Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila

The highly conserved Notch signaling pathway controls a multitude of developmental processes including hematopoiesis. Here, we provide evidence for a novel mechanism of tissue-specific Notch regulation involving phosphorylation of CSL transcription factors within the DNA-binding domain. Earlier we found that a phospho-mimetic mutation of the Drosophila CSL ortholog Suppressor of Hairless [Su(H)] at Ser269 impedes DNA-binding. By genome-engineering, we now introduced phospho-specific Su(H) mutants at the endogenous Su(H) locus, encoding either a phospho-deficient [Su(H)S269A] or a phospho-mimetic [Su(H)S269D] isoform. Su(H)S269D mutants were defective of Notch activity in all analyzed tissues, consistent with impaired DNA-binding. In contrast, the phospho-deficient Su(H)S269A mutant did not generally augment Notch activity, but rather specifically in several aspects of blood cell development. Unexpectedly, this process was independent of the corepressor Hairless acting otherwise as a general Notch antagonist in Drosophila. This finding is in agreement with a novel mode of Notch regulation by posttranslational modification of Su(H) in the context of hematopoiesis. Importantly, our studies of the mammalian CSL ortholog (RBPJ/CBF1) emphasize a potential conservation of this regulatory mechanism: phospho-mimetic RBPJS221D was dysfunctional in both the fly as well as two human cell culture models, whereas phospho-deficient RBPJS221A rather gained activity during fly hematopoiesis. Thus, dynamic phosphorylation of CSL-proteins within the DNA-binding domain provides a novel means to fine-tune Notch signal transduction in a context-dependent manner.

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Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.1922-1934.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 1922-1934 ◽

Cited By ~ 128

Author(s):

Marcel J. M. Schaaf ◽

John A. Cidlowski

Keyword(s):

Dna Binding ◽

Glucocorticoid Receptor ◽

Ligand Binding ◽

Conformational Change ◽

Binding Domain ◽

Ligand Binding Domain ◽

Dependent Manner ◽

Dna Binding Domain ◽

Ligand Affinity ◽

High Affinity

ABSTRACT The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR α-isoform (hGRα) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRα ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRα antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRα mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRα deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRα differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.

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Methylation of RUNX1 by the Nuclear PRMT5/COPR5 Complex Regulates Its Cellular Location in Leukemia Cells,

Blood ◽

10.1182/blood.v118.21.3403.3403 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3403-3403

Author(s):

Xinyang Zhao ◽

Ly P. Vu ◽

Fabiana Perna ◽

Fan Liu ◽

Hao Xu ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Arginine Methylation ◽

Binding Domain ◽

Dependent Manner ◽

Dna Binding Domain ◽

Sm Proteins ◽

Differentiation Potential ◽

Hematopoietic Stem

Abstract Abstract 3403 RUNX1 is a transcription factor that is required for definitive hematopoietic development, and helps regulate long term hematopoietic stem cell self-renewal, platelet production, and lymphocyte development during adult hematopoiesis. RUNX1 is known to be modified via phosphorylation, acetylation, ubiquitination and methylation, for example on R208 and R210 by PRMT1, which activates its activating function. We continue to investigate how the methylation of RUNX1 by other protein arginine methyl transferases (PRMTs) regulates its function. Loop 9 of the DNA binding domain (the Runt domain) of RUNX1 contains an SGRGK sequence that is also present on the tails of histone H2A and H4. The histone tails of H4 and H2A can be methylated by a purified PRMT5 complex in vitro. An enzymatically active in vitro PRMT5 complex capable of methylating histones and SM proteins requires two subunits: both PRMT5 and MEP50, a WD 40 repeat domain protein. Nevertheless, this purified PRMT5/MEP50 complex cannot methylate the DNA binding domain of the RUNX1 protein in vitro. We show that RUNX1 also can be symmetrically methylated at R142 within the SGRGK motif in vitro by a nuclear PRMT5/MEP50 complex which also contains COPR5. We show after RUNX1 is methylated on R142 within the nucleus of HEL cells, RUNX1 is exported to the cytoplasm in a CRM1 dependent manner, as the export of methylated RUNX1 is blocked by lemptomycin B. CRM1 interacts with PRMT5, supporting that PRMT5 mediated arginine methylation tags protein for nuclear export. Therefore, PRMT5 not only involves in epigenetic regulation by methylation of histones but also it can directly controls the level of transcription factor proteins within the nucleus. Polycytocemia Vera patients who express the Jak2V617F mutation have low PRMT5 activity due to JAK2V617F mediated PRMT5 phosphorylation (Liu et al 2011). How Jak2 signaling affects RUNX1 methylation and RUNX1 localization within the nucleus is still under investigation. By controlling the amount of RUNX1 available within the cell nucleus, PRMT5 may regulate lineage differentiation potential and growth potential of hematopoietic stem and progenitor cells. The nuclear localization of RUNX1 can be changed through post translational modification such as arginine methylation in addition to point mutations and translocations involving RUNX1 found patients with leukemia and pre-leukemic diseases. Disclosures: No relevant conflicts of interest to declare.

