Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A.

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Multi-parameter analysis of the kinetics of NF-κB signalling and transcription in single living cells

Journal of Cell Science ◽

10.1242/jcs.115.6.1137 ◽

2002 ◽

Vol 115 (6) ◽

pp. 1137-1148 ◽

Cited By ~ 1

Author(s):

Glyn Nelson ◽

Luminita Paraoan ◽

David G. Spiller ◽

Geraint J. C. Wilde ◽

Mark A. Browne ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Nuclear Export ◽

Mammalian Cells ◽

Nuclear Translocation ◽

Fluorescent Proteins ◽

Parameter Analysis ◽

Nuclear Accumulation ◽

Fusion Protein Expression ◽

Kinetics Of

Proteins of the NF-κB transcription factor family normally reside in the cytoplasm of cells in a complex with IκB inhibitor proteins. Stimulation with TNFα leads to proteosomal degradation of the IκB proteins and nuclear translocation of the NF-κB proteins. Expression of p65 and IκBα fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IκBα-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression,the half-time of IκBα-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IκBα and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IκBα is the true substrate for TNFα stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor,leptomycin B (LMB), there was nuclear accumulation of IκBα-EGFP and p65-dsRed, with IκBα-EGFP accumulating more rapidly. No NF-κB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IκBα-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFα, degradation of IκBα-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-κB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-κB-dependent transcription occur even when nuclear export of NF-κB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

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Nuclear Export of the Transcription Factor NirA Is a Regulatory Checkpoint for Nitrate Induction in Aspergillus nidulans

Molecular and Cellular Biology ◽

10.1128/mcb.00761-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 791-802 ◽

Cited By ~ 60

Author(s):

Andreas Bernreiter ◽

Ana Ramon ◽

Javier Fernández-Martínez ◽

Harald Berger ◽

Lidia Araújo-Bazan ◽

...

Keyword(s):

Transcription Factor ◽

Aspergillus Nidulans ◽

Transcriptional Activation ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Constitutive Expression ◽

Nuclear Accumulation ◽

Nuclear Retention ◽

Nitrate Induction

ABSTRACT NirA, the specific transcription factor of the nitrate assimilation pathway of Aspergillus nidulans, accumulates in the nucleus upon induction by nitrate. NirA interacts with the nuclear export factor KapK, which bridges an interaction with a protein of the nucleoporin-like family (NplA). Nitrate induction disrupts the NirA-KapK interaction in vivo, whereas KapK associates with NirA when this protein is exported from the nucleus. A KpaK leptomycin-sensitive mutation leads to inducer-independent NirA nuclear accumulation in the presence of the drug. However, this does not lead to constitutive expression of the genes controlled by NirA. A nirA c 1 mutation leads to constitutive nuclear localization and activity, remodeling of chromatin, and in vivo binding to a NirA upstream activation sequence. The nirA c 1 mutation maps in the nuclear export signal (NES) of the NirA protein. The NirA-KapK interaction is nearly abolished in NirAc1 and NirA proteins mutated in canonical leucine residues in the NirA NES. The latter do not result in constitutively active NirA protein, which implies that nuclear retention is necessary but not sufficient for NirA activity. The results are consistent with a model in which activation of NirA by nitrate disrupts the interaction of NirA with the NplA/KapK nuclear export complex, thus resulting in nuclear retention, leading to AreA-facilitated DNA binding of the NirA protein and subsequent chromatin remodeling and transcriptional activation.

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Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

Biochemistry and Cell Biology ◽

10.1139/o05-062 ◽

2005 ◽

Vol 83 (4) ◽

pp. 535-547 ◽

Cited By ~ 7

Author(s):

Gareth N Corry ◽

D Alan Underhill

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Current Knowledge ◽

Nuclear Architecture ◽

Subnuclear Localization ◽

Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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Dimeric transcription factor families: it takes two to tango but who decides on partners and the venue?

Journal of Cell Science ◽

10.1242/jcs.103.1.9 ◽

1992 ◽

Vol 103 (1) ◽

pp. 9-14 ◽

Cited By ~ 1

Author(s):

K.A. Lee

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Dna Binding ◽

Experimental Evidence ◽

Cdna Cloning ◽

Transcriptional Activation ◽

Effector Functions ◽

Dna Binding Domains ◽

Binding Domains ◽

Dna Elements

Dimeric transcription factors that bind to DNA are often grouped into families on the basis of dimerization and DNA-binding specificities. cDNA cloning studies have established that members of the same family have structurally related dimerisation and DNA-binding domains but diverge in other regions that are important for transcriptional activation. These features lead to the straightforward suggestion that although all members of a family bind to similar DNA elements, individual members exhibit distinct transcriptional effector functions. This simple view is now supported by experimental evidence from those systems that have proved amenable to study. There are however some largely unaddressed questions that concern the mechanisms that allow family members to go about their business without interference from their highly related siblings. Here I will discuss some insights from studies of the bZIP class of transcription factors.

