scholarly journals The transcription factor PITX1 drives astrocyte differentiation by regulating the SOX9 gene

2020 ◽  
Vol 295 (39) ◽  
pp. 13677-13690
Author(s):  
Jeong Su Byun ◽  
Mihee Oh ◽  
Seonha Lee ◽  
Jung-Eun Gil ◽  
Yeajin Mo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Formation Control ◽  
Synapse Formation ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Mobility Shift ◽  
Differentiation Protocol ◽  
Mature Brain ◽  
Astrocyte Differentiation ◽  
Electrophoretic Mobility Shift Assays

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.

scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

scholarly journals Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.

1995 ◽  
Vol 15 (1) ◽  
pp. 476-487 ◽  
Author(s):  
X Guo ◽  
Y P Zhang ◽  
D A Mitchell ◽  
D T Denhardt ◽  
A F Chambers
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Uv Light ◽  
Metastatic Potential ◽  
Binding Motif ◽  
Binding Assays ◽  
Mobility Shift ◽  
Related Transcription Factor ◽  
Altered Gene ◽  
Alpha V Beta 3

The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.

scholarly journals Persistent NF-κB activation in renal epithelial cells in a mouse model of HIV-associated nephropathy

2006 ◽  
Vol 290 (3) ◽  
pp. F657-F665 ◽  
Author(s):  
Scott Martinka ◽  
Leslie A. Bruggeman
Keyword(s):  
Transcription Factor ◽  
Epithelial Cells ◽  
Cell Types ◽  
Nuclear Accumulation ◽  
Mobility Shift ◽  
Glomerular Epithelial Cells ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Electrophoretic Mobility Shift Assays ◽  
Hiv 1

Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is caused, in part, by direct infection of kidney epithelial cells by HIV-1. In the spectrum of pathogenic host-virus interactions, abnormal activation or suppression of host transcription factors is common. NF-κB is a necessary host transcription factor for HIV-1 gene expression, and it has been shown that NF-κB activity is dysregulated in many naturally infected cell types. We show here that renal glomerular epithelial cells (podocytes) expressing the HIV-1 genome, similar to infected immune cells, also have a dysregulated and persistent activation of NF-κB. Although podocytes produce p50, p52, RelA, RelB, and c-Rel, electrophoretic mobility shift assays and immunocytochemistry showed a predominant nuclear accumulation of p50/RelA-containing NF-κB dimers in HIV-1-expressing podocytes compared with normal. In addition, the expression level of a transfected NF-κB reporter plasmid was significantly higher in HIVAN podocytes. The mechanism of NF-κB activation involved increased phosphorylation of IκBα, resulting in an enhanced turnover of the IκBα protein. There was no evidence for regulation by IκBβ or the alternate pathway of NF-κB activation. Altered activation of this key host transcription factor likely plays a role in the well-described cellular phenotypic changes observed in HIVAN, such as proliferation. Studies with inhibitors of proliferation and NF-κB suggest that NF-κB activation may contribute to the proliferative mechanism in HIVAN. In addition, because NF-κB regulates many aspects of inflammation, this dysregulation may also contribute to disease severity and progression through regulation of proinflammatory processes in the kidney microenvironment.

Fusion of the ets Transcription Factor TEL to Jak2 Results in Constitutive Jak-Stat Signaling

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4354-4364 ◽  
Author(s):  
Jen M.-Y. Ho ◽  
Bryan K. Beattie ◽  
Jeremy A. Squire ◽  
David A. Frank ◽  
Dwayne L. Barber
Keyword(s):  
Transcription Factor ◽  
Fusion Protein ◽  
Janus Kinase ◽  
Chimeric Proteins ◽  
Mobility Shift ◽  
Specific Antibodies ◽  
Hematopoietic Cell Line ◽  
Ets Transcription Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Insight Into

Abstract To study constitutive Janus kinase signaling, chimeric proteins were generated between the pointed domain of the etstranscription factor TEL and the cytosolic tyrosine kinase Jak2. The effects of these proteins on interleukin-3 (IL-3)–dependent proliferation of the hematopoietic cell line, Ba/F3, were studied. Fusion of TEL to the functional kinase (JH1) domain of Jak2 resulted in conversion of Ba/F3 cells to factor-independence. Importantly, fusion of TEL to the Jak2 pseudokinase (JH2) domain or a kinase-inactive Jak2 JH1 domain had no effect on IL-3–dependent proliferation of Ba/F3 cells. Active TEL-Jak2 constructs (consisting of either Jak2 JH1 or Jak2 JH2+JH1 domain fusions) were constitutively tyrosine-phosphorylated but did not affect phosphorylation of endogeneous Jak1, Jak2, or Jak3. TEL-Jak2 activation resulted in the constitutive tyrosine phosphorylation of Stat1, Stat3, and Stat5 as determined by detection of phosphorylation using activation-specific antibodies and by binding of each protein to a preferential GAS sequence in electrophoretic mobility shift assays. Elucidation of signaling events downstream of TEL-Jak2 activation may provide insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.

NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene

2008 ◽  
Vol 86 (1) ◽  
pp. 46-56 ◽  
Author(s):  
José R. Blesa ◽  
Jesús A. Prieto-Ruiz ◽  
Beth A. Abraham ◽  
Bridget L. Harrison ◽  
Anita A. Hegde ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Colorectal Tumors ◽  
Methylation Analysis ◽  
Mobility Shift ◽  
Nuclear Respiratory Factors ◽  
Hela S3 ◽  
Electrophoretic Mobility Shift Assays

The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear–mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5′ region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity.

scholarly journals Variants in a cis-regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xuechao Jiang ◽  
Tingting Li ◽  
Sijie Liu ◽  
Qihua Fu ◽  
Fen Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Regulatory Element ◽  
Expression Patterns ◽  
Human Embryos ◽  
Mobility Shift ◽  
T Box ◽  
Conotruncal Heart Defect ◽  
Velo Cardio Facial Syndrome ◽  
Electrophoretic Mobility Shift Assays

Abstract Background TBX1 (T-box transcription factor 1) is a major candidate gene that likely contributes to the etiology of velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Although the haploinsufficiency of TBX1 in both mice and humans results in congenital cardiac malformations, little has been elucidated about its upstream regulation. We aimed to explore the transcriptional regulation and dysregulation of TBX1. Methods Different TBX1 promoter reporters were constructed. Luciferase assays and electrophoretic mobility shift assays (EMSAs) were used to identify a cis-regulatory element within the TBX1 promoter region and its trans-acting factor. The expression of proteins was identified by immunohistochemistry and immunofluorescence. Variants in the cis-regulatory element were screened in conotruncal defect (CTD) patients. In vitro functional assays were performed to show the effects of the variants found in CTD patients on the transactivation of TBX1. Results We identified a cis-regulatory element within intron 1 of TBX1 that was found to be responsive to GATA6 (GATA binding protein 6), a transcription factor crucial for cardiogenesis. The expression patterns of GATA6 and TBX1 overlapped in the pharyngeal arches of human embryos. Transfection experiments and EMSA indicated that GATA6 could activate the transcription of TBX1 by directly binding with its GATA cis-regulatory element in vitro. Furthermore, sequencing analyses of 195 sporadic CTD patients without the 22q11.2 deletion or duplication identified 3 variants (NC_000022.11:g.19756832C > G, NC_000022.11:g.19756845C > T, and NC_000022.11:g. 19756902G > T) in the non-coding cis-regulatory element of TBX1. Luciferase assays showed that all 3 variants led to reduced transcription of TBX1 when incubated with GATA6. Conclusions Our findings showed that TBX1 might be a direct transcriptional target of GATA6, and variants in the non-coding cis-regulatory element of TBX1 disrupted GATA6-mediated transactivation.

scholarly journals Transcription factor organic cation transporter 1 (OCT-1) affects the expression of porcine Klotho (KL) gene

2016 ◽  
Author(s):  
Yan Li ◽  
Lei Wang ◽  
Jiawei Zhou ◽  
Fenge Li
Keyword(s):  
Transcription Factor ◽  
Organic Cation ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Organic Cation Transporter ◽  
Mobility Shift ◽  
Fragment Analysis ◽  
Intron 1 ◽  
Organic Cation Transporter 1 ◽  
Electrophoretic Mobility Shift Assays