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NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3

Journal of Virology ◽

10.1128/jvi.01202-18 ◽

2018 ◽

Vol 92 (23) ◽

Cited By ~ 6

Author(s):

Jennifer Deborah Wuerth ◽

Matthias Habjan ◽

Julia Wulle ◽

Giulio Superti-Furga ◽

Andreas Pichlmair ◽

...

Keyword(s):

Dna Binding ◽

World War I ◽

Binding Domain ◽

Nuclear Accumulation ◽

Type I ◽

Dependent Manner ◽

Dna Binding Domain ◽

Regulatory Factor ◽

World War ◽

Highly Virulent

ABSTRACT Sandfly fever Sicilian virus (SFSV) is one of the most widespread and frequently identified members of the genus Phlebovirus (order Bunyavirales, family Phenuiviridae) infecting humans. Being transmitted by Phlebotomus sandflies, SFSV causes a self-limiting, acute, often incapacitating febrile disease (“sandfly fever,” “Pappataci fever,” or “dog disease”) that has been known since at least the beginning of the 20th century. We show that, similarly to other pathogenic phleboviruses, SFSV suppresses the induction of the antiviral type I interferon (IFN) system in an NSs-dependent manner. SFSV NSs interfered with the TBK1-interferon regulatory factor 3 (IRF3) branch of the RIG-I signaling pathway but not with NF-κB activation. Consistently, we identified IRF3 as a host interactor of SFSV NSs. In contrast to IRF3, neither the IFN master regulator IRF7 nor any of the related transcription factors IRF2, IRF5, and IRF9 were bound by SFSV NSs. In spite of this specificity for IRF3, NSs did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the IRF3 activation or multimerization state. In further studies, we identified the DNA-binding domain of IRF3 (amino acids 1 to 113) as sufficient for NSs binding and found that SFSV NSs prevented the association of activated IRF3 with the IFN-β promoter. Thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, SFSV modulates type I IFN induction by directly masking the DNA-binding domain of IRF3. IMPORTANCE Phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. Outbreaks of sandfly fever were reported in the 19th century, during World War I, and during World War II. Currently, SFSV is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. We show how the nonstructural NSs protein of SFSV counteracts the upregulation of the antiviral interferon (IFN) system. SFSV NSs specifically inhibits promoter binding by IFN transcription factor 3 (IRF3), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic RNA viruses in general. This IRF3-specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of SFSV.

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The POU domain of SCIP/Tst-1/Oct-6 is sufficient for activation of an acetylcholine receptor promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.5004 ◽

1996 ◽

Vol 16 (9) ◽

pp. 5004-5014 ◽

Cited By ~ 20

Author(s):

D Fyodorov ◽

E Deneris

Keyword(s):

Dna Binding ◽

Acetylcholine Receptor ◽

Transcriptional Activation ◽

Binding Domain ◽

Dependent Manner ◽

Dna Binding Domain ◽

Wild Type ◽

Amino Terminal ◽

Pou Domain ◽

Synergistic Activation

In the PC12 neuroendocrine line, the neuronal nicotinic acetylcholine receptor alpha3 gene promoter is activated by SCIP/Tst-1/Oct-6, a POU domain transcription factor proposed to be important for regulating the development of specific neural cell populations. In this study, we have investigated the SCIP polypeptide domains involved in alpha3 promoter activation. The characteristics of activation by a chimeric effector in which the GAL4 DNA binding domain was substituted for the SCIP POU domain were dramatically different from those of wild-type SCIP. At low effector masses, the chimeric polypeptide weakly activated alpha3 in a GAL4 binding-site-dependent manner but then squelched transcription at higher masses. In contrast, wild-type SCIP activation was not modulated by the presence of multimerized SCIP binding sites, and squelching was not observed. Analysis of wild-type SCIP truncations revealed that deletion of the previously characterized SCIP amino-terminal activation domain did not destroy activity of the factor. Surprisingly, a truncation expressing nothing more than the POU domain was nearly as active as wild-type SCIP. Moreover, cotransfection of a GAL4-VP16 effector with an effector expressing just the SCIP POU domain resulted in synergistic activation of the promoter. Synergistic activation did not depend on an Sp1 motif that is the only functional alpha3 cis element outside the transcription start site region. Our results show that the DNA binding domain of a POU factor is capable of transcriptional activation probably through protein-protein interactions with components of the basal transcription complex.

