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Exclusion of NFAT5 from mitotic chromatin resets its nucleo-cytoplasmic distribution in interphase.

Estrada-Gelonch A, Aramburu J, López-Rodríguez C - PLoS ONE (2009)

Bottom Line: Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase.Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5.The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis.

View Article: PubMed Central - PubMed

Affiliation: Immunology Unit, Department of Experimental and Health Sciences (DCEXS), Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain.

ABSTRACT

Background: The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.

Methodology/principal findings: Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.

Conclusions/significance: Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress.

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Transcriptional activity of NFAT5a CTD mutants.NFAT5-deficient MEF cotransfected with the indicated constructs and the ORE-Luc reporter were cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of DBD5 (transcriptionally inactive) in cells cultured in isotonic medium (arbitrary value of 1). Results are the mean±SD of three independent experiments.
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pone-0007036-g009: Transcriptional activity of NFAT5a CTD mutants.NFAT5-deficient MEF cotransfected with the indicated constructs and the ORE-Luc reporter were cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of DBD5 (transcriptionally inactive) in cells cultured in isotonic medium (arbitrary value of 1). Results are the mean±SD of three independent experiments.

Mentions: We next mapped which regions in the CTD of NFAT5a mediated its exclusion from mitotic chromatin (Figure 8 and Figure S5). Two regions encoded by exon 14 were independently capable of causing a strong exclusion (constructs NFAT51−838 and NFAT5 (1−473) + (839−1377)), whereas the regions encoded by the short exons 13 and 15 had a milder effect (constructs NFAT51−547 and NFAT5 (1−497) + (1378−1455)), and the region encoded by exon 12 did not displace NFAT5 from mitotic chromatin (construct NFAT51−497) (Figure 8). In parallel, we analyzed the transcriptional activity of these deletion mutants in isotonic and hypertonic conditions. This analysis was done in mouse embryo fibroblasts (MEFs) devoid of NFAT5 activity [9], to prevent the contribution of endogenous NFAT5. The experiments showed that some of the regions causing the exclusion of NFAT5 from mitotic chromatin overlapped with transactivation domains, whereas others did not confer transcriptional activity (Figures 8 and 9). This is respectively illustrated by constructs NFAT5(1−473)+(839−1377) and NFAT51−838 (Figures 8 and 9). In addition, it was noticeable that among the mutants spanning different regions of the CTD, none of those able to bind to mitotic DNA were transcriptionally active whereas those with transcriptional activity were all excluded from mitotic chromatin. These results indicated that the CTD of NFAT5 contained two types of regions that could independently cause its exclusion from mitotic chromatin: those encoded by exons 13, 15 and part of exon 14 were not essential for its transcriptional activity, whereas the region encoded by the second half of exon 14 had a hypertonicity-activated transcriptional function in interphase that changed to repressing the binding of NFAT5 to chromatin in mitosis.


Exclusion of NFAT5 from mitotic chromatin resets its nucleo-cytoplasmic distribution in interphase.

Estrada-Gelonch A, Aramburu J, López-Rodríguez C - PLoS ONE (2009)

Transcriptional activity of NFAT5a CTD mutants.NFAT5-deficient MEF cotransfected with the indicated constructs and the ORE-Luc reporter were cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of DBD5 (transcriptionally inactive) in cells cultured in isotonic medium (arbitrary value of 1). Results are the mean±SD of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2737149&req=5

pone-0007036-g009: Transcriptional activity of NFAT5a CTD mutants.NFAT5-deficient MEF cotransfected with the indicated constructs and the ORE-Luc reporter were cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (510 mOsm/kg) during 20 hours. The activity of each construct is represented as relative to that of DBD5 (transcriptionally inactive) in cells cultured in isotonic medium (arbitrary value of 1). Results are the mean±SD of three independent experiments.
Mentions: We next mapped which regions in the CTD of NFAT5a mediated its exclusion from mitotic chromatin (Figure 8 and Figure S5). Two regions encoded by exon 14 were independently capable of causing a strong exclusion (constructs NFAT51−838 and NFAT5 (1−473) + (839−1377)), whereas the regions encoded by the short exons 13 and 15 had a milder effect (constructs NFAT51−547 and NFAT5 (1−497) + (1378−1455)), and the region encoded by exon 12 did not displace NFAT5 from mitotic chromatin (construct NFAT51−497) (Figure 8). In parallel, we analyzed the transcriptional activity of these deletion mutants in isotonic and hypertonic conditions. This analysis was done in mouse embryo fibroblasts (MEFs) devoid of NFAT5 activity [9], to prevent the contribution of endogenous NFAT5. The experiments showed that some of the regions causing the exclusion of NFAT5 from mitotic chromatin overlapped with transactivation domains, whereas others did not confer transcriptional activity (Figures 8 and 9). This is respectively illustrated by constructs NFAT5(1−473)+(839−1377) and NFAT51−838 (Figures 8 and 9). In addition, it was noticeable that among the mutants spanning different regions of the CTD, none of those able to bind to mitotic DNA were transcriptionally active whereas those with transcriptional activity were all excluded from mitotic chromatin. These results indicated that the CTD of NFAT5 contained two types of regions that could independently cause its exclusion from mitotic chromatin: those encoded by exons 13, 15 and part of exon 14 were not essential for its transcriptional activity, whereas the region encoded by the second half of exon 14 had a hypertonicity-activated transcriptional function in interphase that changed to repressing the binding of NFAT5 to chromatin in mitosis.

Bottom Line: Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase.Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5.The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis.

View Article: PubMed Central - PubMed

Affiliation: Immunology Unit, Department of Experimental and Health Sciences (DCEXS), Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain.

ABSTRACT

Background: The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.

Methodology/principal findings: Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.

Conclusions/significance: Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress.

Show MeSH
Related in: MedlinePlus