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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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Association with mitotic chromatin and subcellular localization of an NFAT5c mutant lacking its CTD.(A) Comparison of the relative mass of endogenous NFAT5 in different cell lines. HA-tagged NFAT5c and HA-tagged NFAT5a expressed in HEK293 cells, or endogenous NFAT5 in the indicated cell lines were resolved in SDS-polyacrylamide (6%) gels. HA-tagged NFAT5c and NFAT5a constructs were detected by Western blot with the HA-specific antibody 12CA5, and endogenous NFAT5 was detected with an antibody specific for carboxy-terminal peptide conserved in all isoforms. A detailed description of this experiment is included in Materials and Methods. (B) Confocal microscopy images of mitotic and interphase HEK293 cells expressing full-length NFAT5c (FL5c), a mutant lacking its CTD (ND5c) or a mutant lacking the CTD and unable to bind DNA (ND5cDB1). Cells transfected with a construct of NFAT5a lacking the CTD (ND5) were analyzed in the same experiment for comparison. Cells were cultured in isotonic medium (290 and 310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of four independent transfections (see Figure S4).
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pone-0007036-g007: Association with mitotic chromatin and subcellular localization of an NFAT5c mutant lacking its CTD.(A) Comparison of the relative mass of endogenous NFAT5 in different cell lines. HA-tagged NFAT5c and HA-tagged NFAT5a expressed in HEK293 cells, or endogenous NFAT5 in the indicated cell lines were resolved in SDS-polyacrylamide (6%) gels. HA-tagged NFAT5c and NFAT5a constructs were detected by Western blot with the HA-specific antibody 12CA5, and endogenous NFAT5 was detected with an antibody specific for carboxy-terminal peptide conserved in all isoforms. A detailed description of this experiment is included in Materials and Methods. (B) Confocal microscopy images of mitotic and interphase HEK293 cells expressing full-length NFAT5c (FL5c), a mutant lacking its CTD (ND5c) or a mutant lacking the CTD and unable to bind DNA (ND5cDB1). Cells transfected with a construct of NFAT5a lacking the CTD (ND5) were analyzed in the same experiment for comparison. Cells were cultured in isotonic medium (290 and 310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of four independent transfections (see Figure S4).

Mentions: Until here, we had used constructs corresponding to isoform NFAT5a. Three mRNA isoforms of NFAT5 have been described, all of which encode predicted proteins that are identical along 1455 amino acids and they only differ in that NFAT5b has 29 extra amino acids in its amino-terminus with respect to the shorter NFAT5a, and isoform NFAT5c is 47 amino acids longer than NFAT5b and 76 amino acids longer than NFAT5a [4]. A distinct feature of isoform NFAT5c is that it has a CRM1-dependent canonical nuclear export signal (NES) in its first 15 amino acids, which is absent from isoforms a and b [20]. At present it is unknown whether all isoforms are expressed as proteins in different cell types. To detect the potential presence of various protein isoforms we prepared lysates of several cell types and did Western blot with an antibody specific for the carboxy terminus, which is common to all three isoforms. As controls, we used lysates of HEK293 cells transfected with NFAT5a or NFAT5c constructs tagged with 3 copies of an HA epitope, which added 38 amino acids to each respective NFAT5 isoform. The anti-NFAT5 antibody detected a single band in SDS-polyacrylamide gels of lysates of HEK293, U2OS, HeLa and mouse embryo fibroblasts (MEFs) (Figure 7A). The mobility of endogenous NFAT5 in HEK293 was faster than that of the recombinant HA-NFAT5a expressed in the same cell line and clearly faster than that of HA-NFAT5c (Figure 7A). Similarly, endogenous NFAT5 in the other cell lines (U2OS, HeLa, MEFs) migrated faster than HA-NFAT5a and HA-NFAT5c. Hence, this experiment suggested that the predominant isform in them corresponded to NFAT5a. Nonetheless, we analyzed whether the long NFAT5c isoform also exhibited a CTD-dependent regulation of its subcellular localization in interphase. Full-length NFAT5c (FL5c) expressed in HEK293 cells was predominantly cytosolic in isotonic medium (290–310 mOsm/kg) and completely nuclear in hypertonic conditions (470 mOsm/kg) (Figure 7B and Figure S4). NFAT5c was also excluded from mitotic chromatin in isotonic as well as hypertonic conditions, and removal of its CTD caused it to bind mitotic chromatin (ND5c, Figure 7B and Figure S4). ND5c was more accumulated in the nucleus than full length NFAT5c or NFAT5a in isotonic conditions (290–310 mOsm/kg) (Figure 7B and Figure S4), which suggested that the NES and AED were not sufficient to ensure a complete cytoplasmic localization of an NFAT5c construct lacking the CTD. This observation was in agreement with experiments by Tong et al. using a similar NFAT5c deletion, which showed an appreciable degree of nuclear localization in isotonic medium, although it became cytoplasmic under hypotonic conditions [20]. The nuclear accumulation of ND5c in isotonic conditions was prevented by disrupting its DNA binding capacity (construct ND5cDB1) (Figure 7B and Figure S4). Altogether, our results suggested that the cytoplasmic localization of both NFAT5a and NFAT5c isoforms in isotonic conditions required the CTD-mediated exclusion from mitotic chromatin.