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GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription

Blood ◽

10.1182/blood.v95.8.2543 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2543-2551 ◽

Cited By ~ 230

Author(s):

Claus Nerlov ◽

Erich Querfurth ◽

Holger Kulessa ◽

Thomas Graf

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Direct Interaction ◽

Binding Domain ◽

Dependent Manner ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Ets Domain ◽

And Function

Abstract The GATA-1 transcription factor is capable of suppressing the myeloid gene expression program when ectopically expressed in myeloid cells. We examined the ability of GATA-1 to repress the expression and function of the PU.1 transcription factor, a central regulator of myeloid differentiation. We found that GATA-1 is capable of suppressing the myeloid phenotype without interfering with PU.1 gene expression, but instead was capable of inhibiting the activity of the PU.1 protein in a dose-dependent manner. This inhibition was independent of the ability of GATA-1 to bind DNA, suggesting that it is mediated by protein-protein interaction. We examined the ability of PU.1 to interact with GATA-1 and found a direct interaction between the PU.1 ETS domain and the C-terminal finger region of GATA-1. Replacing the PU.1 ETS domain with the GAL4 DNA-binding domain removed the ability of GATA-1 to inhibit PU.1 activity, indicating that the PU.1 DNA-binding domain, rather than the transactivation domain, is the target for GATA-1–mediated repression. We therefore propose that GATA-1 represses myeloid gene expression, at least in part, through its ability to directly interact with the PU.1 ETS domain and thereby interfere with PU.1 function.

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SUMO-Mediated Inhibition of Glucocorticoid Receptor Synergistic Activity Depends on Stable Assembly at the Promoter But Not on DAXX

Molecular Endocrinology ◽

10.1210/me.2007-0581 ◽

2008 ◽

Vol 22 (9) ◽

pp. 2061-2075 ◽

Cited By ~ 38

Author(s):

Sam R. Holmstrom ◽

Sergey Chupreta ◽

Alex Yick-Lun So ◽

Jorge A. Iñiguez-Lluhí

Keyword(s):

Dna Binding ◽

Glucocorticoid Receptor ◽

Binding Domain ◽

Synergistic Activity ◽

Dependent Manner ◽

Dna Binding Domain ◽

Dimerization Interface ◽

Endogenous Genes ◽

Nuclear Receptor Family ◽

Genomic Regions

Abstract Multiple transcription factors, including members of the nuclear receptor family, harbor one or more copies of a short regulatory motif that limits synergistic transactivation in a context-dependent manner. These synergy control (SC) motifs exert their effects by serving as sites for posttranslational modification by small ubiquitin-like modifier (SUMO) proteins. By analyzing the requirements for both synergy control and SUMOylation in the glucocorticoid receptor (GR), we find that an intact ligand-binding domain and an engaged DNA- binding domain dimerization interface are necessary for effective synergy control. However, these features, which promote stable assembly of GR-DNA complexes, are required downstream of SUMOylation because their disruption or deletion does not interfere with SUMO modification. Remarkably, in the absence of these features, sensitivity to the effects of SUMOylation can be restored simply by stabilization of DNA interactions through a heterologous DNA binding domain. The data indicate that stable interaction with DNA is an important prerequisite for SUMO-dependent transcriptional inhibition. Analysis of genomic regions occupied by GR indicates that the effects of SC motif SUMOylation are most evident at multiple, near-ideal GR binding sites and that SUMOylation selectively affects the induction of linked endogenous genes. Although the SUMO-binding protein DAXX has been proposed to mediate the inhibitory effects of GR SUMOylation, we find that inhibition by DAXX is independent of GR SUMOylation. Furthermore, neither expression nor knockdown of DAXX influences SUMO effects on GR. We therefore propose that stable binding of GR to multiple sites on DNA allows for the SUMO-dependent recruitment of inhibitory factors distinct from DAXX.

