Goblet-cell-specific transcription of mouse intestinal trefoil factor gene results from collaboration of complex series of positive and negative regulatory elements

Hiroshi Itoh, Nagamu Inoue, Daniel K. Podolsky

研究成果: Article

21 引用 (Scopus)

抄録

Intestinal trefoil factor (ITF) is expressed selectively in intestinal goblet cells. Previous studies of the rat ITF gene identified one cis-regulatory element, designated the goblet-cell-response element (GCRE), present in the proximal region of the promoter. To identify additional cis-regulatory elements responsible for goblet-cell-specific expression, a DNA fragment containing 6353 bp of the 5'-flanking region of the mouse ITF gene was cloned and its promoter activity was examined extensively. In human and murine intestinal-derived cell lines (LS174T and CMT-93), the luciferase activities of a 6.3-kb construct were 5- and 2-fold greater than the smaller 1.8-kb construct, respectively. In contrast, the activity in non-intestinal cell lines (HepG2 and HeLa) was 2-4-fold lower than the smaller construct. In the region downstream from the 1.8-kb position, strong luciferase activities in LS174T and HepG2 cells were observed using a 201-bp construct. Interestingly, increased activity was almost completely suppressed in cells transfected with a 391-bp construct. Detailed analyses of this region revealed the existence of a 11-bp positive regulatory element (-181 to -170; ACCTCTTCCTG) and a 9-bp negative regulatory element (-208 to -200; ATTGACAGA) in addition to the GCRE. All three elements were well conserved among human, rat and mouse ITF gene promoters. In addition, a mutant 1.8-kb construct in which the negative regulatory region was deleted yielded the same approximate luciferase activity as a 6.3-kb construct, suggesting binding of a goblet-cell-specific silencer inhibitor (SI) between -6.3 and -1.8 kb. The SI present in goblet cells may block the silencers' binding to the pre-initiation complex and allow increased transcriptional activity driven by specific and non-specific enhancers. High-level expression of the mouse ITF gene specifically in intestinal goblet cells may be achieved through the combined effects of these regulatory elements.

元の言語English
ページ(範囲)461-472
ページ数12
ジャーナルBiochemical Journal
341
発行部数2
DOI
出版物ステータスPublished - 1999 7 15
外部発表Yes

Fingerprint

Goblet Cells
Transcription
Genes
Luciferases
Response Elements
Cells
5' Flanking Region
Nucleic Acid Regulatory Sequences
Genetic Promoter Regions
Rats
Cell Line
Hep G2 Cells
Trefoil Factor-3
DNA

ASJC Scopus subject areas

  • Biochemistry

これを引用

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title = "Goblet-cell-specific transcription of mouse intestinal trefoil factor gene results from collaboration of complex series of positive and negative regulatory elements",
abstract = "Intestinal trefoil factor (ITF) is expressed selectively in intestinal goblet cells. Previous studies of the rat ITF gene identified one cis-regulatory element, designated the goblet-cell-response element (GCRE), present in the proximal region of the promoter. To identify additional cis-regulatory elements responsible for goblet-cell-specific expression, a DNA fragment containing 6353 bp of the 5'-flanking region of the mouse ITF gene was cloned and its promoter activity was examined extensively. In human and murine intestinal-derived cell lines (LS174T and CMT-93), the luciferase activities of a 6.3-kb construct were 5- and 2-fold greater than the smaller 1.8-kb construct, respectively. In contrast, the activity in non-intestinal cell lines (HepG2 and HeLa) was 2-4-fold lower than the smaller construct. In the region downstream from the 1.8-kb position, strong luciferase activities in LS174T and HepG2 cells were observed using a 201-bp construct. Interestingly, increased activity was almost completely suppressed in cells transfected with a 391-bp construct. Detailed analyses of this region revealed the existence of a 11-bp positive regulatory element (-181 to -170; ACCTCTTCCTG) and a 9-bp negative regulatory element (-208 to -200; ATTGACAGA) in addition to the GCRE. All three elements were well conserved among human, rat and mouse ITF gene promoters. In addition, a mutant 1.8-kb construct in which the negative regulatory region was deleted yielded the same approximate luciferase activity as a 6.3-kb construct, suggesting binding of a goblet-cell-specific silencer inhibitor (SI) between -6.3 and -1.8 kb. The SI present in goblet cells may block the silencers' binding to the pre-initiation complex and allow increased transcriptional activity driven by specific and non-specific enhancers. High-level expression of the mouse ITF gene specifically in intestinal goblet cells may be achieved through the combined effects of these regulatory elements.",
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T1 - Goblet-cell-specific transcription of mouse intestinal trefoil factor gene results from collaboration of complex series of positive and negative regulatory elements

AU - Itoh, Hiroshi

AU - Inoue, Nagamu

AU - Podolsky, Daniel K.

