal expression of the GnRHR gene. LHX3, a member of the LIM homeodomain family, binds the −298 ATTA site in vitro as well as to the endogenous GnRHR promoter in vivo. Additionally, LHX3 specifically activates through this −298 ATTA site in transient transfection assays. LHX3 is essential for pituitary development and has been implicated in the regulation of a number of pituitary specific genes; however, this is the first report identifying its role in the regulation of GnRHR.

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