Novel Phosphorylated Forms of E2F-1 Transcription Factor Bind to the Retinoblastoma Protein in Cells Overexpressing an E2F-1 cDNA

1997 ◽  
Vol 232 (2) ◽  
pp. 336-339 ◽  
Author(s):  
Xiao-He Yang ◽  
Todd L. Sladek
Keyword(s):  
Transcription Factor ◽  
Retinoblastoma Protein ◽  
In Cells

Inhibition of NGLY1 inactivates the transcription factor Nrf1 and potentiates proteasome inhibitor cytotoxicity

2017 ◽  
Author(s):  
Carolyn Bertozzi ◽  
Fred Tomlin ◽  
Ulla Gerling-Driessen ◽  
Yi-Chang Liu ◽  
Ryan Flynn ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Proteasome Inhibitor ◽  
Activation Mechanism ◽  
Cancer Drugs ◽  
Small Molecule Library ◽  
Library Screen ◽  
In Cells

We discovered that the proteostasis modulating transcription factor Nrf1 requires cytosolic de-N-glycosylation by the N-glycanase NGly1 as part of its activation mechanism. Through a covalent small molecule library screen, we discovered an inhibitor of NGly1 that blocks Nrf1 activation in cells and potentiates the activity of proteasome inhibitor cancer drugs. The requirement of NGly1 for Nrf1 activity likely underlies several pathologies associated with a rare hereditary deficiency in NGly1.

scholarly journals Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ilaria Frasson ◽  
Paola Soldà ◽  
Matteo Nadai ◽  
Sara Lago ◽  
Sara N. Richter
Keyword(s):  
Transcription Factor ◽  
Early Gene ◽  
Proximity Ligation Assay ◽  
Gene Promoters ◽  
Herpes Simplex Virus 1 ◽  
Direct Binding ◽  
Simplex Virus ◽  
In Cells ◽  
Hsv 1

AbstractG-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.

scholarly journals Clonal sector analysis and cell ablation confirm a function for DORNROESCHEN-LIKE in founder cells and the vasculature in Arabidopsis

Planta ◽  
2021 ◽  
Vol 253 (2) ◽  
Author(s):  
Dorothea Glowa ◽  
Petra Comelli ◽  
John W. Chandler ◽  
Wolfgang Werr
Keyword(s):  
Transcription Factor ◽  
Functional Gene ◽  
Gene Analysis ◽  
Cell Ablation ◽  
Sector Analysis ◽  
Founder Cells ◽  
Conventional Methods ◽  
Functional Gene Analysis ◽  
Lineage Analysis ◽  
In Cells

Abstract Main conclusion Inducible lineage analysis and cell ablation via conditional toxin expression in cells expressing the DORNRÖSCHEN-LIKE transcription factor represent an effective and complementary adjunct to conventional methods of functional gene analysis. Abstract Classical methods of functional gene analysis via mutational and expression studies possess inherent limitations, and therefore, the function of a large proportion of transcription factors remains unknown. We have employed two complementary, indirect methods to obtain functional information for the AP2/ERF transcription factor DORNRÖSCHEN-LIKE (DRNL), which is dynamically expressed in flowers and marks lateral organ founder cells. An inducible, two-component Cre–Lox system was used to express beta-glucuronidase GUS in cells expressing DRNL, to perform a sector analysis that reveals lineages of cells that transiently expressed DRNL throughout plant development. In a complementary approach, an inducible system was used to ablate cells expressing DRNL using diphtheria toxin A chain, to visualise the phenotypic consequences. These complementary analyses demonstrate that DRNL functionally marks founder cells of leaves and floral organs. Clonal sectors also included the vasculature of the leaves and petals, implicating a previously unidentified role for DRNL in provasculature development, which was confirmed in cotyledons by closer analysis of drnl mutants. Our findings demonstrate that inducible gene-specific lineage analysis and cell ablation via conditional toxin expression represent an effective and informative adjunct to conventional methods of functional gene analysis.

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 Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor.

