title: A cell-based high-throughput screening assay to measure cellular histone h3 lys27 trimethylation with a modified dissociation-enhanced lanthanide fluorescent immunoassay.
abstract: Histone proteins are subject to several modifications, including phosphorylation, acetylation, methylation, sumoylation, and ubiquitination. These posttranslational modifications play critical roles in chromatin structure and gene transcription. Because of their involvement in the progression of a variety of diseases, histone modifications are attracting increased attention. We report herein a high-throughput DELFIA assay to quantify H3K27me3 in the prostate cancer cell line, PC3. Using a high binding MaxiSorp plate, we were able to eliminate the need for the capture antibody. We also developed an effective method, a combination of "freeze-thaw" and 0.2 N HCl, to extract histone proteins in PC3 cells cultured in a 384-well plate. To compensate for cell viability change, we normalized H3K27me3 signal to the total amount of H3 in each sample well. As a result, we show that the assay has a good dynamic range with a robust assay window. Using a methlytransferase inhibitor, DZNep, we show that the change of H3K27me3 signal is target specific. This method simplifies the logistics in screening and profiling and reduces the cost per well to an acceptable level for high-throughput screening. The findings presented here should be applicable to other assays involving binding and extraction of histone proteins.
authors.affiliation: Biological Reagents and Assay Development, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA 19426, USA.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Xie
authors.forename: Robert S
authors.initials: RS
authors.lastname: Ames
authors.forename: Hu
authors.initials: H
authors.lastname: Li
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D057166:High-Throughput Screening Assays / Q000191:economics; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors; D006657:Histones / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008239:Lysine / Q000378:metabolism*; D008297:Male; D008745:Methylation; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology
chemical_list: D004791:Enzyme Inhibitors; D006657:Histones; D028581:Lanthanoid Series Elements; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine