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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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Displacement of NFAT5 from mitotic chromatin.(A) Endogenous NFAT5 was detected with an antibody specific for its DNA binding domain and visualized by immunofluorescence and confocal laser microscopy in asynchronous cultures of HEK293 cells after a fresh change of media to either isotonic conditions (290 or 310 mOsm/kg, 6 hours) or hypertonic conditions (470 mOsm/kg, 4 and 6 hours). Pictures at lower magnification show a general view of the culture, and higher magnification images display individual mitotic cells (marked by arrowheads). Scale bar is 20 µm. (B) Schematic representation of NFAT5a constructs FL5, ND5, DBD5 and DBDC5, and the position of their respective tags: FL5, ND5 and DBDC5 were tagged with 6 copies of a Myc epitope at their amino terminus and GFP at their carboxy terminus. DBD5 was tagged with GFP at its amino terminus. Constructs were expressed in HEK293 cells and detected by Western blotting with anti-Myc antibody (FL5, ND5 and DBDC5) or anti-GFP antibody (DBD5). (C) Representative confocal microscopy images of mitotic cells in asynchronous cultures of HEK293 cells expressing the indicated GFP-tagged constructs in isotonic conditions (310 mOsm/kg) or after a 6-hour exposure to hypertonic conditions (470 mOsm/kg).
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pone-0007036-g001: Displacement of NFAT5 from mitotic chromatin.(A) Endogenous NFAT5 was detected with an antibody specific for its DNA binding domain and visualized by immunofluorescence and confocal laser microscopy in asynchronous cultures of HEK293 cells after a fresh change of media to either isotonic conditions (290 or 310 mOsm/kg, 6 hours) or hypertonic conditions (470 mOsm/kg, 4 and 6 hours). Pictures at lower magnification show a general view of the culture, and higher magnification images display individual mitotic cells (marked by arrowheads). Scale bar is 20 µm. (B) Schematic representation of NFAT5a constructs FL5, ND5, DBD5 and DBDC5, and the position of their respective tags: FL5, ND5 and DBDC5 were tagged with 6 copies of a Myc epitope at their amino terminus and GFP at their carboxy terminus. DBD5 was tagged with GFP at its amino terminus. Constructs were expressed in HEK293 cells and detected by Western blotting with anti-Myc antibody (FL5, ND5 and DBDC5) or anti-GFP antibody (DBD5). (C) Representative confocal microscopy images of mitotic cells in asynchronous cultures of HEK293 cells expressing the indicated GFP-tagged constructs in isotonic conditions (310 mOsm/kg) or after a 6-hour exposure to hypertonic conditions (470 mOsm/kg).

Mentions: As the distribution of many chromatin-binding proteins during mitosis can be readily visualized using cell imaging techniques by their exclusion from condensed chromatin between prophase and telophase [24], our first approach consisted on analyzing whether NFAT5 was retained on, or excluded from, mitotic chromatin in cells growing in isotonic or hypertonic conditions. Confocal laser microscopy analysis of endogenous NFAT5 in HEK293 cells showed that it had a variable nucleo-cytoplasmic distribution in individual cells in asynchronous cultures in isotonic media (290–310 mOsm/kg) and accumulated in the nucleus in response to hypertonicity (470 mOsm/kg) (Figure 1A). The same experiment indicated that NFAT5 was excluded from mitotic chromatin in both isotonic and hypertonic conditions, despite that this stimulus induced a substantial nuclear accumulation of NFAT5 (Figure 1A) and, as will be shown later, its recruitment to chromatin in interphase. Next, we determined the regions of NFAT5 involved in the exclusion from mitotic chromatin (Figure 1B). GFP-tagged full-length NFAT5 (isoform a, FL5) [4] exhibited the same behaviour as the endogenous protein in HEK293 cells, being excluded from mitotic chromatin regardless of hypertonic stimulation (Figures 1C, 2 and Figure S1). However, NFAT5 constructs ND5 and DBD5, which lacked the carboxy-terminal domain (CTD), were constitutively associated with mitotic chromatin in either isotonic (290–310 mOsm/kg) or hypertonic conditions (470 mOsm/kg) (Figures 1C, 2 and Figure S1), and the CTD was sufficient to prevent the association of the DBD with mitotic chromatin (DBDC5 construct in Figure S1). The same result was observed in an independent cell line, U2OS (Figure S1). These results showed that the CTD of NFAT5 caused its 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)

