An assessment of histone-modification antibody quality.
Ontology highlight
ABSTRACT:
SUBMITTER: Egelhofer TA
PROVIDER: S-EPMC3017233 | biostudies-literature | 2011 Jan
REPOSITORIES: biostudies-literature
Publications
An assessment of histone-modification antibody quality.
Egelhofer Thea A TA Minoda Aki A Klugman Sarit S Lee Kyungjoon K Kolasinska-Zwierz Paulina P Alekseyenko Artyom A AA Cheung Ming-Sin MS Day Daniel S DS Gadel Sarah S Gorchakov Andrey A AA Gu Tingting T Kharchenko Peter V PV Kuan Samantha S Latorre Isabel I Linder-Basso Daniela D Luu Ying Y Ngo Queminh Q Perry Marc M Rechtsteiner Andreas A Riddle Nicole C NC Schwartz Yuri B YB Shanower Gregory A GA Vielle Anne A Ahringer Julie J Elgin Sarah C R SC Kuroda Mitzi I MI Pirrotta Vincenzo V Ren Bing B Strome Susan S Park Peter J PJ Karpen Gary H GH Hawkins R David RD Lieb Jason D JD
Nature structural & molecular biology 20101205 1
We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posti ...[more]
Similar Datasets
Project description:Histone post-translational modification (PTM) antibodies are essential research reagents in chromatin biology. However, they suffer from variable properties and insufficient documentation of quality. Antibody manufacturers and vendors should provide detailed lot-specific documentation of quality, rendering further quality checks by end-customers unnecessary. A shift from polyclonal antibodies towards sustainable reagents like monoclonal or recombinant antibodies or histone binding domains would help to improve the reproducibility of experimental work in this field.
| S-EPMC4706057 | biostudies-literature
Project description:Commercially available histone antibodies were analyzed using peptide microarrays to assess specificity. For two H3K27me3 antibodies from different vendors, we performed ChIP-seq in WT and EED-null murine ES cells to assess specificity. Cell lines were generated from e3.5 blastocysts of EED fl/fl animals and transfected with plasmid CAG-CRE-ERT2. Null cells were generated by single cell clones from tamoxifen-treated parent cell line. Analysis of the specificity of two H3K27me3 antibodies using WT and EED (Embryonic Ectoderm Develepment)-null (mutants lacking H3K27me3) ES cells. The goal was to compare WT and KO for each antibody with the expectation that if the antibodies were specific, there would remain few peaks and little signal in the knockout. We sequenced 2 replicates of each condition (3 for WT input) and after comparing replicates, we merged data, called peaks, and calculated signal in bins centered on the wild type H3K27me3 peaks. As little to no signal remained either by direct comparison of peaks or looking at 'metagenes' of the peaks, we concluded that the antibodies were specific. Antibodies: Abcam H3K27me3 (ab6002, lot# GR130002-1) Upstate H3K27me3 (07-449, lot# 2455635)
2015-06-21 | E-GEOD-66902 | biostudies-arrayexpress
Project description:Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.
| S-EPMC4530063 | biostudies-literature
Project description:Commercially available histone antibodies were analyzed using peptide microarrays to assess specificity. For two H3K27me3 antibodies from different vendors, we performed ChIP-seq in WT and EED-null murine ES cells to assess specificity. Cell lines were generated from e3.5 blastocysts of EED fl/fl animals and transfected with plasmid CAG-CRE-ERT2. Null cells were generated by single cell clones from tamoxifen-treated parent cell line.
2015-06-21 | GSE66902 | GEO
Project description:Dysregulated epigenetic enzymes and resultant abnormal epigenetic modifications (EMs) have been suggested to be closely related to tumor occurrence and progression. Histone modifications (HMs) can assist in maintaining genome stability, DNA repair, transcription, and chromatin modulation within breast cancer (BC) cells. In addition, HMs are reversible, dynamic processes involving the associations of different enzymes with molecular compounds. Abnormal HMs (e.g. histone methylation and histone acetylation) have been identified to be tightly related to BC occurrence and development, even though their underlying mechanisms remain largely unclear. EMs are reversible, and as a result, epigenetic enzymes have aroused wide attention as anti-tumor therapeutic targets. At present, treatments to restore aberrant EMs within BC cells have entered preclinical or clinical trials. In addition, no existing studies have comprehensively analyzed aberrant HMs within BC cells; in addition, HM-targeting BC treatments remain to be further investigated. Histone and non-histone protein methylation is becoming an attractive anti-tumor epigenetic therapeutic target; such methylation-related enzyme inhibitors are under development at present. Consequently, the present work focuses on summarizing relevant studies on HMs related to BC and the possible mechanisms associated with abnormal HMs. Additionally, we also aim to analyze existing therapeutic agents together with those drugs approved and tested through pre-clinical and clinical trials, to assess their roles in HMs. Moreover, epi-drugs that target HMT inhibitors and HDAC inhibitors should be tested in preclinical and clinical studies for the treatment of BC. Epi-drugs that target histone methylation (HMT inhibitors) and histone acetylation (HDAC inhibitors) have now entered clinical trials or are approved by the US Food and Drug Administration (FDA). Therefore, the review covers the difficulties in applying HM-targeting treatments in clinics and proposes feasible approaches for overcoming such difficulties and promoting their use in treating BC cases.
