Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure

Abstract

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.

CHD3 chromatin-remodeling factor PICKLE regulates floral transition partially via modulating LEAFY expression at the chromatin level in Arabidopsis

“…Therefore, the binding activity of PKL is even not specific to its target genes. Similarly, it has also been previously found that PKL was present at the promoters of either H3K27me3 enriched genes, or actively transcribed genes that were ubiquitously expressed and PKL-independent genes such as ACT7 and UBQ10 (polyubiquitin 10) (Zhang et al, 2012;Harmeyer et al, 2015). Based on these data, it was even thought that PKL facilitates a common chromatin remodeling process that is not restricted to specific regions of the genome (Zhang et al, 2012).…”

Exploring the bZIP regulatory network in the sponge Amphimedon queenslandica: insights into the ancestral animal regulatory genome

“…This time point corresponds to larvae that are competent to settle and undergo metamorphosis (10-12 hours post emergence from the brood chamber) . The entire amino acid sequence of histone H3 is perfectly conserved between A. queenslandica and other eukaryotes where these antibodies have been used successfully (Barraza et al, 2015;Eckalbar et al, 2016;Harmeyer et al, 2015;Liu et al, 2007;Sebe-Pedros et al, 2016) (Appendix 5.3). I also used a custom-made affinity-purified rabbit polyclonal antibody, raised by GenScript Biotechnology (NJ, USA), against a peptide located outside the bZIP domain of AqATF4 (CTPKRTPQGRGGRKS).…”

“…Antibodies against these modifications of the histone H3 tail have also successfully been used in a range of eukaryotic species (Barraza et al, 2015;Eckalbar et al, 2016;Harmeyer et al, 2015;Liu et al, 2007;Sebe-Pedros et al, 2016). Since the amino acid sequence of A. queenslandica histone H3 is identical to its bilaterian orthologues (Appendices 5.3, 5.6) and A. queenslandica also have the relevant histone methyltransferases and acetyltransferases (Appendix 5.7), these antibodies are prone to recognise the correct epitopes in this sponge.…”

Origin and evolution of the metazoan non-coding regulatory genome: Insights from the sponge Amphimedon queenslandica

“…S4.1; Appendix C: Table S4.2, S4.3). These antibodies recognize the correct epitopes in even more distantly related organisms (i.e., non-metazoan eukaryotes) (e.g., (Barraza et al, 2015;Eckalbar et al, 2016;Ercan et al, 2009;Harmeyer et al, 2015;Liu et al, 2007;Sebé-Pedrós et al, 2016).…”

“…The entire amino acid sequence of histone H3 is perfectly conserved between Amphimedon and other eukaryotes where these antibodies have been used successfully (Barraza et al, 2015;Eckalbar et al, 2016;Ercan et al, 2009;Harmeyer et al, 2015;Liu et al, 2007;Sebé-Pedrós et al, 2016) (Appendix C: Fig. S4.1).…”