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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

Identification of PPA1 minimal promoter.(A) Determination of transcription start site of PPA1 gene. Total RNA was isolated from MCF7 cells and analyzed by primer extension analysis using a primer complementary to the -20 to -1 region of exon 1 of PPA1 gene. The sequencing reaction was set using same primer as in primer extension. Both the sequencing reaction products (A, T, G and C) and primer extension product (PE) were run on a 7.5% urea-polyacrylamide gel. (B) Schematic representation of 5’-deletion constructs of PPA1 promoter. All the deletion constructs were generated by PCR using pGL3-1217 construct as template and cloned into the Kpn-I and Xho-I restriction site of pGL3-Basic vector. (C) Promoter activity of all the 5’-deleted promoter constructs was determined by Dual-Luciferase assay. MCF7 cells were transfected with promoter constructs and after 24 h luciferase assay was carried out using Dual-Luciferase assay kit (Promega). Each transfection was conducted in triplicate and the results are represented as mean ± S.E.M.
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pone.0124864.g001: Identification of PPA1 minimal promoter.(A) Determination of transcription start site of PPA1 gene. Total RNA was isolated from MCF7 cells and analyzed by primer extension analysis using a primer complementary to the -20 to -1 region of exon 1 of PPA1 gene. The sequencing reaction was set using same primer as in primer extension. Both the sequencing reaction products (A, T, G and C) and primer extension product (PE) were run on a 7.5% urea-polyacrylamide gel. (B) Schematic representation of 5’-deletion constructs of PPA1 promoter. All the deletion constructs were generated by PCR using pGL3-1217 construct as template and cloned into the Kpn-I and Xho-I restriction site of pGL3-Basic vector. (C) Promoter activity of all the 5’-deleted promoter constructs was determined by Dual-Luciferase assay. MCF7 cells were transfected with promoter constructs and after 24 h luciferase assay was carried out using Dual-Luciferase assay kit (Promega). Each transfection was conducted in triplicate and the results are represented as mean ± S.E.M.

Mentions: Primer extension analysis was carried out using a primer PPA1PER which is complementary to the -20 to -1 region of exon 1 of PPA1 gene. As shown in (Fig 1A), two transcription start sites at 67 bp and 57 bp away from translation start site were found. TATA-less promoters are often known to be associated with multiple transcription start sites [30].


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)

Identification of PPA1 minimal promoter.(A) Determination of transcription start site of PPA1 gene. Total RNA was isolated from MCF7 cells and analyzed by primer extension analysis using a primer complementary to the -20 to -1 region of exon 1 of PPA1 gene. The sequencing reaction was set using same primer as in primer extension. Both the sequencing reaction products (A, T, G and C) and primer extension product (PE) were run on a 7.5% urea-polyacrylamide gel. (B) Schematic representation of 5’-deletion constructs of PPA1 promoter. All the deletion constructs were generated by PCR using pGL3-1217 construct as template and cloned into the Kpn-I and Xho-I restriction site of pGL3-Basic vector. (C) Promoter activity of all the 5’-deleted promoter constructs was determined by Dual-Luciferase assay. MCF7 cells were transfected with promoter constructs and after 24 h luciferase assay was carried out using Dual-Luciferase assay kit (Promega). Each transfection was conducted in triplicate and the results are represented as mean ± S.E.M.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124864.g001: Identification of PPA1 minimal promoter.(A) Determination of transcription start site of PPA1 gene. Total RNA was isolated from MCF7 cells and analyzed by primer extension analysis using a primer complementary to the -20 to -1 region of exon 1 of PPA1 gene. The sequencing reaction was set using same primer as in primer extension. Both the sequencing reaction products (A, T, G and C) and primer extension product (PE) were run on a 7.5% urea-polyacrylamide gel. (B) Schematic representation of 5’-deletion constructs of PPA1 promoter. All the deletion constructs were generated by PCR using pGL3-1217 construct as template and cloned into the Kpn-I and Xho-I restriction site of pGL3-Basic vector. (C) Promoter activity of all the 5’-deleted promoter constructs was determined by Dual-Luciferase assay. MCF7 cells were transfected with promoter constructs and after 24 h luciferase assay was carried out using Dual-Luciferase assay kit (Promega). Each transfection was conducted in triplicate and the results are represented as mean ± S.E.M.
Mentions: Primer extension analysis was carried out using a primer PPA1PER which is complementary to the -20 to -1 region of exon 1 of PPA1 gene. As shown in (Fig 1A), two transcription start sites at 67 bp and 57 bp away from translation start site were found. TATA-less promoters are often known to be associated with multiple transcription start sites [30].

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