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Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckβ promoter via the protein kinase C signaling pathway.

Kuan CS, Yee YH, See Too WC, Few LL - PLoS ONE (2014)

Bottom Line: In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements.PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated.These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

View Article: PubMed Central - PubMed

Affiliation: School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

ABSTRACT

Background: Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.

Methodology/principal findings: In this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.

Conclusion/significance: These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

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Related in: MedlinePlus

Characterization of Ets and GATA binding to the ckβ promoter by EMSA and ChIP.(A) EMSA was performed by using biotinylated ckβ-Ets/GATA probes with or without mutations at the Ets and GATA binding sequence. The blot shown is representative of two independent experiments that produced similar results. (B) Competition assay was performed by using unlabeled Ets and Ets/GATA consensus probes as competitors in the EMSA. The blot shown is representative of three independent experiments that produced similar results. (C) Supershift assay was performed by using GATA2 or GATA3 antibody in the EMSA. The blot shown is representative of two independent experiments that produced similar results. (D) ChIP was performed with GATA2 or GATA3 antibody and pre-immune normal rabbit IgG as control. Lane M: GeneRuler DNA Ladder Mix, Lane T: total input positive control (unprocessed chromatin), Lane N: pre-immune normal rabbit IgG (negative control).
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pone-0113485-g005: Characterization of Ets and GATA binding to the ckβ promoter by EMSA and ChIP.(A) EMSA was performed by using biotinylated ckβ-Ets/GATA probes with or without mutations at the Ets and GATA binding sequence. The blot shown is representative of two independent experiments that produced similar results. (B) Competition assay was performed by using unlabeled Ets and Ets/GATA consensus probes as competitors in the EMSA. The blot shown is representative of three independent experiments that produced similar results. (C) Supershift assay was performed by using GATA2 or GATA3 antibody in the EMSA. The blot shown is representative of two independent experiments that produced similar results. (D) ChIP was performed with GATA2 or GATA3 antibody and pre-immune normal rabbit IgG as control. Lane M: GeneRuler DNA Ladder Mix, Lane T: total input positive control (unprocessed chromatin), Lane N: pre-immune normal rabbit IgG (negative control).

Mentions: EMSAs with a biotin-labeled DNA probe were used to assess the binding of the Ets and GATA repressive elements (between −2000 and −1886 in the ckβ promoter) by transcription factors from nuclear extracts from MCF-7 cells. Fig. 5A shows that the biotin-labeled DNA probe produced two slowly migrating shifted complexes, indicating two different nuclear proteins were bound to the probe. Mutations of either Ets, GATA or both elements in the promoter sequence markedly reduced the formation of the upper shifted complex, suggesting that the protein component of the complex was Ets- and GATA-related.


Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckβ promoter via the protein kinase C signaling pathway.

Kuan CS, Yee YH, See Too WC, Few LL - PLoS ONE (2014)

Characterization of Ets and GATA binding to the ckβ promoter by EMSA and ChIP.(A) EMSA was performed by using biotinylated ckβ-Ets/GATA probes with or without mutations at the Ets and GATA binding sequence. The blot shown is representative of two independent experiments that produced similar results. (B) Competition assay was performed by using unlabeled Ets and Ets/GATA consensus probes as competitors in the EMSA. The blot shown is representative of three independent experiments that produced similar results. (C) Supershift assay was performed by using GATA2 or GATA3 antibody in the EMSA. The blot shown is representative of two independent experiments that produced similar results. (D) ChIP was performed with GATA2 or GATA3 antibody and pre-immune normal rabbit IgG as control. Lane M: GeneRuler DNA Ladder Mix, Lane T: total input positive control (unprocessed chromatin), Lane N: pre-immune normal rabbit IgG (negative control).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260826&req=5

pone-0113485-g005: Characterization of Ets and GATA binding to the ckβ promoter by EMSA and ChIP.(A) EMSA was performed by using biotinylated ckβ-Ets/GATA probes with or without mutations at the Ets and GATA binding sequence. The blot shown is representative of two independent experiments that produced similar results. (B) Competition assay was performed by using unlabeled Ets and Ets/GATA consensus probes as competitors in the EMSA. The blot shown is representative of three independent experiments that produced similar results. (C) Supershift assay was performed by using GATA2 or GATA3 antibody in the EMSA. The blot shown is representative of two independent experiments that produced similar results. (D) ChIP was performed with GATA2 or GATA3 antibody and pre-immune normal rabbit IgG as control. Lane M: GeneRuler DNA Ladder Mix, Lane T: total input positive control (unprocessed chromatin), Lane N: pre-immune normal rabbit IgG (negative control).
Mentions: EMSAs with a biotin-labeled DNA probe were used to assess the binding of the Ets and GATA repressive elements (between −2000 and −1886 in the ckβ promoter) by transcription factors from nuclear extracts from MCF-7 cells. Fig. 5A shows that the biotin-labeled DNA probe produced two slowly migrating shifted complexes, indicating two different nuclear proteins were bound to the probe. Mutations of either Ets, GATA or both elements in the promoter sequence markedly reduced the formation of the upper shifted complex, suggesting that the protein component of the complex was Ets- and GATA-related.

Bottom Line: In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements.PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated.These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

View Article: PubMed Central - PubMed

Affiliation: School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

ABSTRACT

Background: Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.

Methodology/principal findings: In this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.

Conclusion/significance: These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

Show MeSH
Related in: MedlinePlus