Cga Promoter Research Articles

L3MBTL2-mediated CGA transcriptional suppression promotes pancreatic cancer progression through modulating autophagy

SummaryL3MBTL2 is a crucial component of ncPRC1.6 and has been implicated in transcriptional repression and chromatin compaction. However, the repression mechanism of L3MBTL2 and its biological functions are largely undefined. Here, we found that L3MBTL2 plays a distinct oncogenic role in tumor development. We demonstrated that L3MBTL2 repressed downstream CGA through an H2AK119ub1-dependent mechanism. Importantly, the binding of the MGA/MAX heterodimer to the E-box on the CGA promoter enhanced the specific selective repression of CGA by L3MBTL2. CGA encodes the alpha subunit of glycoprotein hormones; however, we showed that CGA plays an individual tumor suppressor role in PDAC. Moreover, CGA-transcript1 (T1) was identified as the major transcript, and the tumor suppression function of CGA-T1 depends on its own glycosylation. Furthermore, glycosylated CGA-T1 inhibited PDAC, partly by repression of autophagy through multiple pathways, including PI3K/Akt/mTOR and TP53INP2 pathways. These findings reveal the important roles of L3MBTL2 and CGA in tumor development.

Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

Force Spectroscopy of Nucleosomes at the Promoters of the Lh Genes Reveals two Distinct Strategies for their Regulation

Luteinizing hormone (LH) is a heterodimeric protein that regulates function of the gonads, and is composed of two subunits, which are encoded by two different genes: chorionic gonadotropin-a (Cga) and Lhs. A sequence-based algorithm designed to predict nucleosome positions on DNA suggests that the promoters of both genes, and in particular the binding regions for important transcription factors, are packed into nucleosomes. Since gene expression requires that the promoter be accessible to the cellular transcription machinery, we hypothesized that nucleosome positioning regulates basal expression of these genes. We are performing single-molecule force spectroscopy with optical tweezers, complemented by MNase mapping, to probe the architecture of chromatin on both gene promoters. In gonadotrope cells, the promoter of Lhs is occupied by a nucleosome containing the variant H2A.Z, while the Cga promoter appears nucleosome-depleted. Optical tweezers experiments show that a nucleosome reconstituted on the Cga promoter is weaker, and poorly localized, as compared to the one on the promoter of LHs. This may suggest that it is evicted, in vivo, by chromatin remodelers. In contrast, the first nucleosome downstream of the transcription start site contains H2A.Z in Cga but H2A in Lhs, and is well localized on both genes, although more weakly bound on the Lhs than the Cga gene. Hence, these two genes are apparently regulated by distinct strategies, tailored to obtain higher expression for Cga, which encodes a subunit shared also by other hormones, and lower for Lhs, which is unique to this hormone. Our results highlight how DNA sequence, the usage of alternative histone variants and, possibly, remodeling machinery act synergistically to modulate gene expression.

Transcription of follicle-stimulating hormone subunit genes is modulated by porcine LIM homeobox transcription factors, LHX2 and LHX3.

The LIM-homeobox transcription factors LHX2 and LHX3s (LHX3a and LHX3b) are thought to be involved in regulating the pituitary glycoprotein hormone subunit genes Cga and Fshβ. These two factors show considerable differences in their amino acid sequences for DNA binding and protein-protein interactions and in their vital function in pituitary development. Hence, we compared the DNA binding properties and transcriptional activities of Cga and Fshβ between LHX2 and LHX3s. A gel mobility shift assay for approximately 1.1 kb upstream of Cga and 2.0 kb upstream of Fshβ varied in binding profiles between LHX2 and LHX3s. DNase I footprinting revealed DNA binding sites in 8 regions of the Cga promoter for LHX2 and LHX3s with small differences in the binding range and strength. In the Fshβ promoter, 14 binding sites were identified for LHX2 and LHX3, respectively. There were alternative binding sites to either gene in addition to similar differences observed in the Cga promoter. The transcriptional activities of LHX2 and LHX3s according to a reporter assay showed cell-type dependent activity with repression in the pituitary gonadotrope lineage LβT2 cells and stimulation in Chinese hamster ovary lineage CHO cells. Reactivity of LHX2 and LHX3s was observed in all regions, and differences were observed in the 5'-upstream region of Fshβ. However, immunohistochemistry showed that LHX2 resides in a small number of gonadotropes in contrast to LHX3. Thus, LHX3 mainly controls Cga and Fshβ expression.

Squelching of ETS2 Transactivation by POU5F1 Silences the Human Chorionic Gonadotropin CGA Subunit Gene in Human Choriocarcinoma and Embryonic Stem Cells

The subunit genes encoding human chorionic gonadotropin, CGA, and CGB, are up-regulated in human trophoblast. However, they are effectively silenced in choriocarcinoma cells by ectopically expressed POU domain class 5 transcription factor 1 (POU5F1). Here we show that POU5F1 represses activity of the CGA promoter through its interactions with ETS2, a transcription factor required for both placental development and human chorionic gonadotropin subunit gene expression, by forming a complex that precludes ETS2 from interacting with the CGA promoter. Mutation of a POU5F1 binding site proximal to the ETS2 binding site does not alter the ability of POU5F1 to act as a repressor but causes a drop in basal promoter activity due to overlap with the binding site for DLX3. DLX3 has only a modest ability to raise basal CGA promoter activity, but its coexpression with ETS2 can up-regulate it 100-fold or more. The two factors form a complex, and both must bind to the promoter for the combination to be transcriptionally effective, a synergy compromised by POU5F1. Similarly, in human embryonic stem cells, which express ETS2 but not CGA, ETS2 does not occupy its binding site on the CGA promoter but is found instead as a soluble complex with POU5F1. When human embryonic stem cells differentiate in response to bone morphogenetic protein-4 and concentrations of POU5F1 fall and hCG and DLX3 rise, ETS2 then occupies its binding site on the CGA promoter. Hence, a squelching mechanism underpins the transcriptional silencing of CGA by POU5F1 and could have general relevance to how pluripotency is maintained and how the trophoblast lineage emerges from pluripotent precursor cells.

Polymorphism of the porcine CGA gene and its association with growth and carcass traits

Glycoprotein hormones in the pituitary gland affect a myriad of biological processes such as development, growth, metabolic control and gametogenesis. The quantitative trait loci (QTL) near their common glycoprotein α subunit gene (CGA) have been reported inconsistently. The aim of this study was to dissect potential genetic factors for these unstable results and validate the association or linkage relationship of CGA gene with growth and carcass traits. By resequencing all the exons and part of the introns of the porcine CGA gene, 22 polymorphisms in total were identified in this study. Five single nucleotide polymorphism (SNP) markers were chosen and evaluated in six pure-bred pig breeds (n = 228). Breed-specific haplotypes were found and a map of the porcine CGA polymorphisms’ evolution history was inferred. A resource family (n = 365) with different genetic backgrounds from those used in other papers was used to perform an association study. The resource family was created based on crosses of Pietrain and Jinhua pigs (Central China type pigs). Results indicated that a low correlation between haplotype blocks may abolish each other’s effects. Moreover, a significant association of SNP C-925T with growth rate and back-fat thickness in this study confirmed the existence of previously reported QTL. SNP C-925T and SNP A+15599G could be useful linkage markers and SNP C-925T may also be a candidate causative SNP for the corresponding traits. Further investigation for variants within of the CGA promoter region and their association with growth rate and back-fat traits is suggested. Keywords: Common glycoprotein α subunit gene, haplotype, SNP, tetra-primer ARMS, swine

GnRH-Regulated Expression of Jun and JUN Target Genes in Gonadotropes Requires a Functional Interaction between TCF/LEF Family Members and β-Catenin

