Looking to CATCH a glimpse of chromatin dynamics? Well CATCH-IT if you can. Covalent attachment of tags to capture histones and identify turnover (CATCH-IT) is used to estimate the rates of assembly, disassembly or turnover of native nucleosomes.
First, cells are treated with the methionine analog azidohomoalanine (AHA). AHA is incorporated into newly-synthesized proteins including nucleosomes. Nuclei are isolated and AHA-containing proteins are pulled down. Chromatin is then digested with micrococcal nuclease to yield mono-nucleosome fragments. Biotin-tags and a series of specific washes are then used to isolate only histone H3/H4 dimers. The DNA is then purified and can be hybridized to microarray, or sequenced. This identifies regions of the genome that were undergoing changes in nucleosome content during the experimental conditions.
CATCH-IT was thrown out there in a 2010 paper (Deal et al., 2010). The authors applied the technique in Drosophila. They found that the fastest turnover was at active gene bodies, replication origins, and epigenetic regulatory elements. Also, turnover was faster at DNA sites bearing the activating trithorax-group protein than those bearing the repressive polycomb-group binding sites (Deal et al., 2010).
CATCH-IT Additional Reading
This review discusses various techniques for assess chromatin dynamics including CATCH-IT. The authors discuss different levels of transcriptional regulation and how different techniques are suited to measure each.
This review discusses ZNF, TALEN and CRISPR nucleases as methods for genome editing in human stem cells. The authors also discuss non-nuclease options for genome editing and the pros and cons of each.
- Deal, R.B., Henikoff, J.G., and Henikoff, S. (2010). Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161-1164.