Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A2A adenosine receptor

Yi Chao Lee, Hsing Lin Lai, Chung Nan Sun, Chen Li Chien, Yijuang Chern

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5′-flanking region and a 0.5-kb 5′-untranslated region of the rat A2A adenosine receptor (A2A-R) gene. Various lengths of the 5′-flanking region of the A2A-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A2A-R/NF1), located between bases -2846 and -2827 of the A2A-R gene, functions as a repressor for A2A-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A2A-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A2A-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A2A-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A2A-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A2A-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A2A-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A2A-R. Moreover, addition of the A2A-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A2A-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A2A-R/NF1) that plays a functional role in modulating the expression of A2A-R.

Original languageEnglish
Pages (from-to)61-73
Number of pages13
JournalMolecular Brain Research
Volume111
Issue number1-2
DOIs
Publication statusPublished - Mar 17 2003
Externally publishedYes

Fingerprint

NFI Transcription Factors
Adenosine A2A Receptors
Cell Line
Proteins
5' Flanking Region
DNA
Genes
Thymidine Kinase
5' Untranslated Regions
Electrophoretic Mobility Shift Assay
Astrocytes

Keywords

  • A adenosine receptor
  • Gene
  • NF1
  • Promoter

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A2A adenosine receptor. / Lee, Yi Chao; Lai, Hsing Lin; Sun, Chung Nan; Chien, Chen Li; Chern, Yijuang.

In: Molecular Brain Research, Vol. 111, No. 1-2, 17.03.2003, p. 61-73.

Research output: Contribution to journalArticle

Lee, Yi Chao ; Lai, Hsing Lin ; Sun, Chung Nan ; Chien, Chen Li ; Chern, Yijuang. / Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A2A adenosine receptor. In: Molecular Brain Research. 2003 ; Vol. 111, No. 1-2. pp. 61-73.
@article{f9df5ba87dba4c7a856cd49d77da96eb,
title = "Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A2A adenosine receptor",
abstract = "By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5′-flanking region and a 0.5-kb 5′-untranslated region of the rat A2A adenosine receptor (A2A-R) gene. Various lengths of the 5′-flanking region of the A2A-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A2A-R/NF1), located between bases -2846 and -2827 of the A2A-R gene, functions as a repressor for A2A-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A2A-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A2A-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A2A-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A2A-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A2A-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A2A-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A2A-R. Moreover, addition of the A2A-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A2A-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A2A-R/NF1) that plays a functional role in modulating the expression of A2A-R.",
keywords = "A adenosine receptor, Gene, NF1, Promoter",
author = "Lee, {Yi Chao} and Lai, {Hsing Lin} and Sun, {Chung Nan} and Chien, {Chen Li} and Yijuang Chern",
year = "2003",
month = "3",
day = "17",
doi = "10.1016/S0169-328X(02)00670-8",
language = "English",
volume = "111",
pages = "61--73",
journal = "Brain Research",
issn = "0006-8993",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A2A adenosine receptor

AU - Lee, Yi Chao

AU - Lai, Hsing Lin

AU - Sun, Chung Nan

AU - Chien, Chen Li

AU - Chern, Yijuang

PY - 2003/3/17

Y1 - 2003/3/17

N2 - By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5′-flanking region and a 0.5-kb 5′-untranslated region of the rat A2A adenosine receptor (A2A-R) gene. Various lengths of the 5′-flanking region of the A2A-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A2A-R/NF1), located between bases -2846 and -2827 of the A2A-R gene, functions as a repressor for A2A-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A2A-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A2A-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A2A-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A2A-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A2A-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A2A-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A2A-R. Moreover, addition of the A2A-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A2A-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A2A-R/NF1) that plays a functional role in modulating the expression of A2A-R.

AB - By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5′-flanking region and a 0.5-kb 5′-untranslated region of the rat A2A adenosine receptor (A2A-R) gene. Various lengths of the 5′-flanking region of the A2A-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A2A-R/NF1), located between bases -2846 and -2827 of the A2A-R gene, functions as a repressor for A2A-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A2A-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A2A-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A2A-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A2A-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A2A-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A2A-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A2A-R. Moreover, addition of the A2A-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A2A-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A2A-R/NF1) that plays a functional role in modulating the expression of A2A-R.

KW - A adenosine receptor

KW - Gene

KW - NF1

KW - Promoter

UR - http://www.scopus.com/inward/record.url?scp=0037451092&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037451092&partnerID=8YFLogxK

U2 - 10.1016/S0169-328X(02)00670-8

DO - 10.1016/S0169-328X(02)00670-8

M3 - Article

C2 - 12654506

AN - SCOPUS:0037451092

VL - 111

SP - 61

EP - 73

JO - Brain Research

JF - Brain Research

SN - 0006-8993

IS - 1-2

ER -