The PROGINS polymorphism of the human progesterone receptor diminishes the response to progesterone

The human progesterone receptor (PR) is a ligand-dependent transcription factor and two isoforms, (PRA and PRB), can be distinguished. PROGINS, a PR polymorphic variant, affects PRA and PRB and acts as a risk-modulating factor in several gynaecological disorders. Little is known about the functional consequences of this variant. Here, we characterise the properties of PROGINS with respect to transcription, mRNA maturation, protein activity and proliferation. PROGINS is characterised by a 320 bp PV/HS-1 Alu insertion in intron G and two point mutations, V660L in exon 4 and H770H (silent substitution) in exon 5. The Alu element contains a half oestrogen-response element/Sp1-binding site (Alu-ERE/Sp1), which acts as an in-cis intronic enhancer leading to increased transcription of the PROGINS allele in response to 17β-oestradiol. Moreover, Alu insertions in the human genome are frequently methylated. Our data indicate that the PROGINS-Alu does not affect gene transcription due to DNA methylation. However, the Alu element reduced the stability of the PROGINS transcript compared with the CP allele and does not generate splice variants. The amino acid substitution (V600L) in exon 4 leads to differences in PR phosphorylation and degradation in the two PR variants upon ligand binding, most likely as a result of differences in the three-dimensional structures of the two PR variants. As a consequence, the PR-L660 (PROGINS) variant (1) displays decreased transactivation activity in a luciferase reporter system and (2) is less efficient in opposing cell proliferation in hamster ovarian cells expressing human PRA, when compared with the PR-V660 (most common variant). Taken together, our results indicate that the PROGINS variant of PR is less responsive to progestin compared with the most common PR because of (i) reduced amounts of gene transcript and (ii) decreased protein activity.

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Development of a High-Throughputin VitroBioassay to Assess Potencies of Progestagenic Compounds Using Chinese Hamster Ovary Cells Stably Transfected with the Human Progesterone Receptor and a Luciferase Reporter System

Analytical Biochemistry ◽

10.1006/abio.1998.2681 ◽

1998 ◽

Vol 261 (2) ◽

pp. 222-224 ◽

Cited By ~ 14

Author(s):

Willem G.E.J. Schoonen ◽

Rob J.H. de Ries ◽

Jan W.H. Joosten ◽

Gerrie J.W. Mathijssen-Mommers ◽

Helenius J. Kloosterboer

Keyword(s):

Progesterone Receptor ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Luciferase Reporter ◽

Reporter System ◽

Ovary Cells ◽

Luciferase Reporter System ◽

Human Progesterone Receptor

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Alu insertion variants alter gene transcript levels

Genome Research ◽

10.1101/gr.261305.120 ◽

2021 ◽

Author(s):

Lindsay M. Payer ◽

Jared P. Steranka ◽

Maria S. Kryatova ◽

Giacomo Grillo ◽

Mathieu Lupien ◽

...

Keyword(s):

Disease Risk ◽

Luciferase Reporter ◽

Regulatory Sequence ◽

Gene Transcript ◽

Genomic Context ◽

Transcript Levels ◽

Expression Of Genes ◽

Alu Insertion ◽

Regulatory Effects ◽

Interspersed Repeats

Alu are high copy number interspersed repeats that have accumulated near genes during primate and human evolution. They are a pervasive source of structural variation in modern humans. Impacts that Alu insertions may have on gene expression are not well understood, although some have been associated with expression quantitative trait loci (eQTLs). Here, we directly test regulatory effects of polymorphic Alu insertions in isolation of other variants on the same haplotype. To screen insertion variants for those with such effects, we used ectopic luciferase reporter assays and evaluated 110 Alu insertion variants, including more than 40 with a potential role in disease risk. We observed a continuum of effects with significant outliers that up- or down-regulate luciferase activity. Using a series of reporter constructs, which included genomic context surrounding the Alu, we can distinguish between instances in which the Alu disrupts another regulator and those in which the Alu introduces new regulatory sequence. We next focused on three polymorphic Alu loci associated with breast cancer that display significant effects in the reporter assay. We used CRISPR to modify the endogenous sequences, establishing cell lines varying in the Alu genotype. Our findings indicate that Alu genotype can alter expression of genes implicated in cancer risk, including PTHLH, RANBP9, and MYC. These data show that commonly occurring polymorphic Alu elements can alter transcript levels and potentially contribute to disease risk.

