Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combined ChIP with label-free quantitative (LFQ) mass spectrometry (ChIP-MS) to interrogate local chromatin compositions. We demonstrate the versality of our approach at telomeres, with transcription factors, in tissue and by dCas9-driven locus-specific enrichment.
Project description:Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combined ChIP with label-free quantitative (LFQ) mass spectrometry (ChIP-MS) to interrogate local chromatin compositions. We demonstrate the versality of our approach at telomeres, with transcription factors, in tissue and by dCas9-driven locus-specific enrichment.
Project description:CHIP is a neuroprotective E3-ubiquitin ligase that supports longevity and healthy ageing. Loss of CHIP function has a major impact on life expectancy in animal models, whilst in humans’ mutations that compromise the E3-ligase activity of CHIP are causative for forms of Spinocerebellar Ataxia (SCA) that are accompanied by cognitive decline and/or dementia. The pathways regulated by CHIP to maintain neuronal health remain to be discovered. Gene-edited neuroblastoma cells were produced and used as a model to study the effects of CHIP loss on the steady state proteome in the absence of proteotoxic stress. Label free quantitative proteomic analysis (SWATH-MS) highlighted VGF, a member of the neuropeptide precursor family of proteins, as being a dominant protein responding to loss of CHIP function. By studying the dependence of VGF expression on CHIP using SILAC and RNA-Seq we have defined a role for the ligase in regulated neuropeptide expression.
