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NFAT1 and NFAT3 cooperate with HDAC4 during regulation of alternative splicing of PMCA isoforms in PC12 cells.

Kosiorek M, Podszywalow-Bartnicka P, Zylinska L, Pikula S - PLoS ONE (2014)

Bottom Line: Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA.RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells.NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland; Department of Neurodegenerative Disorders, Laboratory of Neurogenetics, Mossakowski Medical Research Centre PAS, Warsaw, Poland.

ABSTRACT

Background: The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants.

Results: In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.

Conclusions: We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

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Related in: MedlinePlus

HDAC4-NFAT1/NFAT3 complex contribution to regulation of PMCA alternative splicing in PC12 cells.HDAC/NFAT involvement in regulation of alternative splicing of PMCA transcripts was analyzed by qPCR-chromatin immunoprecipitation. The analysis was performed for the splicing at site A and at site for four PMCA isoforms and the PCR products were migrated in 1% agarose gels (A). The qPCR data, were expressed as fold of change (2−ΔΔC) calculated from the difference: ΔCT of output (immunoprecipitated DNA with HDAC/NFATs) – ΔCT of input (total DNA) and statistics were calculated according to nonparametric paired Wilcoxon signed rank test at 95% confidence, where PMCA2-deficient cells (_2) or PMCA3-deficient cells (_3) were compared to control cells assigned to y = 1 value (B). Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3), n = 3.
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pone-0099118-g005: HDAC4-NFAT1/NFAT3 complex contribution to regulation of PMCA alternative splicing in PC12 cells.HDAC/NFAT involvement in regulation of alternative splicing of PMCA transcripts was analyzed by qPCR-chromatin immunoprecipitation. The analysis was performed for the splicing at site A and at site for four PMCA isoforms and the PCR products were migrated in 1% agarose gels (A). The qPCR data, were expressed as fold of change (2−ΔΔC) calculated from the difference: ΔCT of output (immunoprecipitated DNA with HDAC/NFATs) – ΔCT of input (total DNA) and statistics were calculated according to nonparametric paired Wilcoxon signed rank test at 95% confidence, where PMCA2-deficient cells (_2) or PMCA3-deficient cells (_3) were compared to control cells assigned to y = 1 value (B). Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3), n = 3.

Mentions: Regarding the protein interactions suggested above and formation of protein complexes consisting of NFAT1 and HDAC4 or NFAT3 and HDAC4, in the next step we examined whether these protein complexes may play a role in the regulation of alternative splicing of PMCAs. For that, binding of the NFAT1/NFAT3-HDAC4 complex to the splicing regulatory sites of PMCA isoforms has been studied with the use of chromatin immunoprecipitation technique with some modifications. Cross-linked chromatin and proteins were incubated with anti-NFAT1 and anti-NFAT3 antibodies. The DNA-protein complexes were further immunoprecipitated with anti-HDAC4 antibody. According to this protocol, generation of the PCR products of PMCA alternative splicing variants should be linked with the NFAT1/NFAT3-HDAC4 complex. Among all possible combinations of PMCA splicing variants only the PMCA2x splicing variant was detected under these conditions, suggesting that its generation might be related with HDAC4-NFAT1/NFAT3 binding to the splicing regulatory sites of the gene encoding PMCA2 (Fig. 5A), as shown in the supplementary material 2 (Fig. S2). The qPCR data, were expressed as fold of change (2−ΔΔC) calculated from the difference: ΔCT of output (immunoprecipitated DNA with HDAC/NFATs) – ΔCT of input (total DNA) and revealed that PMCA2x splicing variant generation was statistically significantly related to the NFAT1/NFAT3-HDAC4 complex activity, according to nonparametric paired Wilcoxon signed rank test at 95% confidence (Fig. 5B).


NFAT1 and NFAT3 cooperate with HDAC4 during regulation of alternative splicing of PMCA isoforms in PC12 cells.

Kosiorek M, Podszywalow-Bartnicka P, Zylinska L, Pikula S - PLoS ONE (2014)

HDAC4-NFAT1/NFAT3 complex contribution to regulation of PMCA alternative splicing in PC12 cells.HDAC/NFAT involvement in regulation of alternative splicing of PMCA transcripts was analyzed by qPCR-chromatin immunoprecipitation. The analysis was performed for the splicing at site A and at site for four PMCA isoforms and the PCR products were migrated in 1% agarose gels (A). The qPCR data, were expressed as fold of change (2−ΔΔC) calculated from the difference: ΔCT of output (immunoprecipitated DNA with HDAC/NFATs) – ΔCT of input (total DNA) and statistics were calculated according to nonparametric paired Wilcoxon signed rank test at 95% confidence, where PMCA2-deficient cells (_2) or PMCA3-deficient cells (_3) were compared to control cells assigned to y = 1 value (B). Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3), n = 3.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048221&req=5

pone-0099118-g005: HDAC4-NFAT1/NFAT3 complex contribution to regulation of PMCA alternative splicing in PC12 cells.HDAC/NFAT involvement in regulation of alternative splicing of PMCA transcripts was analyzed by qPCR-chromatin immunoprecipitation. The analysis was performed for the splicing at site A and at site for four PMCA isoforms and the PCR products were migrated in 1% agarose gels (A). The qPCR data, were expressed as fold of change (2−ΔΔC) calculated from the difference: ΔCT of output (immunoprecipitated DNA with HDAC/NFATs) – ΔCT of input (total DNA) and statistics were calculated according to nonparametric paired Wilcoxon signed rank test at 95% confidence, where PMCA2-deficient cells (_2) or PMCA3-deficient cells (_3) were compared to control cells assigned to y = 1 value (B). Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3), n = 3.
Mentions: Regarding the protein interactions suggested above and formation of protein complexes consisting of NFAT1 and HDAC4 or NFAT3 and HDAC4, in the next step we examined whether these protein complexes may play a role in the regulation of alternative splicing of PMCAs. For that, binding of the NFAT1/NFAT3-HDAC4 complex to the splicing regulatory sites of PMCA isoforms has been studied with the use of chromatin immunoprecipitation technique with some modifications. Cross-linked chromatin and proteins were incubated with anti-NFAT1 and anti-NFAT3 antibodies. The DNA-protein complexes were further immunoprecipitated with anti-HDAC4 antibody. According to this protocol, generation of the PCR products of PMCA alternative splicing variants should be linked with the NFAT1/NFAT3-HDAC4 complex. Among all possible combinations of PMCA splicing variants only the PMCA2x splicing variant was detected under these conditions, suggesting that its generation might be related with HDAC4-NFAT1/NFAT3 binding to the splicing regulatory sites of the gene encoding PMCA2 (Fig. 5A), as shown in the supplementary material 2 (Fig. S2). The qPCR data, were expressed as fold of change (2−ΔΔC) calculated from the difference: ΔCT of output (immunoprecipitated DNA with HDAC/NFATs) – ΔCT of input (total DNA) and revealed that PMCA2x splicing variant generation was statistically significantly related to the NFAT1/NFAT3-HDAC4 complex activity, according to nonparametric paired Wilcoxon signed rank test at 95% confidence (Fig. 5B).

Bottom Line: Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA.RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells.NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland; Department of Neurodegenerative Disorders, Laboratory of Neurogenetics, Mossakowski Medical Research Centre PAS, Warsaw, Poland.

ABSTRACT

Background: The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants.

Results: In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.

Conclusions: We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

Show MeSH
Related in: MedlinePlus