scholarly journals Functional characterization of the human phosphodiesterase 7A1 promoter

2003 ◽  
Vol 373 (3) ◽  
pp. 835-843 ◽  
Author(s):  
Mònica TORRAS-LLORT ◽  
Fernando AZORÍN
Keyword(s):  
T Cells ◽  
Promoter Region ◽  
Promoter Activity ◽  
Cpg Island ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Nuclear Factor ◽  
Jurkat T Cells

In this paper, the human phosphodiesterase 7A1 (hPDE7A1) promoter region was identified and functionally characterized. Transient transfection experiments indicated that a 2.9 kb fragment of the hPDE7A1 5′-flanking region, to position −2907, has strong promoter activity in Jurkat T-cells. Deletion analysis showed that the proximal region, up to position −988, contains major cis-regulatory elements of the hPDE7A1 promoter. This minimal promoter region contains a regulatory CpG island which is essential for promoter activity. The CpG island contains three potential cAMP-response-element-binding protein (CREB)-binding sites that, as judged by in vivo dimethyl sulphate (DMS) footprinting, are occupied in Jurkat T-cells. Moreover, over-expression of CREB results in increased promoter activity, but, on the other hand, promoter activity decreases when a dominant-negative form of CREB (KCREB) is over-expressed. In vivo DMS footprinting strongly indicates that other transcription factors, such Ets-2, nuclear factor of activated T-cells 1 (NFAT-1) and nuclear factor κB (NF-κB), might also contribute to the regulation of hPDE7A1 promoter. Finally, hPDE7A1 promoter was found to be induced by treatment with PMA, but not by treatment with dibutyryl cAMP or forskolin. These results provide insights into the factors and mechanisms that regulate expression of the hPDE7A gene.

scholarly journals Identification of cis-acting regulatory elements controlling interleukin-4 gene expression in T cells: roles for NF-Y and NF-ATc.

1993 ◽  
Vol 13 (8) ◽  
pp. 4793-4805 ◽  
Author(s):  
S J Szabo ◽  
J S Gold ◽  
T L Murphy ◽  
K M Murphy
Keyword(s):  
T Cells ◽  
T Cell ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Interleukin 4 ◽  
Specific Factor ◽  
Gene Promoters ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Cis Acting

Activity of the murine interleukin-4 (IL-4) promoter was localized to several cis-acting elements present within the first 300 bp from the transcriptional initiation site. Five repeated elements, P0 to P4, that share the common consensus ATTTTCCNNT were located between -40 and -250, and each was shown to interact with the T-cell-specific factor NF(P). These distinct P sites appear functionally interchangeable and cooperatively confer cyclosporin A-sensitive and ionomycin-inducible promoter activity. NF(P) may be closely related to the cytoplasmic component of NF-AT (nuclear factor of activated T cells), a T-cell-specific factor essential for IL-2 gene transcription, as judged from indistinguishable molecular weights and protease fragmentation patterns of UV-photolabeled factors. Also, we identified an element in the IL-4 promoter with homology to the Y box common to all major histocompatibility complex class II gene promoters. Our data show that the IL-4 promoter Y box -114CTGATTGG-107 significantly enhances overall promoter activity, since point mutations within this element diminish promoter activity by 85%. The factor binding this region is indistinguishable from the cloned nuclear factor NF-Y, as judged from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. Last, we point out the presence of two sites that share sequence identity to the OAP region of the ARRE-1 site within the IL-2 promoter (K. S. Ullman, W. M. Flanagan, C. A. Edwards, and G. R. Crabtree, Science 254:558-562, 1991). These regions, -85GTGTAATA-78 and -245GTGTAATT-238, reside adjacent to the NF(P) binding sites P1 and P4 and bind a distinct nuclear factor.

scholarly journals Mice Lacking the Calcineurin Inhibitor Rcan2 Have an Isolated Defect of Osteoblast Function

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3537-3548 ◽  
Author(s):  
J. H. Duncan Bassett ◽  
John G. Logan ◽  
Alan Boyde ◽  
Moira S. Cheung ◽  
Holly Evans ◽  
...  
Keyword(s):  
T Cells ◽  
Bone Development ◽  
Skeletal Development ◽  
Dominant Negative ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Osteoblast Function ◽  
And Function