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MondoA, a Novel Basic Helix-Loop-Helix–Leucine Zipper Transcriptional Activator That Constitutes a Positive Branch of a Max-Like Network

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8845-8854.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8845-8854 ◽

Cited By ~ 77

Author(s):

Andrew N. Billin ◽

Alanna L. Eilers ◽

Kathryn L. Coulter ◽

Jennifer S. Logan ◽

Donald E. Ayer

Keyword(s):

Transcriptional Activation ◽

Nuclear Export ◽

Mammalian Cells ◽

Leucine Zipper ◽

Functional Characterization ◽

Nuclear Accumulation ◽

Cytoplasmic Localization ◽

Basic Helix Loop Helix ◽

Helix Loop Helix

ABSTRACT Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix–leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

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The impact of cell geometry and the cytoskeleton on the nucleo-cytoplasmic localisation of the SMYD3 methyltransferase suggests that the epigenetic machinery is mechanosensitive

10.1101/2020.03.17.994046 ◽

2020 ◽

Cited By ~ 1

Author(s):

David Pereira ◽

Alain Richert ◽

Souhila Medjkane ◽

Sylvie Hénon ◽

Jonathan B Weitzman

Keyword(s):

Transcription Factors ◽

Nuclear Import ◽

Cell Shape ◽

Nuclear Accumulation ◽

Cell Geometry ◽

Epigenetic Machinery ◽

Lysine Methyltransferase ◽

Epigenetic Regulators ◽

Biochemical Signals ◽

The Impact

Mechanical cues from the cellular microenvironment are converted into biochemical signals controlling diverse cell behaviours, including growth and differentiation. But it is still unclear how mechanotransduction ultimately affects nuclear readouts, genome function and transcriptional programs. Key signaling pathways and transcription factors can be activated, and can relocalize to the nucleus, upon mechanosensing. Here, we tested the hypothesis that epigenetic regulators, such as methyltransferase enzymes, might also contribute to mechanotransduction. We found that the SMYD3 lysine methyltransferase is spatially redistributed dependent on cell geometry (cell shape and aspect ratio) in murine myoblasts. Specifically, elongated rectangles were less permissive than square shapes to SMYD3 nuclear accumulation, via reduced nuclear import. Notably, SMYD3 has both nuclear and cytoplasmic substrates. The distribution of SMYD3 in response to cell geometry correlated with cytoplasmic and nuclear lysine tri-methylation (Kme3) levels, but not Kme2. Moreover, drugs targeting cytoskeletal acto-myosin induced nuclear accumulation of Smyd3. We also observed that square vs rectangular geometry impacted the nuclear-cytoplasmic relocalisation of several mechano-sensitive proteins, notably YAP/TAZ proteins and the SETDB1 methyltransferase. Thus, mechanical cues from cellular geometric shapes are transduced by a combination of transcription factors and epigenetic regulators shuttling between the cell nucleus and cytoplasm.

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Global analysis of the RpaB regulon based on the positional distribution of HLR1 sequences and comparative differential RNA-Seq data

10.1101/443713 ◽

2018 ◽

Author(s):

Matthias Riediger ◽

Taro Kadowaki ◽

Ryuta Nagayama ◽

Jens Georg ◽

Yukako Hihara ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Target Genes ◽

Global Analysis ◽

Affinity Purification ◽

Electron Flow ◽

Positional Distribution ◽

State Transitions ◽

Northern Hybridization

ABSTRACTThe transcription factor RpaB regulates the expression of genes encoding photosynthesis-associated proteins during light acclimation. The binding site of RpaB is the HLR1 motif, a pair of imperfect octameric direct repeats, separated by two random nucleotides. Here, we used high-resolution mapping data of transcriptional start sites (TSSs) in the modelSynechocystissp. PCC 6803 in conjunction with the positional distribution of HLR1 sites for the global prediction of the RpaB regulon. The results demonstrate that RpaB regulates the expression of more than 150 promoters, driving the transcription of protein-coding and non-coding genes and antisense transcripts under low light and upon the shift to high light when DNA binding activity is lost. Transcriptional activation by RpaB is achieved when the HLR1 motif is located 66 to 45 nt upstream, repression occurs when it is close to or overlapping the TSS. Selected examples were validated by multiple experimental approaches, including chromatin affinity purification, reporter gene, northern hybridization and electrophoretic mobility shift assays. We found that RpaB controlsssr2016/pgr5, which is involved in cyclic electron flow and state transitions; six out of nine ferredoxins; three of four FtsH proteases;gcvP/slr0293, encoding a crucial photorespiratory protein; andnirAandisiAfor which we suggest cross-regulation with the transcription factors NtcA or FurA, respectively. In addition to photosynthetic gene functions, RpaB contributes to the control of genes affiliated with nitrogen assimilation, cofactor biosyntheses, the CRISPR system and the circadian clock, making it one of the most versatile regulators in cyanobacteria.Significance StatementRpaB is a transcription factor in cyanobacteria and in the chloroplasts of several lineages of eukaryotic algae. Like other important transcription factors, the gene encoding RpaB cannot be deleted, making the study of deletion mutants impossible. Based on a bioinformatic approach, we increased the number of known genes controlled by RpaB by a factor of 5. Depending on the distance to the TSS, RpaB mediates transcriptional activation or repression. The high number and functional diversity among its target genes and co-regulation with other transcriptional regulators characterize RpaB as a regulatory hub.