GnRH regulates gonadotrope function through a complex transcriptional network that includes three members of the immediate early gene family: Egr1, Jun, and Atf3. These DNA-binding proteins act alone or in pairs to confer hormonal responsiveness to Cga, Lhb, Fshb, and Gnrhr. Herein we suggest that the transcriptional response of Jun requires a functional interaction between the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of DNA-binding proteins and beta-catenin (officially CTNNB1), a coactivator of TCF/LEF. Supporting data include demonstration that GnRH increases activity of TOPflash, a TCF/LEF-dependent luciferase reporter, in LbetaT2 cells, a gonadotrope-derived cell line. Additional cotransfection experiments indicate that a dominant-negative form of TCF7L2 (TCFDN) that binds DNA, but not beta-catenin, blocks GnRH induction of TOPflash. Overexpression of AXIN, an inhibitor of beta-catenin, also reduces GnRH stimulation of TOPflash. Transduction of LbetaT2 cells with TCFDN adenoviruses diminishes GnRH stimulation of Jun mRNA without altering expression of Egr1 and Atf3, two other immediate early genes that confer GnRH responsiveness. Reduction of beta-catenin in LbetaT2 cells, through stable expression of short hairpin RNA, also selectively compromises GnRH regulation of Jun expression and levels of JUN protein. Finally, overexpression of TCFDN attenuates GnRH regulation of Cga promoter activity, a known downstream target of JUN. Together, these results indicate that GnRH regulation of Jun transcription requires a functional interaction between TCF/LEF and beta-catenin and that alteration of either impacts expression of JUN downstream targets such as Cga.

Regulation of Porcine Pituitary Glycoprotein Hormone Alpha Subunit Gene with LIM-Homeobox Transcription Factor Lhx3

The aim of this study was to characterize the promoter activity of the porcine pituitary glycoprotein hormone common alpha gene (Cga) promoter (-1059/+12) and the role of LIM homeodomain transcription factor Lhx3. A transfection assay using Chinese hamster ovary (CHO) cells showed that the -1059/-101 region of the Cga promoter definitely responds to Lhx3 and that the -1059/-240 region exhibits a high basal transcriptional level in a pituitary-derived cell line, LbetaT2. A DNA binding and DNase I footprinting assay demonstrated that Lhx3 has seven binding sites in the -1059/+12 region of Cga, including a pituitary glycoprotein hormone basal element (PGBE) known as a LIM homeodomain factor-binding site. A transfection assay of the sequence of Lhx3-binding sites fused with minimal promoter vector confirmed their Lhx3-dependent stimulations in LbetaT2 cells. RT-PCR analysis of porcine pituitary ontogeny demonstrated that porcine Lhx3 showed striking changes of expression in both sexes during the fetal period but a stable high level of expression after birth. Thus, the porcine Cga promoter is regulated by Lhx3 through seven sites in the distal and proximal regions.

Single-stranded DNA-binding proteins regulate the abundance and function of the LIM-homeodomain transcription factor LHX2 in pituitary cells

A family of single-stranded DNA-binding proteins (or SSBPs) has been shown to augment the function of LIM-homeodomain (LIM-HD) transcription factors in embryogenesis by interaction with LIM domain-binding protein-1 (LDB1). No DNA-binding complex has been described, however, containing a LIM-HD protein, LDB1, and SSBP, and the mechanism by which SSBPs affect LIM-HD function had not been elucidated. Through use of electrophoretic mobility shift, antibody supershift, and ChIP analyses, we show that an Lhx2–Ldb1–Ssbp3 complex binds a specific element in the Lhx2 target gene Cga (encoding the alpha subunit of glycoprotein hormones) in the αT3-1 pituitary cell line. Using overexpression and knockdown approaches, we demonstrate that SSBP3 inhibits Lhx2 and Ldb1 turnover, stimulates assembly of this DNA-binding complex, promotes its recruitment to the Cga promoter, and enhances Cga transcription. These studies provide novel insights into the regulation of pituitary gene expression and LIM-HD function more generally.

Single-Stranded DNA-Binding Proteins (SSBPs) Regulate the Abundance of the LIM-Homeodomain Protein LHX2 and Augment Its Transcriptional Activity.