Calcineurin-nuclear factor of activated T cells signaling controls the differentiation and function of osteoclasts and osteoblasts, and regulator of calcineurin-2 (Rcan2) is a physiological inhibitor of this pathway. Rcan2 expression is regulated by T3, which also has a central role in skeletal development and bone turnover. To investigate the role of Rcan2 in bone development and maintenance, we characterized Rcan2−/− mice and determined its skeletal expression in T3 receptor (TR) knockout and thyroid-manipulated mice. Rcan2−/− mice had normal linear growth but displayed delayed intramembranous ossification, impaired cortical bone formation, and reduced bone mineral accrual during development as well as increased mineralization of adult bone. These abnormalities resulted from an isolated defect in osteoblast function and are similar to skeletal phenotypes of mice lacking the type 2 deiodinase thyroid hormone activating enzyme or with dominant-negative mutations of TRα, the predominant TR isoform in bone. Rcan2 mRNA was expressed in primary osteoclasts and osteoblasts, and its expression in bone was differentially regulated in TRα and TRβ knockout and thyroid-manipulated mice. However, in primary osteoblast cultures, T3 treatment did not affect Rcan2 mRNA expression or nuclear factor of activated T cells c1 expression and phosphorylation. Overall, these studies establish that Rcan2 regulates osteoblast function and its expression in bone is regulated by thyroid status in vivo.

scholarly journals The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

2004 ◽  
Vol 383 (3) ◽  
pp. 529-536 ◽  
Author(s):  
Zoulika KHERROUCHE ◽  
Yvan DE LAUNOIT ◽  
Didier MONTE
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Core Promoter ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Dnase I Footprinting ◽  
Dominant Negative Form

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoreticmobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.

Identification of cis-acting regulatory elements controlling interleukin-4 gene expression in T cells: roles for NF-Y and NF-ATc

1993 ◽  
Vol 13 (8) ◽  
pp. 4793-4805
Author(s):  
S J Szabo ◽  
J S Gold ◽  
T L Murphy ◽  
K M Murphy
Keyword(s):  
T Cells ◽  
T Cell ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Interleukin 4 ◽  
Specific Factor ◽  
Gene Promoters ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Cis Acting

Activity of the murine interleukin-4 (IL-4) promoter was localized to several cis-acting elements present within the first 300 bp from the transcriptional initiation site. Five repeated elements, P0 to P4, that share the common consensus ATTTTCCNNT were located between -40 and -250, and each was shown to interact with the T-cell-specific factor NF(P). These distinct P sites appear functionally interchangeable and cooperatively confer cyclosporin A-sensitive and ionomycin-inducible promoter activity. NF(P) may be closely related to the cytoplasmic component of NF-AT (nuclear factor of activated T cells), a T-cell-specific factor essential for IL-2 gene transcription, as judged from indistinguishable molecular weights and protease fragmentation patterns of UV-photolabeled factors. Also, we identified an element in the IL-4 promoter with homology to the Y box common to all major histocompatibility complex class II gene promoters. Our data show that the IL-4 promoter Y box -114CTGATTGG-107 significantly enhances overall promoter activity, since point mutations within this element diminish promoter activity by 85%. The factor binding this region is indistinguishable from the cloned nuclear factor NF-Y, as judged from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. Last, we point out the presence of two sites that share sequence identity to the OAP region of the ARRE-1 site within the IL-2 promoter (K. S. Ullman, W. M. Flanagan, C. A. Edwards, and G. R. Crabtree, Science 254:558-562, 1991). These regions, -85GTGTAATA-78 and -245GTGTAATT-238, reside adjacent to the NF(P) binding sites P1 and P4 and bind a distinct nuclear factor.

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

2004 ◽  
Vol 287 (4) ◽  
pp. G822-G829 ◽  
Author(s):  
Svetlana M. Nabokina ◽  
Hamid M. Said
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Critical Role ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Minimal Promoter Region

Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5′-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5′-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between −77 and +59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position −48/−45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

scholarly journals In vivo inhibition of Nuclear Factor of Activated T‐cells (NFAT) results in enhanced levels of anti‐inflammatory IL‐10 in the retina of diabetic mice

