BioTAP-XL: Cross-linking/Tandem Affinity Purification to Study DNA Targets, RNA, and Protein Components of Chromatin-Associated Complexes

Abstract

In order to understand how chromatin complexes function in the nucleus, it is important to obtain a comprehensive picture of their protein, DNA, and RNA components, as well as their mutual interactions. This unit presents a chromatin cross-linking approach (BioTAP-XL) that utilizes a special BioTAP-tagged transgenic protein bait along with mass spectrometry to identify protein complex components, and high-throughput sequencing to identify RNA components and DNA binding sites. Full protocols are provided for Drosophila cells and for human cells in culture, along with an additional protocol for Drosophila embryos as the source material. A key element of the approach in all cases is the generation of control data from input chromatin samples.

Keywords: LC-MS/MS; chromatin; formaldehyde cross-linking; next-generation sequencing.

Figure 1

Strategic Planning for BioTAP-XL experiments. This flowchart outlines sample generation and processing stages along with their associated Basic Protocol steps and/or Support or Alternative protocols, and time considerations. Different source materials are processed with the same Basic Protocol. A successful BioTAP-XL experiment takes approximately 5 days of benchwork. There is an optional stopping point after fixing the crude nuclear extract.

Figure 2

Extracted ion chromatograms (XICs) of tandem mass spectra (MS/MS) of precursor ions corresponding to trypsin autolysis peptides. The top XIC (red) depicts MS/MS of the 523.59 m/z precursor ion corresponding to the doubly charged 98–107 LSSPATLNSR peptide. The middle XIC (green) depicts MS/MS of the 422.01 m/z precursor ion corresponding to the doubly charged 108–115 VATVSLPR peptide. The bottom XIC (blue) represents MS/MS of the 738.15 m/z precursor ion corresponding to the triply charged 58–77 LGEHNIDVLEGNEQFINAAK peptide. Expected average precursor m/z and total number of MS/MS spanned by the main peak are provided for each XIC. A run of good quality will display this relative elution order of these three peptides in reverse phase chromatography, with narrow and symmetrical peak widths (spanning approximately 1–2 minutes).

Figure 3

Representative results for a BioTAP-XL experiment. ChIP-seq profiles for BioTAP-tagged Ezh2 (both carboxy- [C] and amino- [N] terminal tagged constructs) from human 293T-REx cells exhibit consistent binding patterns and also track with the PRC2-associated H3K27me3 binding profile. BioTAP-tagged CBX4 and Flag-His-tagged CBX2 display patterns distinct from the PRC2 profiles. Figure from (Alekseyenko et al., 2014a), with H3K27me3 and Flag-His-CBX2 profiles from (Gao et al., 2012). Ezh2 is the catalytic subunit of PRC2 (Polycomb Repressive Complex 2); CBX2 and CBX4 are interchangeable subunits in a related, but distinct complex, PRC1.