A small family of proteins with putative single-stranded DNA-binding activity has been shown to augment the biological actions of LIM-homeodomain (LIM-HD) transcription factors through the mediation of the LIM domain-binding protein LDB1. We recently established that two of these SSBPs, Ssbp2 and Ssbp3, were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing transcription factors Tal1, E2A, and Gata-1 and LIM-only protein Lmo2 and showed that Ssbp2 stimulated E box-GATA DNA-binding activity by inhibiting Ldb1 ubiquitination and Ldb1 and Lmo2 turnover (Genes & Dev. 21:942–955, 2007). Since LIM-HD proteins are substrates of different E3 ubiquitin ligases than LIM-only proteins and have the additional property of binding DNA, we sought to determine the effect of SSBPs on LIM-HD expression and function. Using the prototype LIM-HD protein Lhx2 and one of its best-characterized target genes, Cga, for analysis, we found that an Ssbp3-, Ldb1-, and Lhx2-containing complex associated with an Lhx2 binding element in the Cga promoter in vitro and in mouse pituitary cells (alphaT3-1 cell line) in vivo. We then showed that enforced expression of Ssbp2 and Ssbp3 in alphaT3-1 cells increased Lhx2 and Ldb1 protein abundance, Lhx2 DNA-binding activity, and Cga expression and augmented Lhx2 transcriptional activity in an Ldb1-dependent fashion. While Lhx2-Ldb1-Ssbp3 DNA-binding activity increased in Ssbp3- relative to vector-transfected cells, the affinity of this complex for DNA was unaltered. Similar to the effect of Ssbp2 on Lmo2 in murine erythroleukemia (MEL) cells, overexpressed Ssbp3 reduced Lhx2 protein turnover in cycloheximide-treated alphaT3-1 cells without affecting Lhx2 RNA levels. In contrast, knockdown of endogenous Ssbp3, but not Ssbp2 which is expressed at much lower levels in these cells, reduced Lhx2 and Ldb1 abundance, Lhx2 DNA-binding activity, Lhx2, Ldb1, and Ssbp3 loading onto the Cga promoter, Cga promoter activity, and endogenous Cga gene expression. Significantly, neither overexpression nor knockdown of Ssbp2 in MEL cells, which express both the LIM-only protein Lmo2 and LIM-HD protein Lhx2, affected Lhx2 protein abundance, and Lhx2 DNA-binding activity was undetectable in nuclear extracts from these cells despite the presence of immunoreactive Lhx2. These studies indicate that SSBP augmentation of LIM-HD function results from Ldb1-mediated inhibition of LIM-HD protein turnover and increased assembly of a LIM-HD/LDB1/SSBP DNA-binding complex. The much greater affinity for LDB1 of LIM-only compared to LIM-HD proteins is likely a major determinant of the SSBP effect on LIM-HD protein abundance. Finally, these findings are consistent with cell type-specific contributions of different SSBPs, even for similar LDB1-dependent actions.

The C terminus of the synovial sarcoma-associated SSX proteins interacts with the LIM homeobox protein LHX4

As a result of the synovial sarcoma-associated t(X;18) translocation, the SS18 gene on chromosome 18 is fused to either one of the three closely related SSX genes on the X chromosome. The SS18 protein is thought to act as a transcriptional co-activator, whereas the SSX proteins are thought to act as transcriptional corepressors. The main SSX-repression domain is located in its C terminus, a domain that is retained in the respective SS18-SSX fusion proteins. Both the SS18 and SSX proteins lack DNA-binding domains. Previously, we found that the SS18 and SS18-SSX fusion proteins may be tethered to DNA targets via the SS18-interacting protein AF10. Here, we set out to isolate proteins that interact with the SSX C-terminal repression domain using a yeast two-hybrid interaction trap. Of the positive clones isolated, two corresponded to the LIM homeobox protein LHX4, a DNA-binding protein that is involved in transcription regulation. An endogenous interaction was subsequently established in mammalian cells via colocalization and coimmunoprecipitation of the respective proteins. Interestingly, the LHX4 gene was previously found to be deregulated in various human leukemias. In addition, it was previously found that LIM homeobox proteins may bind to and activate the glycoprotein-alpha (CGA) promoter. Using LHX4 chromatin immunoprecipitation and CGA-promoter assays, we found that endogenous LHX4 binds to the CGA promoter and that LHX4-mediated CGA activation is enhanced by the SS18-SSX protein, but not by the SSX protein. Taken together, we conclude that this novel protein - protein interaction may have direct consequences for the (de)regulation of SSX and/or SS18-SSX target genes and, thus, for the development of human synovial sarcomas.

