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Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells.

Mishra DR, Chaudhary S, Krishna BM, Mishra SK - PLoS ONE (2015)

Bottom Line: Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms.These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter.Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.

ABSTRACT
Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

No MeSH data available.


Related in: MedlinePlus

Sp1 regulates PPA1 expression and alters chromatin accessibility.(A) Schematic representation of Sp1 binding site mutated promoter constructs generated by site-directed mutagenesis. All the mutated promoter constructs were generated by a three step PCR method and cloned into pGL3-Basic vector. (B) Luciferase assay of mutated promoter constructs was carried out using Dual-Luciferase assay kit (Promega). (C) Luciferase assay showing the effect of Sp1 overexpression on the promoter deletion constructs. pGL3-137, pGL3-187 and pGL3-266 have one, two and three Sp1 binding sites respectively. Each transfection was carried in triplicate and the results represent mean ± S.E.M of two independent experiments. (D) Overexpression of Sp1 led to the increased expression of PPA1. MCF7 cells were transiently transfected with empty vector (control), or Sp1 expression vector. After 48 h of transfection, cells lysates were prepared for western blot experiment. (E) Sp1 alters chromatin accessibility across PPA1 promoter. Nuclei from MCF7 cells transfected with empty vector (control) or Sp1 expression vector were subjected to MNase digestion and then the purified DNA was amplified by semi-quantitative PCR using primers for the proximal promoter region of PPA1. β-Actin promoter region was amplified as a representative control.
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pone.0124864.g003: Sp1 regulates PPA1 expression and alters chromatin accessibility.(A) Schematic representation of Sp1 binding site mutated promoter constructs generated by site-directed mutagenesis. All the mutated promoter constructs were generated by a three step PCR method and cloned into pGL3-Basic vector. (B) Luciferase assay of mutated promoter constructs was carried out using Dual-Luciferase assay kit (Promega). (C) Luciferase assay showing the effect of Sp1 overexpression on the promoter deletion constructs. pGL3-137, pGL3-187 and pGL3-266 have one, two and three Sp1 binding sites respectively. Each transfection was carried in triplicate and the results represent mean ± S.E.M of two independent experiments. (D) Overexpression of Sp1 led to the increased expression of PPA1. MCF7 cells were transiently transfected with empty vector (control), or Sp1 expression vector. After 48 h of transfection, cells lysates were prepared for western blot experiment. (E) Sp1 alters chromatin accessibility across PPA1 promoter. Nuclei from MCF7 cells transfected with empty vector (control) or Sp1 expression vector were subjected to MNase digestion and then the purified DNA was amplified by semi-quantitative PCR using primers for the proximal promoter region of PPA1. β-Actin promoter region was amplified as a representative control.

Mentions: To identify which of these Sp1 binding sites are important in the PPA1 gene regulation, pGL3-266 mutant constructs for each of the Sp1 binding sites were generated by site-directed mutagenesis (Fig 3A). Mutations in the Sp1 binding site 2 (-167/-145) and site 3 (-232/-208) resulted approximately 74% and 63% decrease in luciferase activity respectively compared to the wild type promoter construct (Fig 3B). Mutating site 1 (-127/-104) did not significantly affects promoter activity.


Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells.

Mishra DR, Chaudhary S, Krishna BM, Mishra SK - PLoS ONE (2015)

Sp1 regulates PPA1 expression and alters chromatin accessibility.(A) Schematic representation of Sp1 binding site mutated promoter constructs generated by site-directed mutagenesis. All the mutated promoter constructs were generated by a three step PCR method and cloned into pGL3-Basic vector. (B) Luciferase assay of mutated promoter constructs was carried out using Dual-Luciferase assay kit (Promega). (C) Luciferase assay showing the effect of Sp1 overexpression on the promoter deletion constructs. pGL3-137, pGL3-187 and pGL3-266 have one, two and three Sp1 binding sites respectively. Each transfection was carried in triplicate and the results represent mean ± S.E.M of two independent experiments. (D) Overexpression of Sp1 led to the increased expression of PPA1. MCF7 cells were transiently transfected with empty vector (control), or Sp1 expression vector. After 48 h of transfection, cells lysates were prepared for western blot experiment. (E) Sp1 alters chromatin accessibility across PPA1 promoter. Nuclei from MCF7 cells transfected with empty vector (control) or Sp1 expression vector were subjected to MNase digestion and then the purified DNA was amplified by semi-quantitative PCR using primers for the proximal promoter region of PPA1. β-Actin promoter region was amplified as a representative control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414593&req=5

pone.0124864.g003: Sp1 regulates PPA1 expression and alters chromatin accessibility.(A) Schematic representation of Sp1 binding site mutated promoter constructs generated by site-directed mutagenesis. All the mutated promoter constructs were generated by a three step PCR method and cloned into pGL3-Basic vector. (B) Luciferase assay of mutated promoter constructs was carried out using Dual-Luciferase assay kit (Promega). (C) Luciferase assay showing the effect of Sp1 overexpression on the promoter deletion constructs. pGL3-137, pGL3-187 and pGL3-266 have one, two and three Sp1 binding sites respectively. Each transfection was carried in triplicate and the results represent mean ± S.E.M of two independent experiments. (D) Overexpression of Sp1 led to the increased expression of PPA1. MCF7 cells were transiently transfected with empty vector (control), or Sp1 expression vector. After 48 h of transfection, cells lysates were prepared for western blot experiment. (E) Sp1 alters chromatin accessibility across PPA1 promoter. Nuclei from MCF7 cells transfected with empty vector (control) or Sp1 expression vector were subjected to MNase digestion and then the purified DNA was amplified by semi-quantitative PCR using primers for the proximal promoter region of PPA1. β-Actin promoter region was amplified as a representative control.
Mentions: To identify which of these Sp1 binding sites are important in the PPA1 gene regulation, pGL3-266 mutant constructs for each of the Sp1 binding sites were generated by site-directed mutagenesis (Fig 3A). Mutations in the Sp1 binding site 2 (-167/-145) and site 3 (-232/-208) resulted approximately 74% and 63% decrease in luciferase activity respectively compared to the wild type promoter construct (Fig 3B). Mutating site 1 (-127/-104) did not significantly affects promoter activity.

Bottom Line: Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms.These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter.Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.

ABSTRACT
Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

No MeSH data available.


Related in: MedlinePlus