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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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Subcellular distribution of NFAT5a constructs.Summary of subcellular localization analyses of the indicated NFAT5a constructs in interphase HEK293 cells cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg, 4 hours). At least 150 cells with similar GFP fluorescence intensity were counted for each transfected construct in each assay. Results are the mean±SD of three independent experiments.
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pone-0007036-g004: Subcellular distribution of NFAT5a constructs.Summary of subcellular localization analyses of the indicated NFAT5a constructs in interphase HEK293 cells cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg, 4 hours). At least 150 cells with similar GFP fluorescence intensity were counted for each transfected construct in each assay. Results are the mean±SD of three independent experiments.

Mentions: Mutation of the critical DNA contact residues Thr222, Glu223 and Arg226 of NFAT5a to alanine (ND5DB1 and DBD5DB1 mutants) [5] prevented the association of ND5 and DBD5 with mitotic chromatin (Figure 3A) and caused them to become cytoplasmic in interphase in isotonic conditions (Figure 3B). This result indicated that the association of ND5 and DBD5 with mitotic chromatin was due to their direct binding to specific NFAT5-recognized DNA elements. Both FL5 and ND5 DNA binding mutants (FL5DB1 and ND5DB1), which were comparably cytoplasmic in isotonic conditions, could translocate to the nucleus completely in response to hypertonicity (Figure 3B). In contrast, the subcellular localization of DBD5 was entirely dependent on its ability to bind DNA and insensitive to tonicity (see comparison of DBD5 and DBD5DB1 in Figures 3B and Figure S2B). Since this domain lacked import and export signals, we hypothesized that its access to DNA could occur in mitosis, and that the formation of the nuclear envelope after mitosis would keep it trapped in the nucleus in the subsequent interphase. To test this, we expressed the DBD5 in HEK293 and U2OS cells and, immediately after transfection, treated them with 2-hydroxyurea to cause their arrest at the G1/S transition so that they would not transit to mitosis (Figure 3C). Stalling of cells in G1 precluded the nuclear localization of the DBD5 in HEK293 and U2OS cells (Figure 3C), supporting the interpretation that binding to mitotic chromatin was necessary for its nuclear retention later in interphase. A summary of the results on nucleo-cytoplasmic localization of FL5, ND5, DBD5 and their corresponding DNA-binding mutants in isotonic and hypertonic conditions is shown in Figure 4. These results indicated that the constitutive nuclear localization of NFAT5 mutants lacking the CTD was due to their specific binding to DNA 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)

Subcellular distribution of NFAT5a constructs.Summary of subcellular localization analyses of the indicated NFAT5a constructs in interphase HEK293 cells cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg, 4 hours). At least 150 cells with similar GFP fluorescence intensity were counted for each transfected construct in each assay. Results are the mean±SD of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2737149&req=5

pone-0007036-g004: Subcellular distribution of NFAT5a constructs.Summary of subcellular localization analyses of the indicated NFAT5a constructs in interphase HEK293 cells cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg, 4 hours). At least 150 cells with similar GFP fluorescence intensity were counted for each transfected construct in each assay. Results are the mean±SD of three independent experiments.
Mentions: Mutation of the critical DNA contact residues Thr222, Glu223 and Arg226 of NFAT5a to alanine (ND5DB1 and DBD5DB1 mutants) [5] prevented the association of ND5 and DBD5 with mitotic chromatin (Figure 3A) and caused them to become cytoplasmic in interphase in isotonic conditions (Figure 3B). This result indicated that the association of ND5 and DBD5 with mitotic chromatin was due to their direct binding to specific NFAT5-recognized DNA elements. Both FL5 and ND5 DNA binding mutants (FL5DB1 and ND5DB1), which were comparably cytoplasmic in isotonic conditions, could translocate to the nucleus completely in response to hypertonicity (Figure 3B). In contrast, the subcellular localization of DBD5 was entirely dependent on its ability to bind DNA and insensitive to tonicity (see comparison of DBD5 and DBD5DB1 in Figures 3B and Figure S2B). Since this domain lacked import and export signals, we hypothesized that its access to DNA could occur in mitosis, and that the formation of the nuclear envelope after mitosis would keep it trapped in the nucleus in the subsequent interphase. To test this, we expressed the DBD5 in HEK293 and U2OS cells and, immediately after transfection, treated them with 2-hydroxyurea to cause their arrest at the G1/S transition so that they would not transit to mitosis (Figure 3C). Stalling of cells in G1 precluded the nuclear localization of the DBD5 in HEK293 and U2OS cells (Figure 3C), supporting the interpretation that binding to mitotic chromatin was necessary for its nuclear retention later in interphase. A summary of the results on nucleo-cytoplasmic localization of FL5, ND5, DBD5 and their corresponding DNA-binding mutants in isotonic and hypertonic conditions is shown in Figure 4. These results indicated that the constitutive nuclear localization of NFAT5 mutants lacking the CTD was due to their specific binding to DNA 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