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:Determining the genomic localization of chromatin features is an essential aspect of investigating gene expression control, and ChIP-Seq has long been the gold standard technique for interrogating chromatin landscapes. Recently, the development of alternative methods, such as CUT&Tag, have provided researchers with alternative strategies that eliminate the need for chromatin purification, and allow for in situ investigation of histone modifications and chromatin bound factors. Mindful of technical differences, we set out to investigate whether distinct chromatin modifications were equally compatible with these different chromatin interrogation techniques. We found that ChIP-Seq and CUT&Tag performed similarly for modifications known to reside at gene regulatory regions, such as promoters and enhancers, but major differences were observed when we assessed enrichment over heterochromatin-associated loci. Unlike ChIP-Seq, CUT&Tag detects robust levels of H3K9me3 at a substantial number of repetitive elements, with especially high sensitivity over evolutionarily young retrotransposons. IAPEz-int elements for example, exhibited underrepresentation in mouse ChIP-Seq datasets but strong enrichment using CUT&Tag. Additionally, we identified several euchromatin-associated proteins that co-purify with repetitive loci and are similarly depleted when applying ChIP-based methods. This study reveals that our current knowledge of chromatin states across the heterochromatin portions of the mammalian genome is extensively incomplete, largely due to36 limitations of ChIP-Seq. We also demonstrate that newer in situ chromatin fragmentation-based techniques, such as CUT&Tag and CUT&RUN, are more suitable for studying chromatin modifications over repetitive elements and retrotransposons.
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.