1997 ◽  
Vol 17 (1) ◽  
pp. 482-494 ◽  
Author(s):  
L Cavarec ◽  
S Jensen ◽  
J F Casella ◽  
S A Cristescu ◽  
T Heidmann
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Axon Growth ◽  
Expression Vectors ◽  
Recognition Sequence ◽  
Enhancer Sequence ◽  
Cells In Culture ◽  
Copia Retrotransposon ◽  
Gel Shift ◽  
In Cells

By transfection experiments, we previously identified a 72-bp enhancer sequence within the Drosophila copia retrotransposon which is involved in the control of the transcription level of this mobile element in cells in culture. Gel shift assays with nuclear extracts from Drosophila hydei-derived DH-33 cells further demonstrated specific interactions of at least two nuclear factors with this enhancer sequence. Using this sequence as a probe for the screening of an expression cDNA library that we constructed from DH-33 cells RNA, we have isolated a cDNA clone encoding a 110-kDa protein with features common to those of known transcription factors; these include a two-zinc-finger motif at the C terminus, three glutamine-rich domains in the presumptive activation domain of the protein, and an N-terminal domain which shares homology with the Bric-à-brac, Tramtrack, and Broad-Complex BTB boxes. The precise DNA recognition sequence for this transcription factor has been determined by both gel shift assays and footprinting experiments with a recombinant protein made in bacteria. The functionality of the cloned element was demonstrated upon transcriptional activation of copia reporter genes, as well as of a minimal promoter coupled with the identified target DNA sequence, in cotransfection assays in cells in culture with an expression vector for the cloned factor. Southern blot and nucleotide sequence analyses revealed a related gene in Drosophila melanogaster (the lola gene) previously identified by a genetic approach as involved in axon growth and guidance. Transfection assays in cells in culture with lola gene expression vectors and in situ hybridization experiments with lola gene mutants finally provided evidence that the copia retrotransposon is regulated by this neurogenic gene in D.melanogaster, with a repressor effect in the central nervous systems of the embryos.

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.

Activating PTPN11 Mutants Promote Myelomonocytic Differentiation Due to Altered Hematopoietic Lineage-Specific Transcription Factor Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3519-3519
Author(s):  
Rebecca J. Chan ◽  
Cara S. Voorhorst ◽  
Takako Kondo ◽  
Eri Hashino
Keyword(s):  
Tissue Culture ◽  
Transcription Factor ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Macrophage Differentiation ◽  
Hematopoietic Lineage ◽  
Factor Expression ◽  
Tissue Culture Plate ◽  
Transcription Factor Expression ◽  
In Cells

Abstract Juvenile myelomonocytic leukemia (JMML) is a lethal disease of young children characterized by pathologic overproduction of myelomonocytic cells. Mutations in PTPN11, which encodes protein tyrosine phosphatase, Shp-2, occur in 35% of JMML patients. We demonstrated that macrophage progenitors expressing activating PTPN11 mutants hyperproliferate in response to GM-CSF(Chan et al., Blood105, 2005). Based on these findings and the the clinical phenotype of JMML, we hypothesized that in addition to inducing hyperproliferation, activating PTPN11 mutants promote commitment to the myelomonocytic lineage at the expense of other myeloid lineages. To test this hypothesis, we compared the ability of cells expressing either WT or mutant Shp-2 to undergo mast cell differentiation. WT and three mutant Shp-2 cDNAs (E76K, D61V, and D61Y, commonly observed in JMML patients) were subcloned into the retroviral vector pMIEG3 in tandem with EGFP. Murine bone marrow low density mononuclear cells (LDMNCs) or lineage depleted (lin-/lo) cells were transduced, sorted for EGFP positive cells, and cultured in IL-3-containing media commonly used for mast cell development. Morphologically, LDMNCs expressing the activating PTPN11 mutants demonstrated increased adherence to the tissue culture plate, suggestive of monocyte/macrophage differentiation. Phenotypic analysis using flow cytometry demonstrated a significantly higher level of Mac-1 and F4/80 on cells expressing the mutants (Mac-1: MIEG3 24.6+/−8.9, WT Shp-2 19.3+/−7.9, E76K 91.7+/−013, D61V 84.7+/−5, and D61Y 88.5+/−3, n=4, p<0.001 for E76K, D61Y, or D61V v. MIEG3 or WT Shp-2; F4/80: MIEG3 8+/−0.6, WT Shp-2 9.9+/−0.9, E76K 66.7+/−9.7, D61V 60.5+/−4, and D61Y 56.5+/−1.4, n=2, p≤0.03 for E76K, D61Y, or D61V v. MIEG3 or WT Shp-2). In contrast, a significantly lower level of c-kit was expressed on cells bearing the PTPN11 mutants (c-kit: MIEG3 65+/−12.6, WT Shp-2 74.8+/−9.3, E76K 18.6+/−8.1, D61V 26.8+/−8.2, D61Y 19.1+/−8.1, n=4, p≤0.05 for E76K, D61Y, or D61V v. MIEG3 or WT Shp-2. Statistics conducted using Students t test). To test more specifically whether activating PTPN11 mutants alter hematopoietic progenitor differentiation, lin-/lo cells were transduced with MIEG3, WT Shp-2, or D61Y. Similar to that observed with LDMNCs, lin-/lo cells expressing D61Y preferentially adhered to the tissue culture plate and expressed significantly higher levels of F4/80 and Mac-1 following culture in IL-3. These data suggest that activating PTPN11 mutants vary hematopoietic cell fate specification and imply alterations in hematopoietic lineage-specific transcription factor expression. PU.1 is required for both macrophage and mast cell differentiation while GATA-2 is dispensible for macrophage development. We predicted elevated PU.1 and reduced GATA-2 expression in cells bearing activating PTPN11 mutants, thus accounting for preferential macrophage differentiation. Using quantitative RT-PCR, a modest elevation of PU.1 expression was found in cells expressing the activating PTPN11 mutants. However, GATA-2 expression in cells bearing the activating PTPN11 mutants was only 5–10% of that in cells transduced with MIEG3 or WT Shp-2, suggesting that Shp-2 gain-of-function mutants alter signaling pathways such that GATA-2 expression is diminished and monocytic differentiation is permitted at the expense of mast cell development. These data imply that normalization of molecular aberrancies at the transcription factor level may provide novel therapeutic tactics for the improved treatment of JMML.

