Navigating the epigenetic landscape is no easy feat. Clear markings and smooth rides through its canals can quickly give way to a hectic hodgepodge of mixed signals and uncertain routes. A great example is bivalent chromatin, which contains marks of both activation and repression.
Characterized by Bernstein et al. in 2006, the most studied bivalent chromatin landmark consists of large regions of repressive H3K27me3 containing smaller areas of activating H3K4me3.
Overall, bivalent chromatin appears to silence developmentally important genes in embryonic stem cells (ESCs) while leaving them poised for activation during later differentiation. Furthermore, bivalent chromatin appears to have a role in poising tissue-specific genomic imprinting, as exemplified by paternal expression of Grb10 in the brain, and also crosstalks with DNA methylation via the TET enzymes.
A New Technique for Examining Bivalent Chromatin
A study from the lab of Bradley Bernstein at Harvard (Shema et al.) now presents a new technique for single-molecule imaging and sequencing of nucleosomes that offers unprecedented insight into the bivalent state.
The imaging technique involves:
- Isolating nucleosomes from cells using micrococcal nuclease digestion.
- Ligating fluorescent biotinylated oligonucleotide adaptors to the free ends of DNA from the individual nucleosomes.
- Purifying and capturing the labeled nucleosomes on slides.
- TIRF (total internal reflection) microscopy to image the position of the nucleosomes while also cleaving the fluorophore from the adaptor.
- Fluorescent antibodies for different histone modifications that can be scored by time-lapse imaging as they repeatability bind and dissociate from the nucleosomes still bound in place by their adaptors.
The team then demonstrated the utility of their technique by examining ESCs, embryoid bodies derived from ESCs, and fully committed lung fibroblasts. They found that H3K27me3 was present in ~6.5% of ESC nucleosomes and at ‘somewhat’ higher levels in the other cells, while H3K4me3 was relatively constant at ~1.6 – 2%. Overall, bivalent chromatin domains appeared in 0.5% of nucleosomes in ESCs but were significantly depleted in the other cells assayed.
Next, the team examined whether the bivalent modifications occurred on the same histone tail or asymmetrically on opposing histone tails within the nucleosome. Interestingly, they found that 94% of bivalent nucleosomes are labeled asymmetrically and only 6% are labeled on the same histone tail in ESCs.
Analysis of cancer cell lines also demonstrated that bivalent nucleosomes are prevalent in some cancer types. Finally, the group also demonstrated how this proteomic imaging platform can be coupled with single-molecule sequencing-by-synthesis on the slide to determine precisely where the combinatorial modifications of individual nucleosomes lie in the genome. Notably, a number of different antibody combinations can be used, thus presenting us with a novel technique for further bivalent chromatin discovery.
A Different State of Bivalency: DNA Cytosine Methylation & H3K27 Acetylation
Interestingly, while H3K4me3 forms bivalent chromatin with H3K27me3, it has also been shown to form bivalent chromatin with H3K9me3 as well. Further attesting to the diversity of bivalent chromatin is the lab of Peter Jones at the University of Southern California, where Charlet et al. examined DNA methylation alongside activating H3K27ac.
Here’s what they found in human cell lines using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq) and H3K27ac ChIP-seq:
- DNA methylation and H3K27ac co-exist at enhancers and super-enhancers but not promoters.
- This pattern was found in cancer and non-cancer human cell lines. ESCs weren’t examined.
- Looking deeper into the human colon cancer cell line (HCT116) they found that 38% of regular enhancers show high levels of methylation, while only 24% show low levels of methylation.
- Strikingly, 64% of super-enhancers show high levels of methylation, and only 6% show low levels.
- There was also a similar trend in normal human colon cells.
- Interestingly, DNA methylation is depleted at transcription factor (TF) binding sites (as indicated by examining TCF4 and YY-1) and the DNA is more accessible.
- However, the TF AP-1 showed no preference for monovalent or bivalent enhancers, which may be due to a CpG site in its binding motif that is known to bind methylated sequence.
- Further experiments using HCT116, DKO1 (a highly demethylated HCT116 line), and HCT116 cells treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine revealed that DNA methylation is needed globally to maintain the activating H3K27ac that is needed for enhancer function.
Thus, while DNA methylation is needed to shape the larger epigenetic landscape of enhancers, its depletion in select areas is required to form a landing zone for some TFs.
Overall, these two papers highlight the regulatory potential of bivalent chromatin and add further complexity to the histone code. It seems that mixed messages might just be the right messages when it comes to the epigenetic landscape and an examination of genes marked by bivalent chromatin may help prioritize candidates for both developmental and cancer research.