Abstract 3879: High-throughput, homogeneous <i>in cyto</i> assays to monitor histone H3 post-translational modifications
Bibliographic record
Abstract
Abstract Histone proteins are an integral part of DNA packaging into chromatin, a dynamic process partly regulated by post-translation modification of histone N-terminal tails. Aberrant histone acetylation (ac) or methylation (me) levels is associated with a number of diseases. For example, histone deacetylases (HDACs and sirtuins) regulate the expression of many genes involved in neurodegeneration and can be aberrantly expressed in different tumors. In addition, deregulation of the methyl marks H3K4me3 and H3K27me3 are found associated with the development of several types of cancer. This work presents the development of high-throughput AlphaLISA® in cyto assays to monitor four specific histone marks: H3K9ac, H3K4me2, H3K27ac and H3K27me3. These assays were performed using an all-in-one well histone extraction protocol requiring no acid extraction or centrifugation steps. The level of each histone mark was first measured in cell titration experiments, seeding cells from 500 to 10 000 in 384 well plates. The chemical modulation of H3K4me2, H3K9ac and H3K27ac was monitored following overnight treatment of HeLa, HEK293, and Jurkat cells with the non-selective histone deacetylase inhibitors sodium butyrate and trichostatin A. Signal increases were all corroborated by Western blot analysis using the same antibodies as in the AlphaLISA detection assay. In the absence of chemical modulation, measurement of different H3K27me3 mark levels was performed in two B cell lymphoma cell lines: OCI-LY-19 and SU-DHL-6. OCI-LY-19 cells express wild-type EZH2 methyltransferase while SU-DHL-6 cells bear a heterozygous EZH2 mutation (Y641N) that alters its substrate selectivity, resulting in increased H3K27me3 levels. These novel cell-based assays showed suitability for high-throughput screening (HTS) protocols as demonstrated by Z’ factors superior to 0.6 for all four marks. In summary, these homogeneous cell-based AlphaLISA assays allow the simple and rapid monitoring of cellular histone H3 mark levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3879. doi:1538-7445.AM2012-3879
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.010 | 0.008 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".