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N2 - Intestinal trefoil factor (ITF) is expressed selectively in intestinal goblet cells. Previous studies of the rat ITF gene identified one cis-regulatory element, designated the goblet-cell-response element (GCRE), present in the proximal region of the promoter. To identify additional cis-regulatory elements responsible for goblet-cell-specific expression, a DNA fragment containing 6353 bp of the 5'-flanking region of the mouse ITF gene was cloned and its promoter activity was examined extensively. In human and murine intestinal-derived cell lines (LS174T and CMT-93), the luciferase activities of a 6.3-kb construct were 5- and 2-fold greater than the smaller 1.8-kb construct, respectively. In contrast, the activity in non-intestinal cell lines (HepG2 and HeLa) was 2-4-fold lower than the smaller construct. In the region downstream from the 1.8-kb position, strong luciferase activities in LS174T and HepG2 cells were observed using a 201-bp construct. Interestingly, increased activity was almost completely suppressed in cells transfected with a 391-bp construct. Detailed analyses of this region revealed the existence of a 11-bp positive regulatory element (-181 to -170; ACCTCTTCCTG) and a 9-bp negative regulatory element (-208 to -200; ATTGACAGA) in addition to the GCRE. All three elements were well conserved among human, rat and mouse ITF gene promoters. In addition, a mutant 1.8-kb construct in which the negative regulatory region was deleted yielded the same approximate luciferase activity as a 6.3-kb construct, suggesting binding of a goblet-cell-specific silencer inhibitor (SI) between -6.3 and -1.8 kb. The SI present in goblet cells may block the silencers' binding to the pre-initiation complex and allow increased transcriptional activity driven by specific and non-specific enhancers. High-level expression of the mouse ITF gene specifically in intestinal goblet cells may be achieved through the combined effects of these regulatory elements.

AB - Intestinal trefoil factor (ITF) is expressed selectively in intestinal goblet cells. Previous studies of the rat ITF gene identified one cis-regulatory element, designated the goblet-cell-response element (GCRE), present in the proximal region of the promoter. To identify additional cis-regulatory elements responsible for goblet-cell-specific expression, a DNA fragment containing 6353 bp of the 5'-flanking region of the mouse ITF gene was cloned and its promoter activity was examined extensively. In human and murine intestinal-derived cell lines (LS174T and CMT-93), the luciferase activities of a 6.3-kb construct were 5- and 2-fold greater than the smaller 1.8-kb construct, respectively. In contrast, the activity in non-intestinal cell lines (HepG2 and HeLa) was 2-4-fold lower than the smaller construct. In the region downstream from the 1.8-kb position, strong luciferase activities in LS174T and HepG2 cells were observed using a 201-bp construct. Interestingly, increased activity was almost completely suppressed in cells transfected with a 391-bp construct. Detailed analyses of this region revealed the existence of a 11-bp positive regulatory element (-181 to -170; ACCTCTTCCTG) and a 9-bp negative regulatory element (-208 to -200; ATTGACAGA) in addition to the GCRE. All three elements were well conserved among human, rat and mouse ITF gene promoters. In addition, a mutant 1.8-kb construct in which the negative regulatory region was deleted yielded the same approximate luciferase activity as a 6.3-kb construct, suggesting binding of a goblet-cell-specific silencer inhibitor (SI) between -6.3 and -1.8 kb. The SI present in goblet cells may block the silencers' binding to the pre-initiation complex and allow increased transcriptional activity driven by specific and non-specific enhancers. High-level expression of the mouse ITF gene specifically in intestinal goblet cells may be achieved through the combined effects of these regulatory elements.

KW - Colon

KW - Enhancer

KW - Epithelium

KW - Promoter

KW - Silencer

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