Acetylation of an Ets Transcription Factor PU.1 Suppresses Its Transcriptional Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2402-2402
Author(s):  
Fumihiko Mouri ◽  
Junichi Tsukada ◽  
Akiyoshi Fukamizu ◽  
Yoshiya Tanaka
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activity ◽  
Hematopoietic Cells ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Wild Type ◽  
Il1b Gene

Abstract PU.1, a member of the Ets family transcription factors, is expressed restrictively in hematopoietic cells including monocytes and macrophages, and plays critical roles in the inflammatory responses and the development of hematopoietic cells. CREB-binding protein (CBP) regulates transcription by acetylating not only histones but also certain transcription factors. Here, we demonstrated that a specific inhibitor of histone deacetylases, trichostatin A (TSA) inhibits PU.1 transcriptional activity in monocytes and further showed that deletion of a histone acetyltransferase (HAT) domain of CBP resulted in synergistic cooperativity between CBP and PU.1. When human monocytic cells THP-1 were treated with TSA, our immunoprecipitaion and western blot assay showed that TSA enhanced PU.1 acetylation. Next, we investigated the effect of TSA on the transcriptional regulation of PU.1-dependent gene promoters such as the human prointerleukin 1β (IL1B) gene and the human granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene in transient transfection studies. Two distinct luciferase reporter plasmids (Luc) for the IL1B gene promoter and the GM-CSFRα gene promoter, IL1B-Luc and GM-CSFRα-Luc were used. When these plasmids were transiently transfected into THP-1 cells, TSA suppressed LPS-induced activities for the IL1B promoter and the GM-CSFRα promoter in a dose-dependent manner. In contrast, when NF-κB luciferase reporter, NF-κB-Luc was transfected into THP-1 cells, TSA synergistically increased LPS-induced NF-κB activities. Moreover, when a PU.1 expression vector, pECEPU.1 was cotransfected into PU.1-deficient murine thymocytes EL4 along with either IL1B-Luc or GM-CSFRα-Luc. The PU.1-induced promoter activities were strongly suppressed through TSA treatment. FACS analysis further indicated that TSA suppressed LPS-induced expression of IL-1β and GM-CSFRα proteins. In addition, our EMSA data showed that TSA treatment did not affect DNA binding activity of PU.1 to the IL1B promoter. PU.1 has been shown to interact physically with CBP to transactivate their target genes. In our study, expression vectors for CBP wild-type or with a deletion of its HAT domain was cotransfected into EL4 cells along with IL1B-Luc and pECEPU.1. The HAT activity-deficient mutant showed synergistic transcriptional activity with PU.1 more strongly than the wild-type CBP. In this regard, our GST-pulldown assay showed that deletion of CBP HAT domain did not change binding affinity of CBP for PU.1. Our results propose a novel molecular mechanism by which PU.1-dependent genes is negatively regulated by HAT-induced acetylation in monocytes.

scholarly journals Histone Deacetylase 11 (HDAC11) Interaction with Ikaros Represent a Novel Mechanism of Regulation of Essential Transcriptional Factors in CD4+ T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 864-864
Author(s):  
Jie Chen ◽  
Fengdong Cheng ◽  
David Michael Woods ◽  
Edward Seto ◽  
Alejandro Villagra ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
T Cell ◽  
Histone Deacetylase ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Epigenetic Mechanism ◽  
Gene Promoters ◽  
H3 Acetylation

