Methods for Global Characterization of Chromatin Regulators in Human Cells

Abstract

Chromatin is a multi-layered structure composed of DNA, nucleosomes, histone modifications, and associated proteins that critically affects genome function. Recently developed sequencing technologies enable genomewide characterization of certain aspects of chromatin structure, including nucleosome positioning and histone modifications. However, chromatin proteins present several challenges due to their dynamic nature and variable association with DNA. Chromatin proteins such as Polycomb regulators and heterochromatic factors play critical and global roles in epigenetic repression and hence new approaches are needed for their study. We first sought to identify sequences that recruit Polycomb repressive complex 2 (PRC2) in mammalian cells. We combined chromatin immunoprecipitation with sequencing (ChIP-seq) to map the candidate transcription factor YY1, and found that it does not correlate with PRC2 localization, suggesting that YY1 is not directly involved in PRC2 recruitment. We also identified GC-rich sequences that are necessary and sufficient for PRC2 recruitment. Yet attempts to map additional Polycomb proteins and other repressors using ChIP-seq proved difficult. Since chromatin proteins are often broadly, secondarily or transiently bound to DNA, they are difficult to crosslink. Antibody quality also varies, further hampering ChIP-seq technology. Here, we adapt DamID, a method for mapping chromatin regulators that uses a fusion enzyme and that does not rely on crosslinking or antibodies, for high-throughput sequencing. We show that DamID-seq can be used to globally characterize chromatin repressors in human cells. We used DamID-seq to map the binding of 12 chromodomain-containing and related proteins in K562 cells. We found that these proteins cluster into two modules: 1) Polycombrelated and 2) heterochromatin-related. Polycomb proteins bind developmental genes, while heterochromatin proteins bind broad olfactory receptor (OR) and zinc finger (ZNF) domains. Surprisingly, unlike other Polycomb proteins, CBX2 uniquely binds genes involved with modifying proteins. Our findings advance the model that the genome is compartmentalized into domains, and identify the distinct protein components that associate respectively with Polycomb and heterochromatin domains in human cells. We expect that DamID-seq, along with further advancements in characterizing the three-dimensional organization of chromatin, will bring us towards a better understanding of the role of chromatin in differentiation, development, and disease.