| S-EPMC9522521 | biostudies-literature
Project description:IPO-38 is a potential biomarker for early diagnosis of gastric cancer that we recently identified. Although we characterized its chemical nature as a nucleosome histone, we suspected the existence of histone modification for the IPO-38 antibody-labeled protein. Here, we used a commercially available modified histone peptide array to identify the type and site of histone modification labeled by the IPO-38 monoclonal antibody. In protein array analysis, the citrulline modification of histone 3 on arginine 26 (H3R26Cit) yielded the strongest signal. Although peptidyl arginine deiminase-2 and -4 (PADI2 and PADI4, respectively) can catalyze the conversion of arginine to citrulline, we observed that only PADI4 expression correlated with the citrulline histone modification of H3R26Cit. Overexpression of PADI4, via transfection of a eukaryotic expression vector, and knockdown of PADI4 gene expression, by a PADI4 CRISPR/Cas9 vector, confirmed the crucial function of PADI4 on the increased level of H3R26Cit in gastric cancer cell lines. By immunoprecipitation and immunoblotting, we found an interaction between H3R26Cit and H3K27me3. Our study established the first link between the IPO-38 antigen and citrullinated histone 3, and clarified the upstream regulatory enzyme PADI4. The new findings suggest an important role for the citrullination modification of histone in gastric cancer biology, and should help us optimize the development of a sensitive and specific diagnostic reagent.
| S-EPMC6482251 | biostudies-literature
Project description:Background: Cervical cancer is the second most lethal malignancy among women, and histone modification plays a fundamental role in most biological processes, but the prognostic value of histone modification in cervical cancer has not been evaluated. Methods: A total of 594 cervical cancer patients from TCGA-CESC, GSE44001, and GSE52903 cohorts were enrolled in the current study, along with the corresponding clinicopathological features. Patients with a follow-up time less than one month were removed. A total of 122 histone modification-associated signaling pathways were obtained from the MSigDB. The activation scores of these pathways were evaluated using the “GSVA” package, differentially expressed genes were identified by the “limma” package, and pathway enrichment was conducted using the “clusterProfiler 4.0” package. The subsequent least absolute shrinkage and selection operator (LASSO) regression analysis was performed using the “glmnet” package, and a prognostic nomogram was established using the “regplot” package. For the prediction of potential therapeutic drugs, we used the data from GDSC2016 and visualized them via “MOVICS”. Results: Nine of 23 histone modification-associated prognostic genes were identified to construct the prognostic signature by LASSO analysis, named the histone modification-associated gene (HMAG) signature. Cervical patients with HMAG-H in TCGA-CESC cohort showed a 2.68-fold change of death risk, with the 95% CI from 1.533 to 4.671 (p < 0.001), as well as the increased death risk of HMAG-H in the GSE44001 cohort (HR: 2.83, 95% CI: 1.370–5.849, p = 0.005) and GSE44001 cohort (HR: 4.59, 95% CI: 1.658–12.697, p = 0.003). We observed the preferable AUC values of the HMAG signature in TCGA-CESC cohort (1-year: 0.719, 3-year: 0.741, and 5-year: 0.731) and GSE44001 cohort (1-year: 0.850, 3-year: 0.781, and 5-year: 0.755). The C-index of the nomogram showed a prognostic value as high as 0.890, while the C-index for age was only 0.562, and that for grade was only 0.542. Patients with high HMAG scores were more suitable for the treatment of CHIR-99021, embelin, FTI-277, JNK-9L, JQ12, midostaurin, PF-562271, pyrimethamine, and thapsigargin, and patients with low HMAG scores were more suitable for the treatment of BMS-536924, CP466722, crizotinib, PHA-665752, rapamycin, and TAE684. Conclusion: We comprehensively evaluated the histone modification status in cervical cancer patients and revealed histone modification-associated prognostic genes to construct the HMAG signature, aiming to provide a new insight into prognosis prediction and precise clinical treatment.
| S-EPMC9527294 | biostudies-literature
Project description:It is widely accepted that transcriptional regulation of eukaryotic genes is intimately coupled to covalent modifications of the underlying chromatin template, and in certain cases the functional consequences of these modifications have been characterized. Here we present evidence that gene activation in the silent heterochromatin of the yeast Saccharomyces cerevisiae can occur in the context of little, if any, covalent histone modification. Using a SIR-regulated heat shock-inducible transgene, hsp82-2001, and a natural drug-inducible subtelomeric gene, YFR057w, as models we demonstrate that substantial transcriptional induction (>200-fold) can occur in the context of restricted histone loss and negligible levels of H3K4 trimethylation, H3K36 trimethylation and H3K79 dimethylation, modifications commonly linked to transcription initiation and elongation. Heterochromatic gene activation can also occur with minimal H3 and H4 lysine acetylation and without replacement of H2A with the transcription-linked variant H2A.Z. Importantly, absence of histone modification does not stem from reduced transcriptional output, since hsp82-?TATA, a euchromatic promoter mutant lacking a TATA box and with threefold lower induced transcription than heterochromatic hsp82-2001, is strongly hyperacetylated in response to heat shock. Consistent with negligible H3K79 dimethylation, dot1? cells lacking H3K79 methylase activity show unimpeded occupancy of RNA polymerase II within activated heterochromatic promoter and coding regions. Our results indicate that large increases in transcription can be observed in the virtual absence of histone modifications often thought necessary for gene activation.
| S-EPMC3983032 | biostudies-literature
Project description:This study examined how transcriptomics tools can be included in a Triad-based soil quality assessment to assess the toxicity of soils from river banks polluted by metals. To that end we measured chemical soil properties and used the standardized ISO guideline for ecotoxicological tests and a newly developed microarray for gene expression in the indicator soil arthropod, Folsomia candida. Microarray analysis revealed that the oxidative stress response pathway was significantly affected in all soils except one. The data indicate that changes in cell redox homeostasis are a significant signature of metal stress. Finally, 32 genes showed significant dose-dependent expression with metal concentrations. They are promising genetic markers providing an early indication of the need for higher tier testing in soil quality. One of the least polluted soils showed toxicity in the bioassay that could be removed by sterilization. The gene expression profile for this soil did not show a metal-related signature, confirming that another factor than metals (most likely of biological origin) caused the toxicity. This study demonstrates the feasibility and advantages of integrating transcriptomics into Triad-based soil quality assessment. Combining molecular and organismal life-history trait’s stress responses helps identifying causes of adverse effect in bioassays. Further validation is needed for verifying the set of genes with dose-dependent expression patterns linked with toxic stress.
2014-02-01 | GSE52393 | GEO
Project description:This study examined how transcriptomics tools can be included in a Triad-based soil quality assessment to assess the toxicity of soils from river banks polluted by metals. To that end we measured chemical soil properties and used the standardized ISO guideline for ecotoxicological tests and a newly developed microarray for gene expression in the indicator soil arthropod, Folsomia candida. Microarray analysis revealed that the oxidative stress response pathway was significantly affected in all soils except one. The data indicate that changes in cell redox homeostasis are a significant signature of metal stress. Finally, 32 genes showed significant dose-dependent expression with metal concentrations. They are promising genetic markers providing an early indication of the need for higher tier testing in soil quality. One of the least polluted soils showed toxicity in the bioassay that could be removed by sterilization. The gene expression profile for this soil did not show a metal-related signature, confirming that another factor than metals (most likely of biological origin) caused the toxicity. This study demonstrates the feasibility and advantages of integrating transcriptomics into Triad-based soil quality assessment. Combining molecular and organismal life-history traitM-bM-^@M-^Ys stress responses helps identifying causes of adverse effect in bioassays. Further validation is needed for verifying the set of genes with dose-dependent expression patterns linked with toxic stress. We used a one-color microarray design where each sample was hybridized to a single array
2014-02-01 | E-GEOD-52393 | biostudies-arrayexpress