Structural and functional characterization of the mouse tescalcin promoter

Erasmo M. Perera, Yong Bao, Lidia Kos, Gary Berkovitz

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Tescalcin, an EF-hand calcium binding protein that regulates the Na+/H+ exchanger 1 (NHE1), is highly expressed in various mouse tissues such as heart and brain. Despite its potentially important role in cell physiology, the mechanisms that regulate tescalcin gene (Tesc) expression are unknown. In this study, we report two new Tesc mRNA variants (V2 and V3) and characterize the mouse Tesc promoter. The V2 and V3 transcripts result from alternative splicing of intron 5. Our results show that Tesc mRNA variants are expressed in various mouse tissues. Primer extension analysis located the transcription start site at 94 nucleotides upstream of the translation start codon. The DNA nucleotide sequence of the 5'-flanking region contained a CpG island spanning the promoter region from nucleotides -372 to +814, a canonical TATA box (-38/-32), and putative transcription factor binding sites for Sp1, EGR1, ZBP-89, KLF3, MZF1, AP2, ZF5, and CDF-1. Transient transfection of the Y1 and msc-1 cell lines with a series of 5'-deleted promoter constructs indicated that the minimal promoter region was between nucleotides -130 and -40. Electrophoresis mobility shift assays, supershift assays, and mutation studies demonstrated that Sp1 and Sp3 bind to the GC-rich motifs, a CACCC box and three GC boxes, located within the Tesc proximal promoter. Nonetheless, mutations that abolished interaction of Sp1 and Sp3 with the GC-rich motifs located within the minimal promoter region did not abrogate promoter activity in Y1 cells. Mithramycin A, an inhibitor of Sp1-DNA interaction, reduced Tesc promoter activity in msc-1 cells in a dose-dependent manner. Sp3 was a weaker transactivator compared to Sp1 in Drosophila D.mel-2 cells. However, when Sp1 and Sp3 were coexpressed, they transactivated the Tesc promoter in a synergistic manner. In Y1 cells, mutation analysis of a putative ZF5 motif located within the Tesc minimal promoter indicated that this motif was critical for activity of Tesc promoter. Taken together, the data demonstrated that Sp1 and Sp3 transcription factors cooperate positively in the regulation of Tesc promoter, and that the putative ZF5 motif is critical for its activation.

Original languageEnglish
Pages (from-to)50-62
Number of pages13
JournalGene
Volume464
Issue number1-2
DOIs
StatePublished - Sep 1 2010

Fingerprint

Genes
Genetic Promoter Regions
Nucleotides
Mutation
Sp3 Transcription Factor
Sp1 Transcription Factor
EF Hand Motifs
Cell Physiological Phenomena
Messenger RNA
Sodium-Hydrogen Antiporter
TATA Box
CpG Islands
Calcium-Binding Proteins
Trans-Activators
Initiator Codon
5' Flanking Region
Transcription Initiation Site
Alternative Splicing
Electrophoretic Mobility Shift Assay
Introns

Keywords

  • Gene expression
  • Mouse
  • Promoter
  • Splicing
  • Tescalcin

ASJC Scopus subject areas

  • Genetics

Cite this

Structural and functional characterization of the mouse tescalcin promoter. / Perera, Erasmo M.; Bao, Yong; Kos, Lidia; Berkovitz, Gary.

In: Gene, Vol. 464, No. 1-2, 01.09.2010, p. 50-62.

Research output: Contribution to journalArticle

Perera, Erasmo M. ; Bao, Yong ; Kos, Lidia ; Berkovitz, Gary. / Structural and functional characterization of the mouse tescalcin promoter. In: Gene. 2010 ; Vol. 464, No. 1-2. pp. 50-62.
@article{c9bec9b0c8834d1b96e30dfb02edaaa5,
title = "Structural and functional characterization of the mouse tescalcin promoter",
abstract = "Tescalcin, an EF-hand calcium binding protein that regulates the Na+/H+ exchanger 1 (NHE1), is highly expressed in various mouse tissues such as heart and brain. Despite its potentially important role in cell physiology, the mechanisms that regulate tescalcin gene (Tesc) expression are unknown. In this study, we report two new Tesc mRNA variants (V2 and V3) and characterize the mouse Tesc promoter. The V2 and V3 transcripts result from alternative splicing of intron 5. Our results show that Tesc mRNA variants are expressed in various mouse tissues. Primer extension analysis located the transcription start site at 94 nucleotides upstream of the translation start codon. The DNA nucleotide sequence of the 5'-flanking region contained a CpG island spanning the promoter region from nucleotides -372 to +814, a canonical TATA box (-38/-32), and putative transcription factor binding sites for Sp1, EGR1, ZBP-89, KLF3, MZF1, AP2, ZF5, and CDF-1. Transient transfection of the Y1 and msc-1 cell lines with a series of 5'-deleted promoter constructs indicated that the minimal promoter region was between nucleotides -130 and -40. Electrophoresis mobility shift assays, supershift assays, and mutation studies demonstrated that Sp1 and Sp3 bind to the GC-rich motifs, a CACCC box and three GC boxes, located within the Tesc proximal promoter. Nonetheless, mutations that abolished interaction of Sp1 and Sp3 with the GC-rich motifs located within the minimal promoter region did not abrogate promoter activity in Y1 cells. Mithramycin A, an inhibitor of Sp1-DNA interaction, reduced Tesc promoter activity in msc-1 cells in a dose-dependent manner. Sp3 was a weaker transactivator compared to Sp1 in Drosophila D.mel-2 cells. However, when Sp1 and Sp3 were coexpressed, they transactivated the Tesc promoter in a synergistic manner. In Y1 cells, mutation analysis of a putative ZF5 motif located within the Tesc minimal promoter indicated that this motif was critical for activity of Tesc promoter. Taken together, the data demonstrated that Sp1 and Sp3 transcription factors cooperate positively in the regulation of Tesc promoter, and that the putative ZF5 motif is critical for its activation.",
keywords = "Gene expression, Mouse, Promoter, Splicing, Tescalcin",
author = "Perera, {Erasmo M.} and Yong Bao and Lidia Kos and Gary Berkovitz",
year = "2010",
month = "9",
day = "1",
doi = "10.1016/j.gene.2010.06.002",
language = "English",
volume = "464",
pages = "50--62",
journal = "Gene",
issn = "0378-1119",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Structural and functional characterization of the mouse tescalcin promoter

AU - Perera, Erasmo M.

