1. Upstream stimulatory factor 1 activates GATA5 expression through an E-box motif

저자: Bohao CHEN1, Rona HSU, Zhenping LI, Paul C. KOGUT, Qingxia DU, Kelly ROUSER, Blanca CAMORETTI-MERCADO and Julian SOLWAY

저널: Biochem J. 2012 Aug 15;446(1):89-98.

사용한 제품: Dynabeads Streptavidin MyOne C1 (Cat# 65001, 65002)

 

 

Abstract

Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. In the present study, we have demonstrated that an E (enhancer)-box in the GATA5 promoter (bp ㅡ118 to ㅡ113 in mice; bp ㅡ164 to ㅡ159 in humans) positively regulates GATA5 transcription by binding USF1 (upstream stimulatory factor 1). Using site-directed mutagenesis, EMSA (electrophoretic mobility-shift analysis) and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5 -CACGTG-3 ), but not to a mutated Ebox. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with a USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT (reverse transcription)–PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. The present study provides the first evidence that USF1 activates GATA5 gene expression through the E-box motif and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer.

 

제품사용법

Biotin-affinity purification and Western blot analysis of DNA-binding proteins Biotin-labelled sense strand and unlabelled antisense oligonucleotides, including WT ㅡ127 to ㅡ100, Mut ㅡ115/ㅡ114, Mut ㅡ108/ㅡ107, WT ㅡ215 to ㅡ173, and WT ㅡ610 to ㅡ575 (Supplementary Table S1), were purchased from IDT. Biotinylated double-stranded oligonucleotides were prepared by annealing 2.5 nmol of each single-stranded oligonucleotide resuspended in 100 μl of annealing buffer (10 mM Tris/HCl, pH 7.5, 50 mM NaCl and 10 mM MgCl2), heated for 3 min at 80℃ and cooled to room temperature.

Dynabeads MyOne Streptavidin C1 (Invitrogen) were washed three times for 10 min each in washing buffer (10 mM Tris/HCl, pH 7.5, 100 mMNaCl and 2 mMDTT, 0.1%BSA). Biotinylated double-stranded DNA (100 pmol) in 50 μl of coupling buffer (10 mM Tris/HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, 2 mM DTT and 0.1% BSA) was mixed with 1 mg of streptavidin C1-coated Dynabeads and incubated at room temperature for 30 min on a rotating wheel. After incubation, the Dynabeads coated with biotinylated double-stranded DNA were washed three times in coupling buffer and resuspended in 100 μl of TNE buffer (10 mM Tris/HCl, pH 8.0, 100 mMNaCl and 1 mMEDTA) supplemented with 2 mM DTT and 20 μg/ml insulin [22]. DNA-coupled Dynabeads (100 μl) were washed four times with 500 μl of binding buffer [20 mM Tris/HCl, pH 8.0, 10% (v/v) glycerol, 2 mMEDTA, 0.01%Triton X-100, 150 mMNaCl, 1 mMDTT, 10 μg/ml insulin and 0.5 mMPefabloc], resuspendedin 50 μl of 2× binding buffer and incubated with an equal volume of CMT-93 nuclear extract (30 μg of protein) for 15 min at room temperature. After magnetic separation, supernatants were removed and reserved for subsequent electrophoresis analysis. The beads containing the protein–biotinylated fragment– streptavidin complex was washed three times with the binding buffer and boiled in Laemmli sample buffer. The bound proteins were resolved by SDS/PAGE (12% gels) along with the corresponding supernatant described above. Proteins were electroblotted on to nitrocellulose membranes following standard protocols. Each membrane was blocked with 2% BSA in TBS (Tris-buffered saline: 10 mM Tris/HCl, pH 8.0, and 150 mM NaCl) for 1 h at room temperature. The membrane was then incubated overnight at 4◦C with rabbit polyclonal anti-USF1 or anti-Sp1 antibody at a 1/200 dilution in TBS with 2% (w/v) BSA. After five washes in TBST (TBS with 0.1% Tween 20), the membrane was incubated with horseradish-peroxidaseconjugated secondary antibody at a 1/10000 dilution in TBS with 5% (w/v) non-fat dried skimmed milk powder for 1 h at room temperature. The membrane was washed three times with TBST and the specific protein bands were visualized using the SuperSignal Chemiluminescence system (Pierce).

 

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