Displacement of NFAT5 from mitotic chromatin.(A) Endogenous NFAT5 was detected with an antibody specific for its DNA binding domain and visualized by immunofluorescence and confocal laser microscopy in asynchronous cultures of HEK293 cells after a fresh change of media to either isotonic conditions (290 or 310 mOsm/kg, 6 hours) or hypertonic conditions (470 mOsm/kg, 4 and 6 hours). Pictures at lower magnification show a general view of the culture, and higher magnification images display individual mitotic cells (marked by arrowheads). Scale bar is 20 µm. (B) Schematic representation of NFAT5a constructs FL5, ND5, DBD5 and DBDC5, and the position of their respective tags: FL5, ND5 and DBDC5 were tagged with 6 copies of a Myc epitope at their amino terminus and GFP at their carboxy terminus. DBD5 was tagged with GFP at its amino terminus. Constructs were expressed in HEK293 cells and detected by Western blotting with anti-Myc antibody (FL5, ND5 and DBDC5) or anti-GFP antibody (DBD5). (C) Representative confocal microscopy images of mitotic cells in asynchronous cultures of HEK293 cells expressing the indicated GFP-tagged constructs in isotonic conditions (310 mOsm/kg) or after a 6-hour exposure to hypertonic conditions (470 mOsm/kg).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2737149&req=5

pone-0007036-g001: Displacement of NFAT5 from mitotic chromatin.(A) Endogenous NFAT5 was detected with an antibody specific for its DNA binding domain and visualized by immunofluorescence and confocal laser microscopy in asynchronous cultures of HEK293 cells after a fresh change of media to either isotonic conditions (290 or 310 mOsm/kg, 6 hours) or hypertonic conditions (470 mOsm/kg, 4 and 6 hours). Pictures at lower magnification show a general view of the culture, and higher magnification images display individual mitotic cells (marked by arrowheads). Scale bar is 20 µm. (B) Schematic representation of NFAT5a constructs FL5, ND5, DBD5 and DBDC5, and the position of their respective tags: FL5, ND5 and DBDC5 were tagged with 6 copies of a Myc epitope at their amino terminus and GFP at their carboxy terminus. DBD5 was tagged with GFP at its amino terminus. Constructs were expressed in HEK293 cells and detected by Western blotting with anti-Myc antibody (FL5, ND5 and DBDC5) or anti-GFP antibody (DBD5). (C) Representative confocal microscopy images of mitotic cells in asynchronous cultures of HEK293 cells expressing the indicated GFP-tagged constructs in isotonic conditions (310 mOsm/kg) or after a 6-hour exposure to hypertonic conditions (470 mOsm/kg).
Mentions: As the distribution of many chromatin-binding proteins during mitosis can be readily visualized using cell imaging techniques by their exclusion from condensed chromatin between prophase and telophase [24], our first approach consisted on analyzing whether NFAT5 was retained on, or excluded from, mitotic chromatin in cells growing in isotonic or hypertonic conditions. Confocal laser microscopy analysis of endogenous NFAT5 in HEK293 cells showed that it had a variable nucleo-cytoplasmic distribution in individual cells in asynchronous cultures in isotonic media (290–310 mOsm/kg) and accumulated in the nucleus in response to hypertonicity (470 mOsm/kg) (Figure 1A). The same experiment indicated that NFAT5 was excluded from mitotic chromatin in both isotonic and hypertonic conditions, despite that this stimulus induced a substantial nuclear accumulation of NFAT5 (Figure 1A) and, as will be shown later, its recruitment to chromatin in interphase. Next, we determined the regions of NFAT5 involved in the exclusion from mitotic chromatin (Figure 1B). GFP-tagged full-length NFAT5 (isoform a, FL5) [4] exhibited the same behaviour as the endogenous protein in HEK293 cells, being excluded from mitotic chromatin regardless of hypertonic stimulation (Figures 1C, 2 and Figure S1). However, NFAT5 constructs ND5 and DBD5, which lacked the carboxy-terminal domain (CTD), were constitutively associated with mitotic chromatin in either isotonic (290–310 mOsm/kg) or hypertonic conditions (470 mOsm/kg) (Figures 1C, 2 and Figure S1), and the CTD was sufficient to prevent the association of the DBD with mitotic chromatin (DBDC5 construct in Figure S1). The same result was observed in an independent cell line, U2OS (Figure S1). These results showed that the CTD of NFAT5 caused its 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