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The Neandertal Progesterone Receptor

Molecular Biology and Evolution ◽

10.1093/molbev/msaa119 ◽

2020 ◽

Vol 37 (9) ◽

pp. 2655-2660 ◽

Cited By ~ 4

Author(s):

Hugo Zeberg ◽

Janet Kelso ◽

Svante Pääbo

Keyword(s):

Progesterone Receptor ◽

High Frequency ◽

Human Population ◽

Modern Human ◽

The Other ◽

Human Populations ◽

Gene Variants ◽

Gene Encoding ◽

Alu Insertion ◽

Alu Element

Abstract The hormone progesterone is important for preparing the uterine lining for egg implantation and for maintaining the early stages of pregnancy. The gene encoding the progesterone receptor (PGR) carries introgressed Neandertal haplotypes with two missense substitutions and a mobile Alu element. These Neandertal gene variants have reached nearly 20% frequency in non-Africans and have been associated with preterm birth. Here, we show that one of the missense substitutions appears fixed in Neandertals, while the other substitution as well as the Alu insertion were polymorphic among Neandertals. We show that two Neandertal haplotypes carrying the PGR gene entered the modern human population and that present-day carriers of the Neandertal haplotypes express higher levels of the receptor. In a cohort of present-day Britons, these carriers have more siblings, fewer miscarriages, and less bleeding during early pregnancy suggesting that the Neandertal progesterone receptor alleles promote fertility. This may explain their high frequency in modern human populations.

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Cell Cycle Dependent Regulation of Human Progesterone Receptor in Breast Cancer

10.21236/ada433995 ◽

2004 ◽

Author(s):

Lisa K. Mullany ◽

Carol A. Lange

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Progesterone Receptor ◽

Human Progesterone Receptor ◽

Cycle Dependent

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Detection and Functional Evaluation of −262A/T and −188A/G Polymorphisms of SLAM Gene in Patients with Systemic Lupus Erythematosus

The Journal of Rheumatology ◽

10.3899/jrheum.091390 ◽

2010 ◽

Vol 37 (11) ◽

pp. 2268-2272 ◽

Cited By ~ 3

Author(s):

YI YOU ◽

ZHE WANG ◽

GUO-HONG DENG ◽

YI LIU ◽

FEI HAO

Keyword(s):

Systemic Lupus Erythematosus ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Gene Promoter ◽

Chinese Patients ◽

Luciferase Reporter ◽

Functional Evaluation ◽

Reporter System ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear

Objective.Signaling lymphocytic activation molecule (SLAM) has been related to the pathology of systemic lupus erythematosus (SLE) through regulation of T cell-dependent humoral immune responses. We investigated the functional associations of the −262A/T and −188A/G polymorphisms of SLAM in Chinese patients with SLE.Methods.Genotyping of −262A/T (rs2295614) and −188A/G (rs2295613) in SLAM was carried out in 248 cases and 278 controls. Promoter activities of haplotypes on the SLAM gene were evaluated with the dual-luciferase reporter system. The mRNA expressions of SLAM on peripheral blood mononuclear cells (PBMC) of SLE patients with different genotypes were determined by real-time polymerase chain reaction.Results.Frequencies of −262A allele and −188G allele were significantly higher in SLE patients than in controls. Haplotype analysis and multifactorial logistic regression analysis showed that individuals with the AG/AG haplotype had increased susceptibility to SLE (p = 0.002, OR 1.478, 95% CI 1.152–1.897). In response to PHA stimulation, the SLAM mRNA expression on PBMC of SLE patients was significantly higher in −262A-188G haplotype homozygotes compared with −262A-188G heterozygotes and individuals with other genotypes.Conclusion.Our findings suggest that −262A-188G haplotype in the SLAM gene promoter contributes to the risk of SLE by increasing the expression of SLAM.

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The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

Bioscience Reports ◽

10.1042/bsr20120043 ◽

2012 ◽

Vol 32 (6) ◽

pp. 531-537 ◽

Cited By ~ 16

Author(s):

Albert Braeuning ◽

Silvia Vetter

Keyword(s):

Photon Emission ◽

Gene Promoter ◽

Firefly Luciferase ◽

Nuclear Factor Κb ◽

Signalling Pathway ◽

Luciferase Reporter ◽

Nuclear Factor ◽

Reporter System ◽

Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

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miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer

Open Life Sciences ◽

10.1515/biol-2021-0022 ◽

2021 ◽

Vol 16 (1) ◽

pp. 266-276

Author(s):

Zhenfen Wang ◽

Qing Liu ◽

Ping Huang ◽

Guohao Cai

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Western Blot ◽

Luciferase Reporter ◽

Reporter System ◽

Western Blot Assay ◽

Normal Tissues ◽

Cell Progression ◽

Luciferase Reporter System ◽

Proliferation And Invasion

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

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