Abstract Histone deacetylase 11 (HDAC11), the most recently identified histone deacetylase, is the sole member of class IV HDACs [1]. Since its discovery, no biological function was assigned to this HDAC until we demonstrated its central role in negatively regulating IL-10 production in antigen presenting cells (APCs) [2]. More recently, we have found that disruption of HDAC11 in T cells is associated with an enhanced pro-inflammatory cytokine profile and effector molecule production. Furthermore, T-cells lacking HDAC11 were less susceptible to regulatory T-cell (Treg) suppression in vitro, were refractory to tolerance induction in vivo and displayed enhanced allo-reactivity and anti-tumor responses in murine models. Of note, T-cells lacking HDAC11 expressed higher levels of the transcription factors Eomes and Tbet. Conversely, overexpression of HDAC11 in T-cells decreased the expression of both transcription factors. The molecular mechanism(s) by which HDAC11 regulates the expression of these transcription factors have remained unknown. By using chromatin immunoprecipitation (ChIP) assay we found that in resting T-cells HDAC11 is present at the Eomes and Tbet gene promoters where it maintains histone deacetylation, a compacted chromatin and gene repression. Following T-cell stimulation, HDAC11 was largely absent from both promoters, which resulted in increased histone 3 (H3) acetylation and gene transcriptional activity. These findings were confirmed in T-cells isolated from HDAC11 knock out (KO) mice which also displayed an increase in H3 acetylation at the Tbet and Eomes gene promoter regions. Conversely, H3 acetylation was decreased in both gene promoters in T-cells overexpressing HDAC11 as compared to empty-vector transfected cells. Given that HDACs do not bind to DNA, we asked next which transcription factor(s) HDAC11 might be associated with, in order to regulate Tbet and Eomes gene transcriptional activity. In prior studies we have found that HDAC11 form a molecular complex with another member of the HDAC family, HDAC6, which physically interacts with the transcription factor, STAT3 in both the cytoplasmic and nuclear compartments. However, in T-cells no direct interaction of HDAC11 with STAT3 was detected in either compartment. In contrast, we found for the first time that HDAC11 physically associates with Ikaros (Ikzf1), a member of the Ikaros zinc finger transcription factor family that has been previously implicated in the regulation of T-bet gene expression and IFN-g production in T-cells [3-5]. The protein complex HDAC11-Ikaros was mainly detected in the nuclear compartment and both proteins were present at the T-bet gene promoter. Collectively, these results point to the HDAC11-Ikaros complex as a novel epigenetic mechanism of regulation of Tbet and Eomes, transcription factors that are essential for T cell development and function. Disclosures Woods: BMS: Other: Stock; HDAC11: Patents & Royalties: Patent for targeting HDAC11; Lion Biotech: Other: Stock.

Cloning of rat laminin gamma 1-chain gene promoter reveals motifs for recognition of multiple transcription factors

1997 ◽  
Vol 273 (3) ◽  
pp. F411-F420 ◽  
Author(s):  
B. C. O'Neill ◽  
H. Suzuki ◽  
W. P. Loomis ◽  
O. Denisenko ◽  
K. Bomsztyk
Keyword(s):  
Transcription Factors ◽  
Transcription Initiation ◽  
Gene Promoter ◽  
Start Codon ◽  
Response To Treatment ◽  
Luciferase Reporter ◽  
Chain Gene ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Constitutive Activity

We have previously shown that laminin gamma 1 (laminin B2)-chain mRNA levels increase in response to treatment of rat glomerular epithelial cells (GEC) with the cytokine interleukin-1 beta (IL-1 beta) [C. A. Richardson, K. L. Gordon, W. G. Couser, and K. Bomsztyk. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F273-F278, 1995]. IL-1 beta-induced increase in laminin gamma 1-chain gene expression is likely to be transcriptionally regulated. As the laminin gamma 1-chain gene promoter had not previously been cloned in the rat, we cloned the 5'-flanking region of this gene from a rat genomic library. Like the human and murine laminin gamma 1-chain gene promoters, the rat laminin gamma 1-chain gene fragment spanning from nucleotides -1104 to +109, relative to the start codon, is "GC" rich and lacks TATA or CAAT boxes. This rat laminin gamma 1-chain gene promoter region appears to contain at least two transcription initiation sites, i.e., position -169 and -234. In transient transfections in GEC, the -1104/+35 and -1104/-15 fragments cloned upstream of a luciferase reporter gene had very little constitutive activity and were not IL-1 beta responsive. In sharp contrast, a -1104/-234 fragment exhibited constitutive activity and was IL-1 beta responsive. The -1104/-234 fragment contains motifs that recognize Sp1, BCN-1, and ApoE-B1-AP1 DNA-binding activities in GEC nuclear extracts. Collectively, the results of this study suggest that multiple inducible transcription factors may regulate laminin gamma 1-chain gene promoter activity in GEC.

