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.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

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 Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314 ◽  
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders

2016 ◽  
Vol 113 (16) ◽  
pp. 4434-4439 ◽  
Author(s):  
Aoi Wakabayashi ◽  
Jacob C. Ulirsch ◽  
Leif S. Ludwig ◽  
Claudia Fiorini ◽  
Makiko Yasuda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activity ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Targeted Disruption ◽  
Altered Gene Expression ◽  
Whole Exome ◽  
Altered Gene ◽  
Insight Into

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptional cis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.

scholarly journals Cloning of Nrf1, an NF-E2-related transcription factor, by genetic selection in yeast

1993 ◽  
Vol 90 (23) ◽  
pp. 11371-11375 ◽  
Author(s):  
J Y Chan ◽  
X L Han ◽  
Y W Kan
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Leucine Zipper ◽  
Genetic Selection ◽  
Yeast Cells ◽  
Binding Motif ◽  
Related Factor ◽  
Transcriptional Activators ◽  
Basic Leucine Zipper ◽  
Related Transcription Factor

We have devised a complementation assay in yeast to clone mammalian transcriptional activators and have used it to identify a human basic leucine-zipper transcription factor that we have designated Nrf1 for NF-E2-related factor 1. Nrf1 potentially encodes a 742-aa protein and displays marked homology to the mouse and human NF-E2 transcription factors. Nrf1 activates transcription via NF-E2 binding sites in yeast cells. The ubiquitous expression pattern of Nrf1 and the range of promoters containing the NF-E2 binding motif suggest that this gene may play a role in the regulation of heme synthesis and ferritin genes.

Platelet/Megakaryocyte PKC-Θ Is a Transcriptional Target of RUNX1/ CBFA2: Studies in Human RUNX1 Haplodeficiency.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1846-1846
Author(s):  
Gauthami S Jalagadugula ◽  
Gurpreet Kaur ◽  
Guangfen Mao ◽  
Danny Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Platelet Function ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Mrna Levels ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Hel Cells ◽  
Transcriptional Target

Abstract Protein kinase C Θ (PKC-Θ) is an important signaling molecule and regulates platelet responses to activation including aggregation and secretion. In a patient with lifelong thrombocytopenia, impaired platelet aggregation and secretion, we have shown (Gabbeta et al 1996, Blood 87:1368–1376) that phosphorylation of pleckstrin (a PKC substrate) and myosin light chain (MLC) is impaired along with diminished GPIIb-IIIa activation. Platelet protein and mRNA levels of PKC-Θ were decreased with normal levels of other PKC isozymes. These findings were associated with a heterozygous nonsense mutation in transcription factor RUNX1 (also known as CBFA2 or AML1) (Sun et al 2004, Blood 103:948–54). RUNX1 is transcription factor that plays a major role in megakaryopoiesis, megakaryocytic maturation, and platelet production. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. Because of the important role of PKC-Θ in platelet activation and of RUNX1 in hematopoiesis, we addressed the hypothesis that PKC-Θ is a direct transcriptional target of RUNX1. Studies were performed using human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. Chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody revealed RUNX1 binding to chromatin in the PKC-Θ 5’ upstream region −1225/−1056 bp from ATG codon. This region includes a RUNX1 consensus binding site ACCGCA at −1081/−1076 bp identified by TFSEARCH. We performed electrophoretic mobility shift assay (EMSA) using 20-mer probe −1088/−1069 containing the RUNX1 site and nuclear extracts from PMA-treated HEL cells. Protein binding to the probe was observed, which was competed by excess unlabelled probe, and anti-RUNX1 antibody inhibited this binding, indicating that RUNX1 was involved in the DNA binding. Moreover, protein binding to the wild type probe was not competed by an oligo with 4 nucleotides deleted from the RUNX1 consensus site. To determine the functional relevance of RUNX1 binding to PKC-Θ, transient transfections were performed in HEL cells with luciferase reporter constructs. The full length construct −1085/−206 showed ~14-fold activity compared to empty vector. A mutant construct with deletion of the RUNX1 site resulted in a ~50% decrease in activity indicating that the site was functional. siRNA-mediated knockdown of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and PKC-Θ protein. Conclusion: These results and our findings in the patient provide the first evidence that PKC-Θ gene transcription in the megakaryocyte/platelet is regulated by RUNX1. They provide a cogent mechanism for the platelet PKC-Θ downregulation associated with RUNX1 haplodeficiency in our patient. RUNX1 dysregulation of PKC-Θ in megakaryocytic cells is an important aspect of the abnormal platelet function and production associated with human RUNX1 mutations.

scholarly journals The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development

2019 ◽  
Vol 29 (4) ◽  
pp. 591-604 ◽  
Author(s):  
Soma Dash ◽  
Lindy K Brastrom ◽  
Shaili D Patel ◽  
C Anthony Scott ◽  
Diane C Slusarski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Eye Development ◽  
Ocular Tissue ◽  
Binding Motif ◽  
Mobility Shift ◽  
Developmental Defects ◽  
Protein Levels

