Using chromatin immunoprecipitation to monitor 1alpha,25-dihydroxyvitamin D3-dependent chromatin activity on the human CYP24 promoter.
We applied the chromatin immunoprecipitation (ChIP) method for the analysis of 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3))-dependent chromatin activity on the human 24-hydroxylase (CYP24) promoter in MCF-7 human breast cancer cells. In this pilot study we concentrated on the proximal promoter (+22 to -424) of the CYP24 gene, which includes the known 1,25-D(3) response element (VDRE) cluster. A constitutively active region of the human histone 4a gene (-40 to +285) served for normalization. Chromatin activity snapshots were taken 0, 30, 60, 120, 180, 240 and 300 min after the onset of stimulation with 1,25-D(3) and anti-acetylated histone 4 antibodies were used for ChIP. Our results suggest that ChIP is suitable for monitoring 1,25-D(3)-dependent changes of chromatin organization and can be used to reveal information about chromatin activity in living cells.
Väisänen
Sami
S
Department of Biochemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. vaisanen@messi.uku.fi
Dunlop
Thomas W
TW
Frank
Christian
C
Carlberg
Carsten
C
D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers; D006801:Humans; D010865:Pilot Projects; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D013250:Steroid Hydroxylases / Q000235:genetics*; D065668:Vitamin D3 24-Hydroxylase
D002843:Chromatin; D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol