Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues

2000 ◽  
Vol 349 (2) ◽  
pp. 629-634 ◽  
Author(s):  
Tatsuo FURUYAMA ◽  
Toru NAKAZAWA ◽  
Itsuko NAKANO ◽  
Nozomu MORI
Keyword(s):  
Target Genes ◽  
Binding Protein ◽  
Expression Patterns ◽  
Distribution Patterns ◽  
Mobility Shift ◽  
Differential Distribution ◽  
C Elegans ◽  
Core Sequence ◽  
Mobility Shift Assay ◽  
Responsive Element

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.

scholarly journals Roles of the GA-mediated SPL Gene Family and miR156 in the Floral Development of Chinese Chestnut (Castanea mollissima)

2019 ◽  
Vol 20 (7) ◽  
pp. 1577 ◽  
Author(s):  
Guosong Chen ◽  
Jingtong Li ◽  
Yang Liu ◽  
Qing Zhang ◽  
Yuerong Gao ◽  
...  
Keyword(s):  
Target Genes ◽  
Floral Development ◽  
Binding Protein ◽  
Expression Patterns ◽  
Evolutionary Relationship ◽  
Deciduous Tree ◽  
Main Function ◽  
Squamosa Promoter Binding Protein ◽  
Spl Genes ◽  
Castanea Mollissima

Chestnut (Castanea mollissima) is a deciduous tree species with major economic and ecological value that is widely used in the study of floral development in woody plants due its monoecious and out-of-proportion characteristics. Squamosa promoter-binding protein-like (SPL) is a plant-specific transcription factor that plays an important role in floral development. In this study, a total of 18 SPL genes were identified in the chestnut genome, of which 10 SPL genes have complementary regions of CmmiR156. An analysis of the phylogenetic tree of the squamosa promoter-binding protein (SBP) domains of the SPL genes of Arabidopsis thaliana, Populus trichocarpa, and C. mollissima divided these SPL genes into eight groups. The evolutionary relationship between poplar and chestnut in the same group was similar. A structural analysis of the protein-coding regions (CDSs) showed that the domains have the main function of SBP domains and that other domains also play an important role in determining gene function. The expression patterns of CmmiR156 and CmSPLs in different floral organs of chestnut were analyzed by real-time quantitative PCR. Some CmSPLs with similar structural patterns showed similar expression patterns, indicating that the gene structures determine the synergy of the gene functions. The application of gibberellin (GA) and its inhibitor (Paclobutrazol, PP333) to chestnut trees revealed that these exert a significant effect on the number and length of the male and female chestnut flowers. GA treatment significantly increased CmmiR156 expression and thus significantly decreased the expression of its target gene, CmSPL6/CmSPL9/CmSPL16, during floral bud development. This finding indicates that GA might indirectly affect the expression of some of the SPL target genes through miR156. In addition, RNA ligase-mediated rapid amplification of the 5′ cDNA ends (RLM-RACE) experiments revealed that CmmiR156 cleaves CmSPL9 and CmSPL16 at the 10th and 12th bases of the complementary region. These results laid an important foundation for further study of the biological function of CmSPLs in the floral development of C. mollissima.

scholarly journals A Sequence of the CIS Gene Promoter Interacts Preferentially with Two Associated STAT5A Dimers: a Distinct Biochemical Difference between STAT5A and STAT5B

1998 ◽  
Vol 18 (10) ◽  
pp. 5852-5860 ◽  
Author(s):  
Frédérique Verdier ◽  
Raquel Rabionet ◽  
Fabrice Gouilleux ◽  
Christian Beisenherz-Huss ◽  
Paule Varlet ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Prolactin Receptor ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Binding Sequence ◽  
Biochemical Difference

ABSTRACT Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.

A downstream sequence of the rpL32 promoter competes with the glucocorticoid responsive element for a protein factor

1992 ◽  
Vol 70 (9) ◽  
pp. 787-791 ◽  
Author(s):  
Thillainathan Yoganathan ◽  
Bruce H. Sells
Keyword(s):  
Ribosomal Protein ◽  
Cross Linking ◽  
Mobility Shift ◽  
Promoter Sequences ◽  
Protein Factor ◽  
In The Wild ◽  
Glucocorticoid Responsive Element ◽  
Mobility Shift Assay ◽  
Responsive Element ◽  
Gel Mobility Shift

The murine ribosomal protein (rp) L32 gene contains essential promoter sequences located both upstream and downstream of the cap site. A combination of gel mobility shift, UV cross-linking, and cell-free transcription assays were used to analyze the interaction of factors binding to a downstream element (located at position +25 to +37). The rpL32 downstream element identified polypeptides (transcription factors) ranging in size from 45 to 25 kilodaltons (kDa). Four base pair changes in the wild-type sequence of the downstream element eliminated binding. An oligonucleotide containing the glucocorticoid responsive element sequence competed specifically for the 45-kDa protein in both the gel mobility shift assay and in the UV cross-linking studies. Our data also indicate that the downstream binding factors contribute to cell-free transcription of the rpL32 gene.Key words: ribosomal protein L32, transcription factor, glucocorticoid response element.