Abstract Mutations in the key transcription factor, SOX2, alone account for 20% of anophthalmia (no eye) and microphthalmia (small eye) birth defects in humans—yet its regulation is not well understood, especially on the post-transcription level. We report the unprecedented finding that the conserved RNA-binding motif protein, RBM24, positively controls Sox2 mRNA stability and is necessary for optimal SOX2 mRNA and protein levels in development, perturbation of which causes ocular defects, including microphthalmia and anophthalmia. RNA immunoprecipitation assay indicates that RBM24 protein interacts with Sox2 mRNA in mouse embryonic eye tissue. and electrophoretic mobility shift assay shows that RBM24 directly binds to the Sox2 mRNA 3’UTR, which is dependent on AU-rich elements (ARE) present in the Sox2 mRNA 3’UTR. Further, we demonstrate that Sox2 3’UTR AREs are necessary for RBM24-based elevation of Sox2 mRNA half-life. We find that this novel RBM24–Sox2 regulatory module is essential for early eye development in vertebrates. We show that Rbm24-targeted deletion using a constitutive CMV-driven Cre in mouse, and rbm24a-CRISPR/Cas9-targeted mutation or morpholino knockdown in zebrafish, results in Sox2 downregulation and causes the developmental defects anophthalmia or microphthalmia, similar to human SOX2-deficiency defects. We further show that Rbm24 deficiency leads to apoptotic defects in mouse ocular tissue and downregulation of eye development markers Lhx2, Pax6, Jag1, E-cadherin and gamma-crystallins. These data highlight the exquisite specificity that conserved RNA-binding proteins like RBM24 mediate in the post-transcriptional control of key transcription factors, namely, SOX2, associated with organogenesis and human developmental defects.

scholarly journals Transcription of the Sox30 Gene Is Positively Regulated by Dmrt1 in Nile Tilapia

2019 ◽  
Vol 20 (21) ◽  
pp. 5487 ◽  
Author(s):  
Yaohao Tang ◽  
Xiaoyan Li ◽  
Hesheng Xiao ◽  
Minghui Li ◽  
Yueqin Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Nile Tilapia ◽  
Regulatory Element ◽  
Transcriptional Profiling ◽  
Sex Differentiation ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Related Transcription Factor ◽  
Mobility Shift Assay ◽  
Male Specific

The Sox family member Sox30 is highly expressed in the testis of several vertebrate species and has been shown to play key roles in spermiogenesis. However, its transcription regulation remains unclear. Here, we analyzed the Sox30 promoter from the teleost fish Nile tilapia (Oreochromis niloticus) and predicted a putative cis-regulatory element (CRE) for doublesex and mab-3 related transcription factor 1 (Dmrt1), a male-specific transcription factor involved in male sex differentiation. Transcriptional profiling revealed that Sox30 and Dmrt1 similarly exhibited a high expression in tilapia testes from 90 days after hatching (dah) to 300 dah, and the transcription of the Sox30 gene was reduced about one-fold in the testes of male tilapia with Dmrt1 knockdown. Further dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter. Chromatin immunoprecipitation-based PCR (ChIP-PCR) and electrophoretic mobility shift assay (EMSA) demonstrated that Dmrt1 directly binds to putative CRE within the Sox30 promoter. These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to specific CRE within the Sox30 promoter.

scholarly journals The role of SF-1/Ad4BP in the control of the bovine gene for the steroidogenic acute regulatory (StAR) protein

1998 ◽  
Vol 21 (2) ◽  
pp. 189-200 ◽  
Author(s):  
W Rust ◽  
K Stedronsky ◽  
G Tillmann ◽  
S Morley ◽  
N Walther ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Regulatory Protein ◽  
Gene Promoter ◽  
Binding Motif ◽  
Clear Understanding ◽  
Mobility Shift ◽  
Bovine Gene ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

The bovine gene for the steroidogenic acute regulatory protein (StAR) was cloned and sequenced, including 2 kb of the upstream control region of the gene. The gene comprises seven exons arranged similarly to those of the human and mouse gene sequences. The sequence analysis identified three cis elements corresponding to the binding motif for the transcription factor SF-1/Ad4BP, at - 100, - 240 and - 1190 from the transcription start site. Electrophoretic mobility shift analysis (EMSA) using nuclear proteins from bovine corpus luteum and bovine adrenal as well as in vitro transcribed/translated SF-1/Ad4BP consistently showed that only the site at -1190 bound the transcription factor significantly. Very weak binding was detectable also at the - 240 site, but none at the -100 site. Heterologous transfection of StAR promoter deletion-reporter constructs into Hela cells cotransfected with an expression vector for bovine SF-1/Ad4BP, showed that this transcription factor can specifically act on the bovine StAR gene promoter, but preferentially in regions corresponding to the two proximal SF-1/Ad4BP elements at - 100 and - 240, though with only low relative effect. Furthermore, additional cotransfection of a construct expressing a constitutive protein kinase A catalytic subunit to mimic the effects of cAMP stimulation, led to a small SF-1/Ad4BP-dependent increase in reporter activity mediated only by the same proximal sites. Since the bovine StAR gene promoter does not appear to have a functional cAMP responsive element (CRE), either this effect is mediated in this system directly by SF-1/Ad4BP, or by other factors interacting with this transcription factor, but which do not involve CRE-mediated gene activation. Taken together, the results show that there is a discrepancy between the results of the EMSA experiments and those using transfection of promoter-reporter constructs, which needs to be resolved before a clear understanding of SF-1/Ad4BP-mediated regulation of the StAR gene is attained.

scholarly journals Runt-Related Transcription Factor 2 (RUNX2) and RUNX2-Related Osteogenic Genes Are Down-Regulated throughout Osteogenesis in Type 1 Diabetes Mellitus

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1697-1704 ◽  
Author(s):  
John L. Fowlkes ◽  
R. Clay Bunn ◽  
Lichu Liu ◽  
Elizabeth C. Wahl ◽  
Hannah N. Coleman ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 1 Diabetes ◽  
Transcription Factor ◽  
Bone Formation ◽  
Type 1 Diabetes Mellitus ◽  
Insulin Treatment ◽  
Binding Motif ◽  
Skeletal Health ◽  
Related Transcription Factor

Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.

Sign in / Sign up

Export Citation Format

Share Document