Cyclic Adenosine 3′,5′Monophosphate/Protein Kinase A and Mitogen-Activated Protein Kinase 3/1 Pathways Are Involved in Adenylate Cyclase-Activating Polypeptide 1-Induced Common Alpha-Glycoprotein Subunit Gene (Cga) Expression in Mouse Pituitary Gonadotroph LbetaT2 Cells

Adenylate cyclase-activating polypeptide 1 (ADCYAP1) binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathways in pituitary gonadotrophs. In this study, we investigated the cAMP and MAPK3/1 signaling pathways induced by ADCYAP1 stimulation and examined the effects of ADCYAP1 on the expression of gonadotropin subunit genes using a clonal gonadotroph cell line, LbetaT2. ADCYAP1 increased intracellular cAMP accumulation up to 19-fold in LbetaT2 cells. Common alpha-glycoprotein subunit gene (Cga) promoter activity was strongly activated by both ADCYAP1 and the cyclic-AMP analog, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Both had little effect on luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoter activities. Cga promoter activity was significantly increased by transfection with constitutively active cAMP-dependent protein kinase (PKA). Activities of the Lhb and Fshb promoters were only modestly increased. Both ADCYAP1 and CPT-cAMP induced MAPK3/1 activation in LbetaT2 cells. The MEK inhibitor, U0126, and the PKA inhibitors, H89 and cAMP-dependent protein kinase peptide inhibitor (PKI), completely inhibited MAPK3/1 activation by either ADCYAP1 or CPT-cAMP. Using luciferase reporter constructs containing cis-elements, the cAMP response element (Cre) promoter was stimulated about 4-fold by ADCYAP1. ADCYAP1-induced Cre promoter activity was completely inhibited by H89, but not by U0126. ADCYAP1 also increased the activity of the serum response element (Sre) promoter, a target for MAPK3/1, and treatment of the cells with U0126 completely inhibited ADCYAP1-induced Sre promoter activity. ADCYAP1-increased Cga promoter activity was inhibited partially by both H89 and U0126. Although combining the inhibitors showed an additive inhibition effect, it did not result in complete inhibition. These results suggest that in LbetaT2 cells, ADCYAP1 mainly increases Cga through activation of PKA and MAPK3/1, as well as through an additional unknown pathway.

Regulation of chromogranin A transcription and catecholamine secretion by the neuropeptide PACAP. Stimulation and desensitization.

We propose a model depicting putative signal transduction pathways underlying PACAP-induced CGA transcription and catecholamine secretion in PC12 cells (Fig 7). PACAP mediates both secretion and transcription through the PAC 1 receptor, but with quite different post-receptor signaling pathways. PACAP signals to CGA transcription through a Ca2+-independent pathway involving the CGA promoter CRE domain in cis and PKA and the transcription factor CREB in trans. PACAP-evoked secretion and transcription are subject to homologous desensitization in PC 12 cells; however, PACAP also provokes long-lasting secretion, even under dose and time circumstances where acute, DHP-sensitive secretion has been desensitized. While initial secretion is mediated by an L-type VOCC, extended secretion may involve a SOCC activated through a Gq/11/PLC-β/PI signaling pathway. Further characterization of PACAP signaling pathways will require definitive identification of the SOCC channel involved in the sustained catecholamine release.