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318 ◽  
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Abstract Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

scholarly journals Genetic Variants and Functional Analyses of the ATG16L1 Gene Promoter in Acute Myocardial Infarction

2021 ◽  
Vol 12 ◽  
Author(s):  
Falan Han ◽  
Shuchao Pang ◽  
Zhaoqing Sun ◽  
Yinghua Cui ◽  
Bo Yan
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Transcription Factors ◽  
Electrophoretic Mobility ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Genotype Frequencies ◽  
Promoter Mutations

BackgroundAcute myocardial infarction (AMI), a common complex disease caused by an interaction between genetic and environmental factors, is a serious type of coronary artery disease and is also a leading cause of death worldwide. Autophagy-related 16-like 1 (ATG16L1) is a key regulatory factor of autophagy and plays an important role in induced autophagy. In the cardiovascular system, autophagy is essential to preserve the homeostasis and function of the heart and blood vessels. No studies have hitherto examined the association between AMI and ATG16L1 gene promoter.MethodsWe conducted a case-control study, using polymerase chain reaction and sequencing techniques, dual luciferase reporter assay, and electrophoretic mobility shift assay, to analyze genetic and functional variation in the ATG16L1 gene promoter between AMI and controls. A variety of statistical analyses were used to analyze the allele and genotype frequencies and the relationship between single-nucleotide polymorphisms (SNPs) and AMI.ResultsIn all, 10 SNPs and two DNA-sequence variants (DSVs) were identified in 688 subjects, and three ATG16L1 gene promoter mutations [g.233250693 T > C (rs185213911), g.233250946 G > A (rs568956599), g.233251133 C > G (rs1301744254)] that were identified in AMI patients significantly altered the transcriptional activity of ATG16L1 gene promoter in HEH2, HEK-293, and H9c2 cells (P < 0.05). Further electrophoretic mobility shift assays indicated that the SNPs affected the binding of transcription factors (P < 0.01).ConclusionATG16L1 gene promoter mutations in AMI patients may affect the binding of transcription factors and change the transcriptional activity of the ATG16L1 gene, changing the level of autophagy and contributing to the occurrence and development of AMI as rare and low-frequency risk factors.

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

GATA-1, but Not GATA-2, Antagonizes PU.1 Mediated Transcriptional Activity at the CBFA2T3 (ETO2, MTG16) Promoter through a Mechanism Dependent on GATA DNA Binding.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1749-1749
Author(s):  
Yogenthiran Saunthararajah ◽  
SiJun Yang ◽  
ShriHari Kadkol ◽  
Marie Baraoidan ◽  
Vinzon Ibanez ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Hematopoietic Cells ◽  
Chromosomal Translocation ◽  
Erythroid Differentiation ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Dna Binding Sites

Abstract CBFA2T3 (ETO2, MTG16), a target of chromosomal translocation in acute myeloid leukemia, has its highest expression in hematopoietic cells compared to other tissues. This suggests that its expression is regulated by major hematopoietic transcription factors. The proximal promoter from −171 to −65 bp has greater than 90% identity between mouse and human and contains recognition sites for major hematopoietic transcription factors PU.1, GATA-1 and GATA-2. Using chromatin immuno-precipitation and the MPD hematopoietic cell-line, this segment was pulled down with endogenous PU.1, GATA-1 and GATA-2. In luciferase reporter gene assays, PU.1 and GATA-2, but not GATA-1, activated the promoter. As would be expected from these findings, CBFA2T3 levels declined during terminal erythroid differentiation of primary hematopoietic cells. GATA-1, but not GATA-2, antagonized PU.1 mediated activation but this effect of GATA-1 was abrogated by mutation of the GATA DNA binding sites. Both GATA-1 and GATA-2 have been reported to antagonize PU.1 transcriptional activity by antagonizing PU.1 interactions with c-Jun (Zhang et al, Proc Natl Acad Sci USA1999;96:8705–8710); however, the DNA binding dependent mechanism reported here allows GATA-2 and GATA-1 to have contrasting relationships with PU.1 and may be the basis for the co-operation of GATA proteins with PU.1 in some contexts yet antagonism of PU.1 activity in others.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