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

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

Association with mitotic chromatin and subcellular localization of an NFAT5c mutant lacking its CTD.(A) Comparison of the relative mass of endogenous NFAT5 in different cell lines. HA-tagged NFAT5c and HA-tagged NFAT5a expressed in HEK293 cells, or endogenous NFAT5 in the indicated cell lines were resolved in SDS-polyacrylamide (6%) gels. HA-tagged NFAT5c and NFAT5a constructs were detected by Western blot with the HA-specific antibody 12CA5, and endogenous NFAT5 was detected with an antibody specific for carboxy-terminal peptide conserved in all isoforms. A detailed description of this experiment is included in Materials and Methods. (B) Confocal microscopy images of mitotic and interphase HEK293 cells expressing full-length NFAT5c (FL5c), a mutant lacking its CTD (ND5c) or a mutant lacking the CTD and unable to bind DNA (ND5cDB1). Cells transfected with a construct of NFAT5a lacking the CTD (ND5) were analyzed in the same experiment for comparison. Cells were cultured in isotonic medium (290 and 310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of four independent transfections (see Figure S4).
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Related In: Results  -  Collection

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pone-0007036-g007: Association with mitotic chromatin and subcellular localization of an NFAT5c mutant lacking its CTD.(A) Comparison of the relative mass of endogenous NFAT5 in different cell lines. HA-tagged NFAT5c and HA-tagged NFAT5a expressed in HEK293 cells, or endogenous NFAT5 in the indicated cell lines were resolved in SDS-polyacrylamide (6%) gels. HA-tagged NFAT5c and NFAT5a constructs were detected by Western blot with the HA-specific antibody 12CA5, and endogenous NFAT5 was detected with an antibody specific for carboxy-terminal peptide conserved in all isoforms. A detailed description of this experiment is included in Materials and Methods. (B) Confocal microscopy images of mitotic and interphase HEK293 cells expressing full-length NFAT5c (FL5c), a mutant lacking its CTD (ND5c) or a mutant lacking the CTD and unable to bind DNA (ND5cDB1). Cells transfected with a construct of NFAT5a lacking the CTD (ND5) were analyzed in the same experiment for comparison. Cells were cultured in isotonic medium (290 and 310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours. Scale bar is 20 µm. Results shown are representative of four independent transfections (see Figure S4).
Mentions: Until here, we had used constructs corresponding to isoform NFAT5a. Three mRNA isoforms of NFAT5 have been described, all of which encode predicted proteins that are identical along 1455 amino acids and they only differ in that NFAT5b has 29 extra amino acids in its amino-terminus with respect to the shorter NFAT5a, and isoform NFAT5c is 47 amino acids longer than NFAT5b and 76 amino acids longer than NFAT5a [4]. A distinct feature of isoform NFAT5c is that it has a CRM1-dependent canonical nuclear export signal (NES) in its first 15 amino acids, which is absent from isoforms a and b [20]. At present it is unknown whether all isoforms are expressed as proteins in different cell types. To detect the potential presence of various protein isoforms we prepared lysates of several cell types and did Western blot with an antibody specific for the carboxy terminus, which is common to all three isoforms. As controls, we used lysates of HEK293 cells transfected with NFAT5a or NFAT5c constructs tagged with 3 copies of an HA epitope, which added 38 amino acids to each respective NFAT5 isoform. The anti-NFAT5 antibody detected a single band in SDS-polyacrylamide gels of lysates of HEK293, U2OS, HeLa and mouse embryo fibroblasts (MEFs) (Figure 7A). The mobility of endogenous NFAT5 in HEK293 was faster than that of the recombinant HA-NFAT5a expressed in the same cell line and clearly faster than that of HA-NFAT5c (Figure 7A). Similarly, endogenous NFAT5 in the other cell lines (U2OS, HeLa, MEFs) migrated faster than HA-NFAT5a and HA-NFAT5c. Hence, this experiment suggested that the predominant isform in them corresponded to NFAT5a. Nonetheless, we analyzed whether the long NFAT5c isoform also exhibited a CTD-dependent regulation of its subcellular localization in interphase. Full-length NFAT5c (FL5c) expressed in HEK293 cells was predominantly cytosolic in isotonic medium (290–310 mOsm/kg) and completely nuclear in hypertonic conditions (470 mOsm/kg) (Figure 7B and Figure S4). NFAT5c was also excluded from mitotic chromatin in isotonic as well as hypertonic conditions, and removal of its CTD caused it to bind mitotic chromatin (ND5c, Figure 7B and Figure S4). ND5c was more accumulated in the nucleus than full length NFAT5c or NFAT5a in isotonic conditions (290–310 mOsm/kg) (Figure 7B and Figure S4), which suggested that the NES and AED were not sufficient to ensure a complete cytoplasmic localization of an NFAT5c construct lacking the CTD. This observation was in agreement with experiments by Tong et al. using a similar NFAT5c deletion, which showed an appreciable degree of nuclear localization in isotonic medium, although it became cytoplasmic under hypotonic conditions [20]. The nuclear accumulation of ND5c in isotonic conditions was prevented by disrupting its DNA binding capacity (construct ND5cDB1) (Figure 7B and Figure S4). Altogether, our results suggested that the cytoplasmic localization of both NFAT5a and NFAT5c isoforms in isotonic conditions required the CTD-mediated exclusion from mitotic chromatin.

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