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GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription

Blood ◽

10.1182/blood.v95.8.2543.008k19_2543_2551 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2543-2551 ◽

Cited By ~ 11

Author(s):

Claus Nerlov ◽

Erich Querfurth ◽

Holger Kulessa ◽

Thomas Graf

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Direct Interaction ◽

Binding Domain ◽

Dependent Manner ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Ets Domain ◽

And Function

The GATA-1 transcription factor is capable of suppressing the myeloid gene expression program when ectopically expressed in myeloid cells. We examined the ability of GATA-1 to repress the expression and function of the PU.1 transcription factor, a central regulator of myeloid differentiation. We found that GATA-1 is capable of suppressing the myeloid phenotype without interfering with PU.1 gene expression, but instead was capable of inhibiting the activity of the PU.1 protein in a dose-dependent manner. This inhibition was independent of the ability of GATA-1 to bind DNA, suggesting that it is mediated by protein-protein interaction. We examined the ability of PU.1 to interact with GATA-1 and found a direct interaction between the PU.1 ETS domain and the C-terminal finger region of GATA-1. Replacing the PU.1 ETS domain with the GAL4 DNA-binding domain removed the ability of GATA-1 to inhibit PU.1 activity, indicating that the PU.1 DNA-binding domain, rather than the transactivation domain, is the target for GATA-1–mediated repression. We therefore propose that GATA-1 represses myeloid gene expression, at least in part, through its ability to directly interact with the PU.1 ETS domain and thereby interfere with PU.1 function.

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Modulation of Runx2 Activity by Estrogen Receptor-α: Implications for Osteoporosis and Breast Cancer

Endocrinology ◽

10.1210/en.2008-0680 ◽

2008 ◽

Vol 149 (12) ◽

pp. 5984-5995 ◽

Cited By ~ 65

Author(s):

Omar Khalid ◽

Sanjeev K. Baniwal ◽

Daniel J. Purcell ◽

Nathalie Leclerc ◽

Yankel Gabet ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Dna Binding ◽

Transcriptional Activation ◽

Target Genes ◽

Estrogen Receptor Α ◽

Binding Domain ◽

Breast Epithelial Cell ◽

Dependent Manner ◽

Dna Binding Domain

The transcription factors Runx2 and estrogen receptor-α (ERα) are involved in numerous normal and disease processes, including postmenopausal osteoporosis and breast cancer. Using indirect immunofluorescence microscopy and pull-down techniques, we found them to colocalize and form complexes in a ligand-dependent manner. Estradiol-bound ERα strongly interacted with Runx2 directly through its DNA-binding domain and only indirectly through its N-terminal and ligand-binding domains. Runx2’s amino acids 417–514, encompassing activation domain 3 and the nuclear matrix targeting sequence, were sufficient for interaction with ERα’s DNA-binding domain. As a consequence of the interaction, Runx2’s transcriptional activation activity was strongly repressed, as shown by reporter assays in COS7 cells, breast cancer cells, and late-stage MC3T3-E1 osteoblast cultures. Metaanalysis of gene expression in 779 breast cancer biopsies indicated negative correlation between the expression of ERα and Runx2 target genes. Selective ER modulators (SERM) induced ERα-Runx2 interactions but led to various functional outcomes. The regulation of Runx2 by ERα may play key roles in osteoblast and breast epithelial cell growth and differentiation; hence, modulation of Runx2 by native and synthetic ERα ligands offers new avenues in selective ER modulator evaluation and development.

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The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis

Development ◽

10.1242/dev.124.3.693 ◽

1997 ◽

Vol 124 (3) ◽

pp. 693-702 ◽

Cited By ~ 23

Author(s):

D.A. Wettstein ◽

D.L. Turner ◽

C. Kintner

Keyword(s):

Dna Binding ◽

Notch Signaling ◽

Signaling Pathway ◽

Cell Interaction ◽

Notch Signaling Pathway ◽

Notch 1 ◽

Xenopus Embryos ◽

Suppressor Of Hairless ◽

Primary Neurogenesis ◽

Enhancer Of Split

The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, we isolated the Xenopus homolog of Suppressor of Hairless (X-Su(H)), a component of the Notch signaling pathway in Drosophila. Using animal cap assays, we show that X-Su(H) induces ESR-1 expression, perhaps directly, when modified by the addition of ankyrin repeats. Using a DNA binding mutant of X-Su(H), we show that X-Su(H) activity is required for induction of ESR-1. Finally, expression of the DNA binding mutant in embryos leads to a neurogenic phenotype as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos.

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