The C-Terminal Zinc Finger of GATA2 Is Necessary for Normalization of Aberrant Myelomonocytic Differentiation Induced by Activating PTPN11 Mutations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3177-3177
Author(s):  
Zhenyun Yang ◽  
Cara S. Voorhorst ◽  
Leila Ndong ◽  
Fuqin Yin ◽  
Takako Kondo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Mast Cell ◽  
Monocytic Differentiation ◽  
Rt Pcr ◽  
Hematopoietic Progenitors ◽  
Murine Bone Marrow ◽  
Factor Expression ◽  
Transcription Factor Expression ◽  
In Cells

Abstract Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation despite being subjected to conditions that typically support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression revealed that GATA2 expression, needed for mast cell differentiation, is dramatically reduced, while, surprisingly, PU.1 expression is unchanged in cells expressing activating Shp2 mutants. In addition to lineage-specific transcription factors such as PU.1, however, c-jun also promotes monocytic differentiation by functioning as a co-activator of PU.1. Thus, we hypothesized that activating Shp2 mutations (Shp2D61Y or Shp2 E76K) induce increased c-jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression in hematopoietic progenitors. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (GATA2del330–407), which is needed for disruption of the PU.1-c-jun interaction, would normalize aberrant myelomonocytic differentiation induced by activating Shp2 mutants. Consistent with our hypothesis, quantitative RT-PCR studies revealed 5-fold higher c-jun levels in cells expressing Shp2D61Y or Shp2E76K compared to cells expressing WT Shp2. We next utilized retroviral co-transduction of murine bone marrow LDMNCs to generate six groups: pMIEG3-Shp2WT plus pCD4 (empty vector); pMIEG3-Shp2WT plus pCD4-GATA2; pMIEG3-Shp2WT plus pCD4-GATA2del330-407; pMIEG3-Shp2E76K plus pCD4; pMIEG3-Shp2E76K plus pCD4-GATA2; and pMIEG3-Shp-2E76K plus pCD4-GATA2del330-407. Transduced cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2E76K plus pCD4 produced significantly more CFU-M than cells co-transduced with WT Shp2 plus pCD4. Upon co-transduction with GATA2, the number of CFU-M generated from Shp2E76K-expressing cells was significantly reduced and was similar to that observed in cells expressing WT Shp2. In contrast, co-transduction of GATA2del330-407 failed to normalize the number of CFU-M produced by Shp2E76K-expressing cells. Quantitative RT-PCR verified ectopic GATA2 and GATA2del330-407 expression in the co-transduced cells. These findings demonstrate that restoration of GATA2 expression normalizes the propensity toward monocytic differentiation induced by Shp2E76K. The lack of correction conferred by GATA2del330-407 in combination with the observed increased c-jun expression support a model in which GATA2 and c-jun compete for binding to PU.1 to direct cell differentiation decisions in hematopoietic progenitors bearing activating Shp2 mutants. Collectively, these findings imply that normalization of transcription factor expression may provide a novel approach to differentiation-mediated therapy in JMML.

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