Abstract
BackgroundUnderstanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking.ResultsHere, we present carbon copy-ChIP (2C-ChIP), a versatile, inexpensive, and high-throughput technique to quantitatively measure the abundance of DNA sequences in ChIP samples. This method combines ChIP with ligation-mediated amplification (LMA) and deep sequencing to probe large genomic regions of interest. 2C-ChIP recapitulates results from benchmark ChIP approaches. We applied 2C-ChIP to the HOXA cluster to find that a region where H3K27me3 and SUZ12 linger encodes HOXA-AS2, a long non-coding RNA that enhances gene expression during cellular differentiation.Conclusions2C-ChIP fills the need for a robust molecular biology tool designed to probe dedicated genomic regions in a high-throughput setting. The flexible nature of the 2C-ChIP approach allows rapid changes in experimental design at relatively low cost, making it a highly efficient method for chromatin analysis.
Highlights
Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies
A Chromatin immunoprecipitation (ChIP) experiment typically yields a complex array of DNA sequences that are selectively immunoprecipitated from entire genomes in a population of cells (‘purified ChIPed genomic DNA (gDNA)’)
2C-ChIP analysis identifies a HOXA cluster region slow to demethylate From the 2C-ChIP analysis of our differentiation time course, we found that rapidly activated genes (HOXA1 and HOTAIRM1) acquire higher levels of H3K4me3 at their promoters and along gene bodies compared to those at the proximal region induced later by retinoic acid (RA) (Fig. 4c)
Summary
Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking. Understanding why genes are expressed at a given time will require full knowledge of how transcription occurs. Acquiring this knowledge, in turn, will demand the integration of many different types of information, including genomic sequence, epigenomic traits, and three-dimensional chromatin organization. Epigenomic information can be gained from robust complementary methodologies, one of which is chromatin immunoprecipitation (ChIP), which is considered a staple in gene expression studies. The resulting associated DNA fragments are purified before sequence and abundance are determined
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