Dataset Information

Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity.

ABSTRACT: In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using the broadly used AGO2 11A9 antibody to determine interactions with chromatin and nuclear proteins. We found a previously described interaction between AGO2 and SWI/SNF on chromatin with ChIP-MS and observed enrichment at enhancers and transcription start sites using ChIP-seq. However, antibody specificity issues can produce misleading results for ChIP, RNA-seq and Mass spectrometry. Therefore, we developed a CRISPR/Cas9 engineered AGO2-/- HEK293T cell line to validate our findings. ChIP-qPCR and immunoprecipitation combined with MS (IP-MS) showed that the 11A9 antibody associates with chromatin and SWI/SNF in the absence of AGO2. Furthermore, stoichiometry, IP-MS and co-IP analysis suggests a direct interaction of this antibody with SMARCC1, a component of the SWI/SNF complex. For this reason, particular care should be taken in performing and interpreting experiments in which the 11A9 antibody is used to study a nuclear role of AGO2.

SUBMITTER: van Eijl RAPM

PROVIDER: S-EPMC5544689 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity.

van Eijl Roderick A P M RAPM van den Brand Teun T Nguyen Luan N LN Mulder Klaas W KW

Scientific reports 20170804 1

In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using the broadly used AGO2 11A9 antibody to determine interactions with chromatin and nuclear proteins. We found a previously described interaction between AGO2 and SWI/SNF on chromatin with ChIP-MS and observed enri ...[more]

PMID: 28779093

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Project description:UNLABELLED:SWI/SNF is a major regulator of gene expression. Its role is to facilitate the shifting and exposure of DNA segments within the promoter and other key domains to transcription factors and other essential cellular proteins. This complex interacts with a wide range of proteins and does not function within a single, specific pathway; thus, it is involved in a multitude of cellular processes, including DNA repair, differentiation, development, cell adhesion, and growth control. Given SWI/SNF's prominent role in these processes, many of which are important for blocking cancer development, it is not surprising that the SWI/SNF complex is targeted during cancer initiation and progression both by mutations and by non-mutational mechanisms. Currently, the understanding of the types of alterations, their frequency, and their impact on the SWI/SNF subunits is an area of intense research that has been bolstered by a recent cadre of NextGen sequencing studies. These studies have revealed mutations in SWI/SNF subunits, indicating that this complex is thus important for cancer development. The purpose of this review is to put into perspective the role of mutations versus other mechanisms in the silencing of SWI/SNF subunits, in particular, BRG1 and BRM. In addition, this review explores the recent development of synthetic lethality and how it applies to this complex, as well as how BRM polymorphisms are becoming recognized as potential clinical biomarkers for cancer risk. SIGNIFICANCE:Recent reviews have detailed the occurrence of mutations in nearly all SWI/SNF subunits, which indicates that this complex is an important target for cancer. However, when the frequency of mutations in a given tumor type is compared to the frequency of subunit loss, it becomes clear that other non-mutational mechanisms must play a role in the inactivation of SWI/SNF subunits. Such data indicate that epigenetic mechanisms that are known to regulate BRM may also be involved in the loss of expression of other SWI/SNF subunits. This is important since epigenetically silenced genes are inducible, and thus, the reversal of the silencing of these non-mutationally suppressed subunits may be a viable mode of targeted therapy.

Dataset Information

Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity.

Publications

Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity.

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