Isolation of extrachromosomal elements by histone immunoprecipitation.
Here, we describe a gentle and effective method for the rapid and reproducible isolation of histone-bound extrachromosomal DNA molecules called extrachromosomal elements (EEs). This method facilitates the harvest of a specific population of EEs following their isolation from cultured cells, primary tissues, and tumor cells. Active EEs are bound to histone proteins, and these histone-bound EEs carry actively transcribing genes such as c-myc. Our method exploits the presence of histones on EEs and serves as a first-step purification procedure, allowing for the cloning or multivariant analysis of an immunopurified sample of EEs. We isolated EEs from 4-hydroxytamoxifen (4-HT)-activated Myc-ER-regulatable Pre-B ABM cells. Following one round of immunoprecipitation, we demonstrate the purification of histone-bound EEs. We confirmed that our purification enriched for EEs that carry genes by fluorescent in situ hybridization of EEs (FISH-EEs), and we probed non-enriched and immunopurified EEs with a dihydrofolate reductase (DHFR) cDNA probe that is known to detect extrachromosomal amplification in Myc-activated cells. We demonstrate the enrichment of immunoprecipitated DHFR-containing extrachromosomal DNA molecules.
Kuschak
T I
TI
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, MB, Canada.
Kuschak
B C
BC
Smith
G M
GM
Wright
J A
JA
Mai
S
S
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D005111:Extrachromosomal Inheritance; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006657:Histones / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007163:Immunosorbent Techniques; D017404:In Situ Hybridization, Fluorescence; D007211:Indoles; D051379:Mice; D009419:Nerve Tissue Proteins; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012685:Sepharose; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D000906:Antibodies; D005456:Fluorescent Dyes; C029152:G-substrate; D006657:Histones; D007211:Indoles; D009419:Nerve Tissue Proteins; D016271:Proto-Oncogene Proteins c-myc; C007293:DAPI; D004247:DNA; D012685:Sepharose; D013762:Tetrahydrofolate Dehydrogenase; D016650:Fluorescein-5-isothiocyanate