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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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Regulation of the localization of NFAT5a by its CTD, nuclear localization signal (NLS), the auxiliary export domain (AED) and DNA binding.(A) Confocal microscopy images of HEK293 cells (whole culture or mitotic cells) expressing wild-type NFAT5a (FL5), an NLS mutant (FL5NLS), or an AED mutant (FL5AED). Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. (B) Confocal microscopy images of HEK293 cells expressing DNA-binding-competent (upper panel) or DNA-binding-disabled (DB1, bottom panel) versions of ND5 with intact NLS and AED motifs or with the NLS, AED or both mutated. Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of three independent transfections.
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pone-0007036-g006: Regulation of the localization of NFAT5a by its CTD, nuclear localization signal (NLS), the auxiliary export domain (AED) and DNA binding.(A) Confocal microscopy images of HEK293 cells (whole culture or mitotic cells) expressing wild-type NFAT5a (FL5), an NLS mutant (FL5NLS), or an AED mutant (FL5AED). Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. (B) Confocal microscopy images of HEK293 cells expressing DNA-binding-competent (upper panel) or DNA-binding-disabled (DB1, bottom panel) versions of ND5 with intact NLS and AED motifs or with the NLS, AED or both mutated. Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of three independent transfections.

Mentions: The results described above indicated that dissociation of NFAT5 from mitotic DNA could influence the equilibrium between nuclear import and export mechanisms in interphase. We examined the relative contribution of import, export and DNA binding to the subcellular localization of NFAT5 in isotonic conditions. These experiments, as those in previous figures, were done with isoform NFAT5a, which has a nuclear localization sequence (NLS) and an auxiliary export domain (AED), but lacks the nuclear export signal (NES) exclusive of the longer isoform NFAT5c [20]. Mutation of the NLS impaired the translocation of full length NFAT5a in response to hypertonicity, whereas removal of its AED caused it to localize in the nucleus in isotonic conditions (Figure 6A), indicating that the NLS and AED were sufficient to control the nucleo-cytoplasmic transport of NFAT5a in isotonic or hypertonic conditions. However, since full-length NFAT5a was naturally excluded from mitotic chromatin (Figures 1C and 2), we tested the effect of the NLS and AED mutations in the ND5 construct to take into account the specific contribution of mitotic DNA binding on import and export in interphase. We then observed that the NLS mutant was cytoplasmic and unable to translocate in response to hypertonicity, whereas the AED mutant was constitutively nuclear in isotonic conditions (Figure 6B). Both mutants were capable of binding to mitotic chromatin (Figure S3). The result that inactivating the NLS of ND5 suppressed its nuclear localization in isotonic conditions was intriguing, since in view of our previous results (Figures 1C and 2) we would have expected that this mutant (ND5NLS) should have been retained to some extent in the nucleus due to its ability to associate with chromatin during mitosis. These results suggested that the nuclear retention of ND5 in isotonic conditions was due to both its binding to chromatin during mitosis and nuclear import in interphase, and that lack of either allowed the AED to become dominant and cause its export. Consistent with this, a double mutant lacking both the NLS and AED (ND5NLS+AED) displayed a pancellular distribution in interphase, which was dependent only on its DNA binding capacity and was insensitive to hypertonicity (comparison between ND5NLS+AED and ND5DB1+NLS+AED in Figure 6B and Figure S3). These results indicated that both the NLS and AED of NFAT5a were active in isotonic conditions, and that the CTD-mediated exclusion from mitotic chromatin was required to shift its export/import balance towards a cytoplasmic localization.


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

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

Regulation of the localization of NFAT5a by its CTD, nuclear localization signal (NLS), the auxiliary export domain (AED) and DNA binding.(A) Confocal microscopy images of HEK293 cells (whole culture or mitotic cells) expressing wild-type NFAT5a (FL5), an NLS mutant (FL5NLS), or an AED mutant (FL5AED). Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. (B) Confocal microscopy images of HEK293 cells expressing DNA-binding-competent (upper panel) or DNA-binding-disabled (DB1, bottom panel) versions of ND5 with intact NLS and AED motifs or with the NLS, AED or both mutated. Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of three independent transfections.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2737149&req=5

pone-0007036-g006: Regulation of the localization of NFAT5a by its CTD, nuclear localization signal (NLS), the auxiliary export domain (AED) and DNA binding.(A) Confocal microscopy images of HEK293 cells (whole culture or mitotic cells) expressing wild-type NFAT5a (FL5), an NLS mutant (FL5NLS), or an AED mutant (FL5AED). Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. (B) Confocal microscopy images of HEK293 cells expressing DNA-binding-competent (upper panel) or DNA-binding-disabled (DB1, bottom panel) versions of ND5 with intact NLS and AED motifs or with the NLS, AED or both mutated. Cells were grown in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of three independent transfections.
Mentions: The results described above indicated that dissociation of NFAT5 from mitotic DNA could influence the equilibrium between nuclear import and export mechanisms in interphase. We examined the relative contribution of import, export and DNA binding to the subcellular localization of NFAT5 in isotonic conditions. These experiments, as those in previous figures, were done with isoform NFAT5a, which has a nuclear localization sequence (NLS) and an auxiliary export domain (AED), but lacks the nuclear export signal (NES) exclusive of the longer isoform NFAT5c [20]. Mutation of the NLS impaired the translocation of full length NFAT5a in response to hypertonicity, whereas removal of its AED caused it to localize in the nucleus in isotonic conditions (Figure 6A), indicating that the NLS and AED were sufficient to control the nucleo-cytoplasmic transport of NFAT5a in isotonic or hypertonic conditions. However, since full-length NFAT5a was naturally excluded from mitotic chromatin (Figures 1C and 2), we tested the effect of the NLS and AED mutations in the ND5 construct to take into account the specific contribution of mitotic DNA binding on import and export in interphase. We then observed that the NLS mutant was cytoplasmic and unable to translocate in response to hypertonicity, whereas the AED mutant was constitutively nuclear in isotonic conditions (Figure 6B). Both mutants were capable of binding to mitotic chromatin (Figure S3). The result that inactivating the NLS of ND5 suppressed its nuclear localization in isotonic conditions was intriguing, since in view of our previous results (Figures 1C and 2) we would have expected that this mutant (ND5NLS) should have been retained to some extent in the nucleus due to its ability to associate with chromatin during mitosis. These results suggested that the nuclear retention of ND5 in isotonic conditions was due to both its binding to chromatin during mitosis and nuclear import in interphase, and that lack of either allowed the AED to become dominant and cause its export. Consistent with this, a double mutant lacking both the NLS and AED (ND5NLS+AED) displayed a pancellular distribution in interphase, which was dependent only on its DNA binding capacity and was insensitive to hypertonicity (comparison between ND5NLS+AED and ND5DB1+NLS+AED in Figure 6B and Figure S3). These results indicated that both the NLS and AED of NFAT5a were active in isotonic conditions, and that the CTD-mediated exclusion from mitotic chromatin was required to shift its export/import balance towards a cytoplasmic localization.

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