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Cytosolic Hsp70 and co-chaperones constitute a novel system for tRNA import into the nucleus

eLife ◽

10.7554/elife.04659 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 10

Author(s):

Akira Takano ◽

Takuya Kajita ◽

Makoto Mochizuki ◽

Toshiya Endo ◽

Tohru Yoshihisa

Keyword(s):

Saccharomyces Cerevisiae ◽

Quality Control ◽

Nuclear Import ◽

Nuclear Export ◽

Substrate Recognition ◽

Structural Features ◽

Nuclear Accumulation ◽

Nucleotide Binding Domain ◽

Trna Import ◽

Trna Binding

tRNAs are unique among various RNAs in that they shuttle between the nucleus and the cytoplasm, and their localization is regulated by nutrient conditions. Although nuclear export of tRNAs has been well documented, the import machinery is poorly understood. Here, we identified Ssa2p, a major cytoplasmic Hsp70 in Saccharomyces cerevisiae, as a tRNA-binding protein whose deletion compromises nuclear accumulation of tRNAs upon nutrient starvation. Ssa2p recognizes several structural features of tRNAs through its nucleotide-binding domain, but prefers loosely-folded tRNAs, suggesting that Ssa2p has a chaperone-like activity for RNAs. Ssa2p also binds Nup116, one of the yeast nucleoporins. Sis1p and Ydj1p, cytoplasmic co-chaperones for Ssa proteins, were also found to contribute to the tRNA import. These results unveil a novel function of the Ssa2p system as a tRNA carrier for nuclear import by a novel mode of substrate recognition. Such Ssa2p-mediated tRNA import likely contributes to quality control of cytosolic tRNAs.

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Nucleo-cytoplasmic distribution of human hnRNP proteins: a search for the targeting domains in hnRNP A1

Journal of Cell Science ◽

10.1242/jcs.108.2.545 ◽

1995 ◽

Vol 108 (2) ◽

pp. 545-555 ◽

Cited By ~ 4

Author(s):

F. Weighardt ◽

G. Biamonti ◽

S. Riva

Keyword(s):

Nuclear Import ◽

Nuclear Export ◽

Rna Binding ◽

Subcellular Localisation ◽

Hnrnp A1 ◽

Reporter Protein ◽

Binding Domains ◽

Rich Domain ◽

Hnrnp Proteins ◽

Active Transcription

hnRNP A1 (34 kDa) is an RNA binding protein consisting of two tandemly arranged RNA binding domains C-terminally linked to a glycine-rich auxiliary domain (2 × RBD-Gly). A1 belongs to the set of polypeptides that bind nascent hnRNA in the nucleus to form the so called hnRNP complexes. These complexes seem to be involved both in pre-mRNA processing and in the nuclear export of mRNA. In fact A1, along with other hnRNP proteins, is exported from the nucleus probably bound to mRNA and is immediately re-imported. A1 nuclear re-import, which requires active transcription, is not mediated by a canonical nuclear localisation signal (NLS). To identify the determinants of A1 subcellular localisation we developed an expression vector for studying the localisation, in transiently transfected cells, of the different structural motifs of A1 fused to a small reporter protein (chloramphenicol acetyltransferase, CAT; 26 kDa). We demonstrate that a 30 amino acid sequence in the glycine-rich domain (YNDFGNYNNQSSNFGPMKGGNFGGRSSGPY), which bears no resemblance to canonical NLS, is necessary and sufficient to target the protein to the nucleus. Our data suggest that this targeting sequence might act by mediating the interaction of A1 with a NLS-containing nuclear import complex. On the other hand, the nuclear export of A1 requires at least one RNA binding domain in accord with the hypothesis that A1 exits from the nucleus bound to mRNA. We propose a mechanism for the nucleo-cytoplasmic shuttling of A1 that envisages a specific role for the different structural domains and can explain the dependence of nuclear import from active transcription.

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Inhibition of CRM1-Mediated Nuclear Export of Transcription FACTORS by Leukemogenic NUP98 Fusion Proteins.

Blood ◽

10.1182/blood.v114.22.1959.1959 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1959-1959

Author(s):

Akiko Takeda ◽

Anmaar M Abdul-Nabi ◽

Nabeel R. Yaseen

Keyword(s):

Transcription Factors ◽

Nuclear Export ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Nuclear Accumulation ◽

Human Leukemia ◽

Dependent Manner ◽

Nucleocytoplasmic Trafficking ◽

Nuclear Retention

Abstract Abstract 1959 Poster Board I-982 NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin (NPMc) and HIV-1 Rev. In-vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG-repeat motif in a Ran-GTP dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of myeloid cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFΚB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFΚB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators. Disclosures: No relevant conflicts of interest to declare.

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