Mutual activation of Ets-1 and AML1 DNA binding by direct interaction of their autoinhibitory domains

Abstract Lineage-specific transcription factors play crucial roles in the development of hematopoietic cells. In a previous study, it was demonstrated that Ras activation was involved in thrombopoietin-induced megakaryocytic differentiation. In this study, constitutive Ras activation by H-rasG12V evoked megakaryocytic maturation of erythroleukemia cell lines F-36P and K562, but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the introduction of GATA-1 led to reprogramming of 32D cl3 toward erythrocytic/megakaryocytic lineage and enabled it to undergo megakaryocytic differentiation in response to H-rasG12V. In contrast, the overexpression of PU.1 and c-Myb changed the phenotype of K562 from erythroid to myeloid/monocytic lineage and rendered K562 to differentiate into granulocytes and macrophages in response to H-rasG12V, respectively. In GATA-1–transfected 32D cl3, the endogenous expression of PU.1 and c-Myb was easily detectable, but their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb–transfected K562. As for the mechanisms of these reciprocal inhibitions, GATA-1 and PU.1 were found to associate through their DNA-binding domains and to inhibit the respective DNA-binding activities of each other. In addition, c-Myb bound to GATA-1 and inhibited its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their own transcriptional activities but could not inhibit the reciprocal partner were less effective in changing the lineage phenotype of 32D cl3 and K562. These results suggested that GATA-1 activities may be crucial for Ras-mediated megakaryocytic differentiation and that its activities may be regulated by the direct interaction with other lineage-specific transcription factors such as PU.1 and c-Myb.

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Targeting NF-κB Signaling by Calebin A, a Compound of Turmeric, in Multicellular Tumor Microenvironment: Potential Role of Apoptosis Induction in CRC Cells

Biomedicines ◽

10.3390/biomedicines8080236 ◽

2020 ◽

Vol 8 (8) ◽

pp. 236 ◽

Cited By ~ 9

Author(s):

Constanze Buhrmann ◽

Parviz Shayan ◽

Kishore Banik ◽

Ajaikumar B. Kunnumakkara ◽

Peter Kubatka ◽

...

Keyword(s):

Tumor Microenvironment ◽

Dna Binding ◽

Binding Assay ◽

Nuclear Translocation ◽

Hct116 Cell ◽

Direct Interaction ◽

Dependent Manner ◽

Natural Agents ◽

Iκb Kinase ◽

Dna Binding Assay

Increasing lines of evidence suggest that chronic inflammation mediates most chronic diseases, including cancer. The transcription factor, NF-κB, has been shown to be a major regulator of inflammation and metastasis in tumor cells. Therefore, compounds or any natural agents that can inhibit NF-κB activation have the potential to prevent and treat cancer. However, the mechanism by which Calebin A, a component of turmeric, regulates inflammation and disrupts the interaction between HCT116 colorectal cancer (CRC) cells and multicellular tumor microenvironment (TME) is still poorly understood. The 3D-alginate HCT116 cell cultures in TME were treated with Calebin A, BMS-345541, and dithiothreitol (DTT) and examined for invasiveness, proliferation, and apoptosis. The mechanism of TME-induced malignancy of cancer cells was confirmed by phase contrast, Western blotting, immunofluorescence, and DNA-binding assay. We found through DNA binding assay, that Calebin A inhibited TME-induced NF-κB activation in a dose-dependent manner. As a result of this inhibition, NF-κB phosphorylation and NF-κB nuclear translocation were down-modulated. Calebin A, or IκB-kinase (IKK) inhibitor (BMS-345541) significantly inhibited the direct interaction of nuclear p65 to DNA, and interestingly this interaction was reversed by DTT. Calebin A also suppressed the expression of NF-κB-promoted anti-apoptotic (Bcl-2, Bcl-xL, survivin), proliferation (Cyclin D1), invasion (MMP-9), metastasis (CXCR4), and down-regulated apoptosis (Caspase-3) gene biomarkers, leading to apoptosis in HCT116 cells. These results suggest that Calebin A can suppress multicellular TME-promoted CRC cell invasion and malignancy by inhibiting the NF-κB-promoting inflammatory pathway associated with carcinogenesis, underlining the potential of Calebin A for CRC treatment.

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The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70 and Core

Journal of Bacteriology ◽

10.1128/jb.188.4.1279-1285.2006 ◽

2006 ◽

Vol 188 (4) ◽

pp. 1279-1285 ◽

Cited By ~ 8

Author(s):

Deborah M. Hinton ◽

Srilatha Vuthoori ◽

Rebecca Mulamba

Keyword(s):

Escherichia Coli ◽

Dna Binding ◽

Rna Polymerase ◽

Dynamic Equilibrium ◽

Bacteriophage T4 ◽

Direct Interaction ◽

Binding Properties ◽

E Coli ◽

Dna Binding Properties ◽

Two Hybrid

ABSTRACT The N-terminal region (region 1.1) of σ70, the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70 regions 2 and 4. Region 1.1 prevents the interaction of free σ70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70 region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P uvsX . However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ70 that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70 and then the AsiA/σ70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ70 plus core, yielding more free σ70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ70 arises through an indirect effect.

