While we’ve asked before how nucleosomes find their way to the right spot on DNA, sometimes we’re left staring at sea monsters in the most tantalizing unexplored areas on our genome-wide maps. When it comes to nucleosomes, positioning is everything since it affects DNA accessibility for replication, repair, and gene expression. In order to understand how nucleosome positions are determined, we need a reliable way to map them. Previously, nucleosome mapping has relied upon H4S47C cleavage, which utilizes chemical cleavage at a mutated cysteine (S47C) in histone 4 (H4), but unfortunately it produces high background noise. Luckily, a talented team from the lab of of Steven Henikoff at the Fred Hutchinson Cancer Research Center have developed a new technique for precision mapping of nucleosomes in the yeast genome.
The authors have improved upon the H4S47C cleavage mapping method by relocating the cysteine mutant to residue 85 (H85C) on histone 3 (H3). Both methods exploit the ability of phenanthroline to chelate copper to the cysteine residue. Addition of peroxide produces free radicals at the conjugated copper, resulting in adjacent DNA backbone cleavage. The resulting DNA fragments are then sequenced. Although H3Q85C and H4S47C cleavage mapping operate on the same principles, H3Q85C has several advantages over its parental method:
- The H3Q85C cleavage site is further from the center of the nucleosome
- A 51-base pair fragment is released upon cleavage, which is long enough for sequencing
- The center of each 51-base pair fragment directly corresponds to nucleosome position and consequently, H3Q85C mapping does not rely on predictive averages
- There is less cleavage within linker regions and reduced non-specific cleavage, resulting in a higher signal-to-noise ratio
These ambitious authors weren’t content to simply develop this improved technique for nucleosome mapping—they immediately put this new methodology to work. Here’s a look at how they applied this powerful technique:
- H3Q85C mapping can detect H3 containing nucleosomes
- This feature was used to disprove a previous hypothesis of a Cse4-H3 heterotypic nucleosome at yeast centromeres
- They also showed that tRNA genes are depleted of nucleosomes
- The improved signal-to-noise ratio allowed for identification of rotational phasing across all nucleosomes, confirming the preference for A/T dinucleotides in the minor groves of the DNA that contacts the histones, and G/C dinucleotides in the major grooves.
- Thanks to the lack of cleavage within linker regions, the authors were able to map nucleosome depleted regions, as well as the nearest flanking nucleosomes
- They ranked yeast genes based on nucleosome crowding and showed that the most frequently transcribed genes have decreased nucleosome spacing and larger nucleosome depleted regions
The combination of nucleosome phasing data and accurate nucleosome mapping led the authors to develop a simple biophysical model for nucleosome positioning. This exciting work begins to dissect the complex relationship between chromatin features and nucleosome position, and the role these play in gene expression.
Searching for more? No map needed. Just click over to the full article in Genome Biology, February 2018.