scholarly journals cAMP-Responsive Element Binding Protein: A Vital Link in Embryonic Hormonal Adaptation

Endocrinology ◽  
2013 ◽  
Vol 154 (6) ◽  
pp. 2208-2221 ◽  
Author(s):  
Maria Schindler ◽  
Sünje Fischer ◽  
René Thieme ◽  
Bernd Fischer ◽  
Anne Navarrete Santos
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Binding Protein ◽  
Cell Lineage ◽  
Element Binding Protein ◽  
A Cell ◽  
Responsive Element ◽  
Camp Responsive Element Binding

Abstract The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.

scholarly journals Localization of Xenobiotic-responsive Element Binding Protein in Rat Hepatocyte Nuclei After Methylcholanthrene Administration as Revealed by In Situ Southwestern Hybridization

1998 ◽  
Vol 46 (7) ◽  
pp. 825-832 ◽  
Author(s):  
Youko Asaka ◽  
Jun Watanabe ◽  
Shinsuke Kanamura
Keyword(s):  
Laser Scanning ◽  
Binding Protein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mobility Shift ◽  
Competitive Binding Assay ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Gel Mobility Shift

Xenobiotic-responsive element binding protein (XRE-BP), a heterodimer of aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt), regulates the transcription of cytochrome P-450 1A1 gene (CYP1A1) through XRE in response to xenobiotic inducers. For a better understanding of the regulatory mechanism of CYP1A1 through XRE, localization of XRE-BP was examined in liver sections or isolated hepatocyte nuclei from control and 3-methylcholanthrene (MC)-treated rats by in situ Southwestern hybridization, using synthetic XRE as a probe, and was observed by confocal laser scanning microscopy and electron microscopy. Gel mobility shift assay and competitive binding assay showed specificity of the synthetic XRE probe. XRE-BP was exclusively localized in hepatocyte nuclei in liver sections from animals 3 hr after MC injection, whereas the protein was absent in hepatocyte cytoplasm in MC-treated animals and in hepatocyte nuclei and cytoplasm in control animals. In isolated hepatocyte nuclei, XRE-BP began to accumulate in the central region between 0.5 and 3 hr, showed a peak between 3 and 6 hr, decreased gradually between 6 and 72 hr, and disappeared at 72 hr after MC injection. The protein was scarce in peripheral and nucleolar regions of the nucleus. Therefore, XRE-BP is formed in the nuclei of hepatocytes after MC stimulation. In addition, XRE-BP was found in isolated hepatocyte nuclei from control animals after preincubation with cytoplasmic lysate from MC-treated animals, although the protein was absent in the nuclei before the preincubation. These findings strongly suggest that AhR translocates from hepatocyte cytoplasm to the nucleus and forms XRE-BP in the nucleus after MC stimulation.

scholarly journals Cloning of human early B-cell factor and identification of target genes suggest a conserved role in B-cell development in man and mouse

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1457-1464 ◽  
Author(s):  
Ramiro Gisler ◽  
Sten Erik W. Jacobsen ◽  
Mikael Sigvardsson
Keyword(s):  
B Cell ◽  
Binding Sites ◽  
Target Genes ◽  
Cell Development ◽  
B Cell Development ◽  
Human Homologue ◽  
Mobility Shift ◽  
Amino Acid Homology ◽  
Mobility Shift Assay ◽  
Early B Cell Factor

Early B-cell factor (EBF) is a helix–loop–helix transcription factor suggested to be essential for B-cell development in the mouse. Several genetic targets for EBF have been identified in mice, among these the surrogate light chain λ5 and the signal-transducing molecules Igα (mb-1) and Igβ (B29). This article reports cloning of the human homologue of EBF, hEBF. This protein has 93% sequence and 98.8% amino acid homology with mouse EBF. The encoded protein binds DNA and is expressed in cells of the B lineage, but not in cell populations representing T lymphocytes or myeloid cells. It is also shown that EBF-binding sites are functionally conserved in the humanmb-1 and B29 promoters because hEBF interacts with these in the electrophoretic mobility shift assay (EMSA) and have the ability to increase the activity of reporter constructs under the control of these promoters in nonlymphoid HeLa cells. A third genetic target for hEBF is the promoter of the human surrogate light chain14.1. This promoter contains 5 independent binding sites capable of interacting with hEBF in the EMSA, and the activity of the promoter was induced 24-fold in co-transfection experiments. These findings suggest that the human homologue of mouse EBF displays conserved biochemical features as well as genetic targets, indicating that this protein also has an important role in human B-cell development.

scholarly journals Cloning of human early B-cell factor and identification of target genes suggest a conserved role in B-cell development in man and mouse

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1457-1464 ◽  
Author(s):  
Ramiro Gisler ◽  
Sten Erik W. Jacobsen ◽  
Mikael Sigvardsson
Keyword(s):  
B Cell ◽  
Binding Sites ◽  
Target Genes ◽  
Cell Development ◽  
B Cell Development ◽  
Human Homologue ◽  
Mobility Shift ◽  
Amino Acid Homology ◽  
Mobility Shift Assay ◽  
Early B Cell Factor

Abstract Early B-cell factor (EBF) is a helix–loop–helix transcription factor suggested to be essential for B-cell development in the mouse. Several genetic targets for EBF have been identified in mice, among these the surrogate light chain λ5 and the signal-transducing molecules Igα (mb-1) and Igβ (B29). This article reports cloning of the human homologue of EBF, hEBF. This protein has 93% sequence and 98.8% amino acid homology with mouse EBF. The encoded protein binds DNA and is expressed in cells of the B lineage, but not in cell populations representing T lymphocytes or myeloid cells. It is also shown that EBF-binding sites are functionally conserved in the humanmb-1 and B29 promoters because hEBF interacts with these in the electrophoretic mobility shift assay (EMSA) and have the ability to increase the activity of reporter constructs under the control of these promoters in nonlymphoid HeLa cells. A third genetic target for hEBF is the promoter of the human surrogate light chain14.1. This promoter contains 5 independent binding sites capable of interacting with hEBF in the EMSA, and the activity of the promoter was induced 24-fold in co-transfection experiments. These findings suggest that the human homologue of mouse EBF displays conserved biochemical features as well as genetic targets, indicating that this protein also has an important role in human B-cell development.

scholarly journals Runx3 negatively regulates Osterix expression in dental pulp cells

2007 ◽  
Vol 405 (1) ◽  
pp. 69-75 ◽  
Author(s):  
Li Zheng ◽  
Koichiro Iohara ◽  
Masaki Ishikawa ◽  
Takeshi Into ◽  
Teruko Takano-Yamamoto ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Dental Pulp ◽  
Tooth Development ◽  
Tooth Germ ◽  
Dental Pulp Cells ◽  
Mobility Shift ◽  
Pulp Cells ◽  
Mobility Shift Assay ◽  
Responsive Element

Osterix, a zinc-finger-containing transcription factor, is required for osteoblast differentiation and bone formation. Osterix is also expressed in dental mesenchymal cells of the tooth germ. However, transcriptional regulation by Osterix in tooth development is not clear. Genetic studies in osteogenesis place Osterix downstream of Runx2 (Runt-related 2). The expression of Osterix in odontoblasts overlaps with Runx3 during terminal differentiation in vivo. Runx3 down-regulates Osterix expression in mouse DPCs (dental pulp cells). Therefore the regulatory role of Runx3 on Osterix expression in tooth development was investigated. Enforced expression of Runx3 down-regulated the activity of the Osterix promoter in the human embryonic kidney 293 cell line. When the Runx3 responsive element on the Osterix promoter, located at −713 to −707 bp (site 3, AGTGGTT) relative to the cap site, was mutated, this down-regulation was abrogated. Furthermore, electrophoretic mobility-shift assay and chromatin immunoprecipitation assays in mouse DPCs demonstrated direct functional binding of Runx3 to the Osterix promoter. These results demonstrate the transcriptional regulation of Osterix expression by Runx3 during differentiation of dental pulp cells into odontoblasts during tooth development.

scholarly journals Ubiquitin specific peptidase Usp53 regulates osteoblast versus adipocyte lineage commitment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hadla Hariri ◽  
William N. Addison ◽  
René St-Arnaud
Keyword(s):  
Stromal Cells ◽  
Target Gene ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Maturation Stage ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Immunocompromised Mice ◽  
The Impact

AbstractWe have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (− 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.

Definition of a lipopolysaccharide-responsive element in the 5'-flanking regions of MuRantes and crg-2

1994 ◽  
Vol 14 (5) ◽  
pp. 2914-2925
Author(s):  
H S Shin ◽  
B E Drysdale ◽  
M L Shin ◽  
P W Noble ◽  
S N Fisher ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Immediate Early ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Natural Immunity ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Responsive Element ◽  
Stimulation Of

Macrophages are stimulated by lipopolysaccharide (LPS) of gram-negative organisms. The changes in LPS-stimulated macrophages include transcriptional activation of multiple immediate-early genes, which may contribute to the natural immunity to microorganisms. We have defined by deletion and mutational analysis LPS-responsive elements (LREs) in two chemokine genes, MuRantes and crg-2, which are activated in an immediate-early manner. LRE consists of two motifs, TCAYR, which is an AP-1 half site with two flanking bases, and (A/T) (G/C)NTTYC(A/T)NTTY, which resembles in part the interferon-stimulated responsive element (ISRE). The orientation of these two motifs relative to each other in MuRantes differed from that in crg-2. These two motifs are separated by 10 and 6 nonconsensus nucleotides in the MuRantes and crg-2 LREs, respectively. Stimulation of macrophage-like RAW 264.7 cells with alpha/beta interferon did not activate MuRantes, indicating that the ISRE-like motif in MuRantes does not have ISRE activity. Upon stimulation of RAW 264.7 cells with LPS, proteins capable of binding to LRE accumulate in the nuclei as measured by electrophoretic mobility shift assay. These LRE-binding proteins include c-Jun and CREB.

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