scholarly journals The AML-associated K313 mutation enhances C/EBPα activity by leading to C/EBPα overexpression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Ian Edward Gentle ◽  
Isabel Moelter ◽  
Mohamed Tarek Badr ◽  
Konstanze Döhner ◽  
Michael Lübbert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activity ◽  
Target Gene ◽  
Wild Type ◽  
Elevated Expression ◽  
Factor C ◽  
Acute Myeloid

AbstractMutations in the transcription factor C/EBPα are found in ~10% of all acute myeloid leukaemia (AML) cases but the contribution of these mutations to leukemogenesis is incompletely understood. We here use a mouse model of granulocyte progenitors expressing conditionally active HoxB8 to assess the cell biological and molecular activity of C/EBPα-mutations associated with human AML. Both N-terminal truncation and C-terminal AML-associated mutations of C/EBPα substantially altered differentiation of progenitors into mature neutrophils in cell culture. Closer analysis of the C/EBPα-K313-duplication showed expansion and prolonged survival of mutant C/EBPα-expressing granulocytes following adoptive transfer into mice. C/EBPα-protein containing the K313-mutation further showed strongly enhanced transcriptional activity compared with the wild-type protein at certain promoters. Analysis of differentially regulated genes in cells overexpressing C/EBPα-K313 indicates a strong correlation with genes regulated by C/EBPα. Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors. Strikingly, the K313 mutation caused strongly elevated expression of C/EBPα-protein, which could also be seen in primary K313 mutated AML blasts, explaining the enhanced C/EBPα activity in K313-expressing cells.

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

1997 ◽  
Vol 19 (2) ◽  
pp. 163-172 ◽  
Author(s):  
K Chu ◽  
HH Zingg
Keyword(s):  
Receptor Binding ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Fine Tuning ◽  
Orphan Receptor ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Interaction Sites

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

Two novel mutations in Gli-similar 3 in patients with congenital hypothyroidism and thyroid dysgenesis

2021 ◽  
Author(s):  
Jie Lan ◽  
Chunhui Sun ◽  
Xinping Liang ◽  
Ruixin Ma ◽  
Yuhua Ji ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Transcriptional Activation ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Thyroid Dysgenesis ◽  
Type Protein ◽  
Wild Type Protein

Abstract Background: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). As variants of the transcription factor Gli-similar 3 (GLIS3) have been associated with CH and GLIS3 is one of candidate genes of TD, we screened and characterized GLIS3 mutations in Chinese patients with CH and TD.Methods: To detect mutations, we sequenced all GLIS3 exons in the peripheral blood genomic DNA isolated from 50 patients with TD and 100 healthy individuals. Wild-type and mutant expression vectors of Glis3 were constructed. Quantitative real-time PCR, western blotting, and double luciferase assay were performed to investigation the effect of the mutations on GLIS3 protein function and transcriptional activation.Results: Two novel heterozygous missense mutations, c.2710G>A (p.G904R) and c.2507C>A (p.P836Q), were detected in two unrelated patients. Functional studies revealed that p.G904R expression was 59.95% lower and p.P836Q was 31.23% lower than wild-type GLIS3 mRNA expression. The p.G904R mutation also resulted in lower GLIS3 protein expression compared with that encoded by wild-type GLIS3. Additionally, the luciferase reporter assay revealed that p.G904R mediated impaired transcriptional activation compared with the wild-type protein (p < 0.05) but did not have a dominant-negative effect on the wild-type protein.Conclusions: We for the first time screened and characterized the function of GLIS3 mutations in Chinese individuals with CH and TD. Our study not only broadens the GLIS3 mutation spectrum, but also provides further evidence that GLIS3 defects cause TD.

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