Don’t you wish you could just look into the nucleus of a cell and watch what’s happening? With SiTomics, Peng Chen’s lab (Peking University) gets close—they can see what modifications are being placed on a particular histone, what proteins bind to it, and where that histone is on the DNA, all in living cells.
To do this now, you’d need some good binders, like antibodies or chromatin readers, but there aren’t enough specific ones out there. And just as sitting in a grassland with a camera can affect how lions behave in the wild, the techniques can interfere with cellular structures, so you may not get an accurate picture.
That’s why Chen’s team developed SiTomics, a general, yet programmable, approach. It’s a natural ecosystem of three parts; the first one trains the binoculars on histone mods:
- Site-Profiling: Mass spectrometry using SILAC with cells under normal conditions, then after short chain fatty acid treatment, shows the dynamics of histone modifications, as well as their stoichiometries.
Genetic code expansion then enabled the team to modify histones in the way they wanted (with a photoaffinity lysine that has a specific modification, such as an acylation, on it). They showed that these histones behave just like native ones, then they went on to develop the other two parts, which let them peer in at proteomes and genomes:
- Site-Link: Cells with the photoaffinity lysine are UV-irradiated, which crosslinks the histone modification to whatever proteins are bound to it. Mass spectrometry shows the proteomic differences between non-irradiated cells and those exposed to UV light.
- Site-Seq: ChIP-seq shows where histones are located on the genome, comparing cells with modified and unmodified lysines.
Using the method, the researchers identified known modification sites, as well as four previously unknown ones that occur in response to short chain fatty acid precursors. Proteomes bound to crotonylated and β-hydroxybutyrylated (bhb) lysines were very different, with many unique proteins in each group. Because cell cycle proteins were specific to bhb, this mod might have a role in regulating division. The experiments also showed that H3K56cr and H3K4me3 could work cooperatively, making the nucleosome more accessible. In addition, site-specific lysine bhb correlated with super-enhancers, which could help explain how ketone bodies respond to metabolic stress.
Channel your inner Sir David Attenborough and use SiTomics to watch your favorite histones in the wild at Cell, March 2023.