AU - Bao, Yong

AU - Kos, Lidia

AU - Berkovitz, Gary

PY - 2010/9/1

Y1 - 2010/9/1

N2 - Tescalcin, an EF-hand calcium binding protein that regulates the Na+/H+ exchanger 1 (NHE1), is highly expressed in various mouse tissues such as heart and brain. Despite its potentially important role in cell physiology, the mechanisms that regulate tescalcin gene (Tesc) expression are unknown. In this study, we report two new Tesc mRNA variants (V2 and V3) and characterize the mouse Tesc promoter. The V2 and V3 transcripts result from alternative splicing of intron 5. Our results show that Tesc mRNA variants are expressed in various mouse tissues. Primer extension analysis located the transcription start site at 94 nucleotides upstream of the translation start codon. The DNA nucleotide sequence of the 5'-flanking region contained a CpG island spanning the promoter region from nucleotides -372 to +814, a canonical TATA box (-38/-32), and putative transcription factor binding sites for Sp1, EGR1, ZBP-89, KLF3, MZF1, AP2, ZF5, and CDF-1. Transient transfection of the Y1 and msc-1 cell lines with a series of 5'-deleted promoter constructs indicated that the minimal promoter region was between nucleotides -130 and -40. Electrophoresis mobility shift assays, supershift assays, and mutation studies demonstrated that Sp1 and Sp3 bind to the GC-rich motifs, a CACCC box and three GC boxes, located within the Tesc proximal promoter. Nonetheless, mutations that abolished interaction of Sp1 and Sp3 with the GC-rich motifs located within the minimal promoter region did not abrogate promoter activity in Y1 cells. Mithramycin A, an inhibitor of Sp1-DNA interaction, reduced Tesc promoter activity in msc-1 cells in a dose-dependent manner. Sp3 was a weaker transactivator compared to Sp1 in Drosophila D.mel-2 cells. However, when Sp1 and Sp3 were coexpressed, they transactivated the Tesc promoter in a synergistic manner. In Y1 cells, mutation analysis of a putative ZF5 motif located within the Tesc minimal promoter indicated that this motif was critical for activity of Tesc promoter. Taken together, the data demonstrated that Sp1 and Sp3 transcription factors cooperate positively in the regulation of Tesc promoter, and that the putative ZF5 motif is critical for its activation.

AB - Tescalcin, an EF-hand calcium binding protein that regulates the Na+/H+ exchanger 1 (NHE1), is highly expressed in various mouse tissues such as heart and brain. Despite its potentially important role in cell physiology, the mechanisms that regulate tescalcin gene (Tesc) expression are unknown. In this study, we report two new Tesc mRNA variants (V2 and V3) and characterize the mouse Tesc promoter. The V2 and V3 transcripts result from alternative splicing of intron 5. Our results show that Tesc mRNA variants are expressed in various mouse tissues. Primer extension analysis located the transcription start site at 94 nucleotides upstream of the translation start codon. The DNA nucleotide sequence of the 5'-flanking region contained a CpG island spanning the promoter region from nucleotides -372 to +814, a canonical TATA box (-38/-32), and putative transcription factor binding sites for Sp1, EGR1, ZBP-89, KLF3, MZF1, AP2, ZF5, and CDF-1. Transient transfection of the Y1 and msc-1 cell lines with a series of 5'-deleted promoter constructs indicated that the minimal promoter region was between nucleotides -130 and -40. Electrophoresis mobility shift assays, supershift assays, and mutation studies demonstrated that Sp1 and Sp3 bind to the GC-rich motifs, a CACCC box and three GC boxes, located within the Tesc proximal promoter. Nonetheless, mutations that abolished interaction of Sp1 and Sp3 with the GC-rich motifs located within the minimal promoter region did not abrogate promoter activity in Y1 cells. Mithramycin A, an inhibitor of Sp1-DNA interaction, reduced Tesc promoter activity in msc-1 cells in a dose-dependent manner. Sp3 was a weaker transactivator compared to Sp1 in Drosophila D.mel-2 cells. However, when Sp1 and Sp3 were coexpressed, they transactivated the Tesc promoter in a synergistic manner. In Y1 cells, mutation analysis of a putative ZF5 motif located within the Tesc minimal promoter indicated that this motif was critical for activity of Tesc promoter. Taken together, the data demonstrated that Sp1 and Sp3 transcription factors cooperate positively in the regulation of Tesc promoter, and that the putative ZF5 motif is critical for its activation.

KW - Gene expression

KW - Mouse

KW - Promoter

KW - Splicing

KW - Tescalcin

UR - http://www.scopus.com/inward/record.url?scp=77955275035&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77955275035&partnerID=8YFLogxK

U2 - 10.1016/j.gene.2010.06.002

DO - 10.1016/j.gene.2010.06.002

M3 - Article

C2 - 20540995

AN - SCOPUS:77955275035

VL - 464

SP - 50

EP - 62

JO - Gene

JF - Gene

SN - 0378-1119

IS - 1-2

ER -