Basal promoter of the rat connexin 32 gene: Identification and characterization of an essential element and its DNA-binding protein

S. Bai, A. Schoenfeld, A. Pietrangelo, Robert D. Burk

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32- B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.

Original languageEnglish (US)
Pages (from-to)1439-1445
Number of pages7
JournalMolecular and Cellular Biology
Volume15
Issue number3
StatePublished - 1995

Fingerprint

DNA-Binding Proteins
Genes
Nuclear Proteins
Liver
connexin 32
Hepatocellular Carcinoma
DNA
Cell Line
Liver Extracts
DNA Probes
Electrophoretic Mobility Shift Assay
Multigene Family
Methylation
Exons
Proteins
Nucleotides
Binding Sites

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Basal promoter of the rat connexin 32 gene : Identification and characterization of an essential element and its DNA-binding protein. / Bai, S.; Schoenfeld, A.; Pietrangelo, A.; Burk, Robert D.

In: Molecular and Cellular Biology, Vol. 15, No. 3, 1995, p. 1439-1445.

Research output: Contribution to journalArticle

@article{a2ee407d8a2d4ce1961857c6335abe6c,
title = "Basal promoter of the rat connexin 32 gene: Identification and characterization of an essential element and its DNA-binding protein",
abstract = "The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32- B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.",
author = "S. Bai and A. Schoenfeld and A. Pietrangelo and Burk, {Robert D.}",
year = "1995",
language = "English (US)",
volume = "15",
pages = "1439--1445",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "3",

}

TY - JOUR

T1 - Basal promoter of the rat connexin 32 gene

T2 - Identification and characterization of an essential element and its DNA-binding protein

AU - Bai, S.

AU - Schoenfeld, A.

AU - Pietrangelo, A.

AU - Burk, Robert D.

PY - 1995

Y1 - 1995

N2 - The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32- B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.

AB - The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32- B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.

UR - http://www.scopus.com/inward/record.url?scp=0028798825&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028798825&partnerID=8YFLogxK

M3 - Article

C2 - 7862137

AN - SCOPUS:0028798825

VL - 15

SP - 1439

EP - 1445

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 3

ER -