Proximity Labeling Techniques to Study Chromatin

Abstract

Mammals contain over 200 different cell types, yet nearly all have the same genomic DNA sequence. It is a key question in biology how the genetic instructions in DNA are selectively interpreted by cells to specify various transcriptional programs and therefore cellular identity. The structural and functional organization of chromatin governs the transcriptional state of individual genes. To understand how genomic loci adopt different levels of gene expression, it is critical to characterize all local chromatin factors as well as long-range interactions in the 3D nuclear compartment. Much of our current knowledge regarding protein interactions in a chromatin context is based on affinity purification of chromatin components coupled to mass spectrometry (AP-MS). AP-MS has been invaluable to map strong protein-protein interactions in the nucleus. However, the interaction is detected after cell lysis and biochemical enrichment, allowing for loss or gain of false positive or negative interaction partners. Recently, proximity-dependent labeling methods have emerged as powerful tools for studying chromatin in its native context. These methods take advantage of engineered enzymes that are fused to a chromatin factor of interest and can directly label all factors in proximity. Subsequent pull-down assays followed by mass spectrometry or sequencing approaches provide a comprehensive snapshot of the proximal chromatin interactome. By combining this method with dCas9, this approach can also be extended to study chromatin at specific genomic loci. Here, we review and compare current proximity-labeling approaches available for studying chromatin, with a particular focus on new emerging technologies that can provide important insights into the transcriptional and chromatin interaction networks essential for cellular identity.

Keywords: APEX2; BioID; ChIP; affinity purification; dCas9; mass spectrometry; protein-protein interactions; proxisome.

FIGURE 1

General workflow of proximity labeling followed by mass spectrometry with biotin ligase (A) or peroxidase (B). The protein of interest (bait) is fused to the reporter enzyme and expressed in cells. Supplying the enzymes with their substrates creates reactive intermediates that target amino acid side chains of proteins in proximity (prey). The covalently biotinylated proteins can be enriched by streptavidin beads. Subsequent on-bead digestion and identification of resulting peptides with mass spectrometry provides a candidate list of proteins in the vicinity of the bait.

FIGURE 2

Nuclear compartments that were successfully targeted by proximity labeling. A detailed compiled list of studies is provided in Supplementary Table S1.

FIGURE 3

Venn diagrams showing the number of shared identifications in BioID studies with the same bait. (A) Comparison of three studies with the MYC protein as bait. (B) Comparison of two studies with Lamin A as bait.