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Heterodimers Formed Via Leucine Zipper Interaction between C/EBPα and c-Jun or c-Fos Induce Monocytic Lineage Commitment.

Blood ◽

10.1182/blood.v108.11.1170.1170 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1170-1170 ◽

Cited By ~ 1

Author(s):

Dong Hong Cai ◽

Dehua Wang ◽

Jeffrey R. Keefer ◽

Kortney Hensley ◽

Alan D. Friedman

Keyword(s):

Dna Binding ◽

Leucine Zipper ◽

Biological Effects ◽

Direct Interaction ◽

Salt Bridge ◽

Dna Binding Sites ◽

Intermediate Size ◽

Gel Shift Assay ◽

Cross Inhibition ◽

Myeloid Progenitors

Abstract Mice lacking C/EBPα have markedly reduced granulocyte-monocyte progenitors (GMP). However, the role of C/EBPα in the commitment of GMP to the monocyte versus granulocyte lineages is poorly defined. We recently reported that exogenous C/EBPα favors monocytic commitment of myeloid progenitors but that this activity is obviated if the C/EBPα leucine zipper (LZ) is replaced with the GCN4 LZ. The GCN4 LZ allows homodimerization and DNA-binding via the adjacent basic region (BR) but does not allow heterodimerization. As both AP-1 and Maf proteins have been shown to induce monocytic development, we have sought to determine whether C/EBPα can zipper with these BR-LZ proteins and whether such heterodimers contribute to monopoiesis. Direct interaction between C/EBPα or C/EBPβ and c-Jun, dependent upon their respective LZs, has been previously demonstrated, but whether these interactions represent interaction of the hydrophobic surfaces of their LZs is not known. If their interaction is via zippering, then the respective BRs would be expected to be in position to bind DNA. The LZs of c-Jun, JunB, c-Fos, c-Maf, or MafB were swapped into C/EBPα. In a gel shift assay, the C/EBPα variants containing the c-Jun, JunB, or c-Fos LZs formed bands of intermediate size when co-expressed with truncated C/EBPα or C/EBPβ containing only their BR-LZ domains. In addition, when expressed in 293T cells, wild-type c-Jun, JunB, or c-Fos co-iped with C/EBPα, but not with a variant of C/EBPα in which two leucines within its LZ where changed to valine. C/EBPβ co-iped with higher affinity. Co-ip of endogenous C/EBPα and c-Jun has been previously observed, and we have coiped endogenous C/EBPα and JunB. Confocal microscopy of U937 cells also suggests direct interaction between C/EBPα and c-Jun or c-Fos. One potential consequence of C/EBPα:AP-1 interaction is cross-inhibition. However, given the lower affinity of such heterodimers, we predicted that a positive effect of C/EBP:AP-1 heterodimers, via unique DNA-binding sites, was more likely. To study the biochemical and biological effects of heterodimers independent of C/EBP or Jun homodimers, we modified the GCN4 LZ to contain either glutamic acid or lysine at the salt bridge positions, generating LZE or LZK, and introduced these into C/EBPα, c-Jun, or c-Fos. Neither C/EBPα-LZE nor C/EBPα-LZK bound DNA, whereas the combination did, prefering a C/EBP site to an AP-1 or hybrid site. When C/EBPα-LZE was expressed in 293T cells with c-Jun-LZK or JunB-LZK, the heterodimers that formed had greatest affinity for a hybrid DNA site. When C/EBPα-LZE-ER in pBabePuro and C/EBPα-LZK-ER in MIGR1 were transduced into murine marrow cells, followed by puromycin selection, lineage-depeletion and culture +/− estradiol, only minimal induction of monocytic development was observed in the GFP+ cells, reminiscent of the inactivity of C/EBPα containing the GCN4 LZ. In sharp contrast, the combination of C/EBPα-LZE-ER with either c-Jun-LZK-ER or c-Fos-LZK-ER markedly induced monocytic development in liquid culture or in CFU assays, even in G-CSF/SCF. Also of note, the C/EBPα homodimer slowed proliferation whereas the C/EBPα:c-Jun heterodimer did not. The C/EBPα-LZE-ER:JunB-LZK-ER combination mildly induced monopoiesis and retained the ability to slow proliferation in the same assay. Overall, these data indicate that C/EBPs can zipper with AP-1 proteins and that C/EBPα:c-Jun or C/EBPα:c-Fos heterodimers direct monocytic commitment of myeloid progenitors.

