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.

RNA Biogenesis Instructs Functional Inter-Chromosomal Genome Architecture

“…A potential alternative is the application of in situ proximity labeling, for instance by relying on promiscuous biotinylating enzymes to covalently tag nearby proteins for subsequent streptavidin-based purification and identification through mass spectrometry. These methods have been successfully used to determine protein-protein interactions, and their implementation to study locus-specific regulations are also emerging (reviewed in Ummethum and Hamperl, 2020 ).…”

Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

“…ntricate networks of direct and coordinated interactions between proteins and nucleic acids are of vital importance in the regulation of numerous cellular processes, such as gene expression, DNA replication or DNA repair 1 . Robust methods that can interrogate these interaction networks in a native chromatin context are key to understand the underlying molecular mechanisms 2,3 . Chromatin immunoprecipitation (ChIP) has been coupled with mass spectrometry (MS)-based proteomics analysis to characterize the composition of particular chromatin-associated protein complexes [4][5][6] .…”

The emerging role of chromatin remodelers in neurodevelopmental disorders: a developmental perspective

“…Recent advances in spatial proximity labelling, such as promiscuous biotinylation targeted via dCas9 [427], allow for a precise snapshot of protein complexes assembled in spatial proximity to a single genomic region. Importantly, this technique can be applied in living cells and in vivo in the developing brain [428], making it applicable to the neurodevelopmental questions that we have detailed here. This technique allows detailed insights site-specific complex dynamics, a largely unexplored feature of the genetic landscape.…”