Isolation of transcriptionally active chromatin from human breast cancer cells using Sulfolink coupling gel chromatography.
The process of transcription unfolds the nucleosome. The unfolded nucleosome structure will be maintained as long as the histones are in a highly acetylated state. Typically the cysteine residue at position 110 of histone H3 is buried in the interior of the nucleosome. However, the transcribed unfolded nucleosome has its H3 cysteine exposed, offering a tag to isolate and study transcribed nucleosomes. In this study, we applied Sulfolink Coupling Gel chromatography to isolate unfolded nucleosomes from estrogen dependent human cancer T5 cells. Inhibition of histone deacetylase activity did not enhance the yield of unfolded nucleosomes from these cells. We show that the estrogen receptor and c-myc transcribed DNA sequences are associated with unfolded nucleosomes. In chromatin immunoprecipitation (ChIPs) assays, we found that the coding regions of the estrogen receptor and c-myc genes are bound to highly acetylated H3 and H4 in cultured T5 Cells. We conclude that in cultured T5 breast cancer cells H3 and H4 are in highly acetylated states maintaining the unfolded structure of the transcribed nucleosome and facilitating subsequent rounds of elongation.
Sun
Jian-Min
JM
Manitoba Institute of Cell Biology, 675 McDermot Avenue, Winnipeg, Manitoba, R3E 0V9, Canada.
Chen
Hou Yu
HY
Davie
James R
JR
D000107:Acetylation; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D004958:Estradiol / Q000502:physiology; D005260:Female; D006657:Histones / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D009707:Nucleosomes / Q000378:metabolism; D011233:Precipitin Tests; D016271:Proto-Oncogene Proteins c-myc / Q000502:physiology; D011960:Receptors, Estrogen / Q000502:physiology; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004273:DNA, Neoplasm; D006657:Histones; D009707:Nucleosomes; D016271:Proto-Oncogene Proteins c-myc; D011960:Receptors, Estrogen; D013439:Sulfhydryl Reagents; D004958:Estradiol; D003545:Cysteine