2010 ◽  
Vol 24 (S1) ◽  
Author(s):  
Anna V Zetterqvist ◽  
Jenny Nilsson‐Öhman ◽  
Olga Kotova ◽  
Lisa M Nilsson‐Berglund ◽  
Sergio Frutos Garcia ◽  
...  
Keyword(s):  
T Cells ◽  
Nuclear Factor ◽  
Diabetic Mice ◽  
Activated T Cells ◽  
Anti Inflammatory

scholarly journals Functional characterization of Lilium lancifolium cold-responsive Zinc Finger Homeodomain (ZFHD) gene in abscisic acid and osmotic stress tolerance

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11508
Author(s):  
Yubing Yong ◽  
Yue Zhang ◽  
Yingmin Lyu
Keyword(s):  
Abscisic Acid ◽  
Transgenic Plants ◽  
Zinc Finger ◽  
Promoter Region ◽  
Salt Water ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
Response Analysis ◽  
Lilium Lancifolium

Background. We have previously performed an analysis of the cold-responsive transcriptome in the mature leaves of tiger lily (Lilium lancifolium) by gene co-expression network identification. The results has revealed that a ZFHD gene, notated as encoding zinc finger homeodomain protein, may play an essential regulating role in tiger lily response to cold stress. Methods. A further investigation of the ZFHD gene (termed as LlZFHD4) responding to osmotic stresses, including cold, salt, water stresses, and abscisic acid (ABA) was performed in this study. Based on the transcriptome sequences, the coding region and 5′ promoter region of LlZFHD4 were cloned from mature tiger lily leaves. Stress response analysis was performed under continuous 4 °C, NaCl, PEG, and ABA treatments. Functional characterization of LlZFHD4 was conducted in transgenic Arabidopsis, tobacco, and yeast. Results. LlZFHD4 encodes a nuclear-localized protein consisting of 180 amino acids. The N-terminal region of LlZFHD4 has transcriptional activation activity in yeast. The 4 °C, NaCl, PEG, and ABA treatments induced the expression of LlZFHD4. Several stress- or hormone-responsive cis-acting regulatory elements (T-Box, BoxI. and ARF) and binding sites of transcription factors (MYC, DRE and W-box) were found in the core promoter region (789 bp) of LlZFHD4. Also, the GUS gene driven by LlZFHD4 promoter was up-regulated by cold, NaCl, water stresses, and ABA in Arabidopsis. Overexpression of LlZFHD4 improved cold and drought tolerance in transgenic Arabidopsis; higher survival rate and better osmotic adjustment capacity were observed in LlZFHD4 transgenic plants compared to wild type (WT) plants under 4 °C and PEG conditions. However, LlZFHD4 transgenic plants were less tolerant to salinity and more hypersensitive to ABA compared to WT plants. The transcript levels of stress- and ABA-responsive genes were much more up-regulated in LlZFHD4 transgenic Arabidopsis than WT. These results indicate LlZFHD4 is involved in ABA signaling pathway and plays a crucial role in regulating the response of tiger lily to cold, salt and water stresses.

scholarly journals Runx proteins regulate Foxp3 expression

2009 ◽  
Vol 206 (11) ◽  
pp. 2329-2337 ◽  
Author(s):  
Ludovica Bruno ◽  
Luca Mazzarella ◽  
Maarten Hoogenkamp ◽  
Arnulf Hertweck ◽  
Bradley S. Cobb ◽  
...  
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cd4 T Cells ◽  
Target Genes ◽  
De Novo ◽  
Foxp3 Expression ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Downstream Target ◽  
Runx Proteins

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.

Structural and functional characterization of the promoter region of the mouse c-Ki-ras gene

1987 ◽  
Vol 7 (7) ◽  
pp. 2592-2596
Author(s):  
E K Hoffman ◽  
S P Trusko ◽  
N Freeman ◽  
D L George
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Functional Characterization ◽  
Genetic Material ◽  
Bidirectional Promoter ◽  
Adrenocortical Tumor ◽  
Ras Gene ◽  
Untranslated Exon ◽  
Size Heterogeneity

We isolated and characterized the 5' region of the mouse c-Ki-ras gene, including a 5' untranslated exon (exon 0). These studies used genetic material from Y1 mouse adrenocortical tumor cells in which the c-Ki-ras gene is amplified and overexpressed. Our data demonstrate that transcription initiates at multiple sites, predicting size heterogeneity at the 5' ends of the c-Ki-ras mRNAs. Using transient expression assays, we identified a genomic fragment within the 5' region which exhibits bidirectional promoter activity.

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