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YY1 can inhibit c-Myc function through a mechanism requiring DNA binding of YY1 but neither its transactivation domain nor direct interaction with c-Myc

Oncogene ◽

10.1038/sj.onc.1201968 ◽

1998 ◽

Vol 17 (4) ◽

pp. 511-520 ◽

Cited By ~ 52

Author(s):

Matthias Austen ◽

Christa Cerni ◽

Juliane M Lüscher-Firzlaff ◽

Bernhard Lüscher

Keyword(s):

Dna Binding ◽

Direct Interaction ◽

Transactivation Domain

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The DNA Architectural Protein HMGB1 Displays Two Distinct Modes of Action That Promote Enhanceosome Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.4390-4401.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 4390-4401 ◽

Cited By ~ 55

Author(s):

Katherine Mitsouras ◽

Ben Wong ◽

Charina Arayata ◽

Reid C. Johnson ◽

Michael Carey

Keyword(s):

Dna Binding ◽

Mutational Analysis ◽

Regulatory Protein ◽

Specific Interaction ◽

Regulatory Region ◽

Direct Interaction ◽

Single Amino Acid ◽

Target Sequence ◽

Mobility Shift ◽

Epstein Barr

ABSTRACT HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.

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Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522372113 ◽

2016 ◽

Vol 113 (16) ◽

pp. 4326-4331 ◽

Cited By ~ 25

Author(s):

Anja Reintjes ◽

Julian E. Fuchs ◽

Leopold Kremser ◽

Herbert H. Lindner ◽

Klaus R. Liedl ◽

...

Keyword(s):

Dna Binding ◽

Stress Responses ◽

Structural Changes ◽

Target Genes ◽

Direct Interaction ◽

Type I ◽

Critical Residue ◽

The Stability ◽

Dynamics Simulations ◽

Protein Arginine Methyltransferase 1

Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB–activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA–NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling.

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C/EBPɛ Directly Interacts With the DNA Binding Domain of c-myb and Cooperatively Activates Transcription of Myeloid Promoters

Blood ◽

10.1182/blood.v93.10.3327 ◽

1999 ◽

Vol 93 (10) ◽

pp. 3327-3337 ◽

Cited By ~ 55

Author(s):

Walter Verbeek ◽

Adrian F. Gombart ◽

Alexey M. Chumakov ◽

Carsten Müller ◽

Alan D. Friedman ◽

...

Keyword(s):

Dna Binding ◽

Cell Line ◽

Transcriptional Activation ◽

Neutrophil Elastase ◽

Myeloid Cells ◽

Direct Interaction ◽

Binding Domain ◽

Jurkat Cells ◽

Dna Binding Domain ◽

Granulocytic Differentiation

C/EBPɛ is essential for granulocytic differentiation. We investigated the role of C/EBPɛ in the transcriptional activation of various myeloid-specific genes. We found that two C/EBPɛ isoforms, p32 and p30, possessing transcriptional activation domains were coexpressed in myeloid cells. Interestingly, isoform C/EBPɛ p30 but not p32 was differentially upregulated in NB-4 promyelocytic leukemia cells treated with retinoids. Both isoforms bound specifically to C/EBP sites in myeloid promoters. The kd for C/EBPɛ binding to the C/EBP site of the neutrophil elastase promoter was 4.2 nmol/L. In transfection assays using the nonhematopoietic cell line, CV-1, the p32 isoform activated promoters from the myeloid-specific mim-1, neutrophil elastase, and granulocyte colony-stimulating factor (G-CSF) receptor genes by 2.5-, 1.8-, and 1.6-fold, respectively. The p30 isoform lacked significant transcriptional activity, suggesting that other hematopoietic-specific factors were required for its function. Consistent with this prediction, transfections into the hematopoietic cell line Jurkat showed a 9.0- and 2.5-fold activation of the mim-1 promoter by the p32 and p30 isoforms, respectively. The additional 32 NH2-terminal residues made p32 a significantly more potent transcriptional activator than p30. T lymphoblasts (Jurkat cells) and immature myeloid cells (eg, Kcl22 cells) expressed high levels of the c-myb hematopoietic transcription factor. Cotransfection of c-myb with either the p32 or p30 isoform of C/EBPɛ in CV-1 cells cooperatively transactivated the mim-1 promoter by 20- and 16-fold, respectively, and the neutrophil elastase promoter by 10-and 7-fold, respectively. Pulldown assays showed that each C/EBPɛ isoform interacted directly with the DNA binding domain of the c-myb protein. Further studies showed that Kcl22 myeloid cells only contained active C/EBPɛ, but not C/EBP, C/EBPβ, or C/EBPδ. A mutation of the C/EBP site in the neutrophil elastase promoter markedly decreased the transactivation of the promoter in Kcl22 myeloblasts. These results demonstrate a role for C/EBPɛ in regulating myeloid promoters, such as neutrophil elastase, probably through a direct interaction with c-myb.

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Novel Antifungal Drug Discovery Based on Targeting Pathways Regulating the Fungus-Conserved Upc2 Transcription Factor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01677-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 258-266 ◽

Cited By ~ 24

Author(s):

Christina Gallo-Ebert ◽

Melissa Donigan ◽

Ilana L. Stroke ◽

Robert N. Swanson ◽

Melissa T. Manners ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Fungal Pathogens ◽

Direct Interaction ◽

Antifungal Drugs ◽

Sterol Biosynthesis ◽

Content Type ◽

Stressed Cells

ABSTRACTInfections byCandida albicansand related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level andUPC2-LacZgene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expressionin vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.

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