Klotho (KL), originally discovered as an aging suppressor, was a membrane protein that shared sequence similarity with the β-glucosidase enzymes. Recent reports showed Klotho might have a role in adipocyte maturation and systemic glucose metabolism. However, little is known about the transcription factors involved in regulating the expression of porcine KL gene. Deletion fragment analysis identified KL-D2 (-418 bp to -3 bp) as the porcine KL core promoter. MARC0022311 in KL intron 1 appeared a polymorphism (A or G) in Landrace × DIV pigs, and relative luciferase activity of pGL3-D2-G was significantly higher than pGL3-D2-A. This was possibly the result of a change in KL binding ability with transcription factor organic cation transporter 1 (OCT-1), which was confirmed using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, OCT-1 regulated endogenous KL expression by RNA interference. Our study indicates SNP MARC0022311 affects porcine KL expression by regulating its promoter activity via OCT-1.

scholarly journals DIMR, a Yeast-Based Synthetic Reporter System for Probing Oligomeric Transcription Factor DNA Binding

2019 ◽  
Author(s):  
Zachary A. Myers ◽  
Swadhin Swain ◽  
Shannan Bialek ◽  
Samuel Keltner ◽  
Ben F. Holt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Reporter System ◽  
Mobility Shift ◽  
Genomic Locus ◽  
Biological Regulation ◽  
Transcriptional Regulatory ◽  
Regulatory Capacity ◽  
Electrophoretic Mobility Shift Assays ◽  
Y Family

AbstractTranscription factors (TFs) are fundamental components of biological regulation, facilitating the basal and differential gene expression necessary for life. TFs exert transcriptional regulation through interactions with both DNA and other TFs, ultimately influencing the action of RNA polymerase at a genomic locus. Current approaches are proficient at identification of binding site requirements for individual TFs, but few methods have been adapted to study oligomeric TF complexes. Further, many approaches that have been turned toward understanding DNA binding of TF complexes, such as electrophoretic mobility shift assays, require protein purification steps that can be burdensome or scope-limiting when considering more exhaustive experimental design. In order to address these shortfalls and to facilitate a more streamlined approach to understanding DNA binding by TF complexes, we developed the DIMR (Dynamic, Interdependent TF binding Molecular Reporter) system, a modular, yeast-based synthetic transcriptional activity reporter. As a proof of concept, we focused on the NUCLEAR FACTOR-Y (NF-Y) family of obligate heterotrimeric TFs in Arabidopsis thaliana. The DIMR system was able to reproduce the strict DNA-binding requirements of an experimentally validated NF-YA2/B2/C3 complex with high fidelity, including recapitulation of previously characterized mutations in subunits that either break NF-Y complex interactions or are directly involved in DNA binding. The DIMR system is a novel, powerful, and easy-to-use approach to address questions regarding the binding of oligomeric TFs to DNA.One sentence summaryThe DIMR system provides an accessible and easy-to-use platform to elucidate DNA binding and transcriptional regulatory capacity of oligomeric transcription factor complexes

Fusion of the ets Transcription Factor TEL to Jak2 Results in Constitutive Jak-Stat Signaling

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4354-4364 ◽  
Author(s):  
Jen M.-Y. Ho ◽  
Bryan K. Beattie ◽  
Jeremy A. Squire ◽  
David A. Frank ◽  
Dwayne L. Barber
Keyword(s):  
Transcription Factor ◽  
Fusion Protein ◽  
Janus Kinase ◽  
Chimeric Proteins ◽  
Mobility Shift ◽  
Specific Antibodies ◽  
Hematopoietic Cell Line ◽  
Ets Transcription Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Insight Into

To study constitutive Janus kinase signaling, chimeric proteins were generated between the pointed domain of the etstranscription factor TEL and the cytosolic tyrosine kinase Jak2. The effects of these proteins on interleukin-3 (IL-3)–dependent proliferation of the hematopoietic cell line, Ba/F3, were studied. Fusion of TEL to the functional kinase (JH1) domain of Jak2 resulted in conversion of Ba/F3 cells to factor-independence. Importantly, fusion of TEL to the Jak2 pseudokinase (JH2) domain or a kinase-inactive Jak2 JH1 domain had no effect on IL-3–dependent proliferation of Ba/F3 cells. Active TEL-Jak2 constructs (consisting of either Jak2 JH1 or Jak2 JH2+JH1 domain fusions) were constitutively tyrosine-phosphorylated but did not affect phosphorylation of endogeneous Jak1, Jak2, or Jak3. TEL-Jak2 activation resulted in the constitutive tyrosine phosphorylation of Stat1, Stat3, and Stat5 as determined by detection of phosphorylation using activation-specific antibodies and by binding of each protein to a preferential GAS sequence in electrophoretic mobility shift assays. Elucidation of signaling events downstream of TEL-Jak2 activation may provide insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.

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