Sometimes, when you’re dealt the right hand, doubling down is a sound strategy. Although it can be a risky move in blackjack, a new ChIP-seq method makes it a sure thing when you’re dealing with bivalent chromatin. The strategy, which includes important preparation steps and two rounds of chromatin immunoprecipitation (ChIP), provides sensitive genome-wide profiles of bivalent chromatin from low-input sample sources.
Being poised for a situation can help you win in both high stakes gambling and epigenetics. One way a cell can adapt to changing conditions during development is through bivalent chromatin with both active (H3K4me3) and inactive (H3K27me3) histone modifications on opposite tails of a nucleosome. But it’s challenging to distinguish true bivalency from marks on different alleles, or on different cells in mixed populations. Protocols that include separate ChIPs and in silico analyses have high false positive rates, and sequential H3K4me3 and H3K27me3 ChIPs require tens of millions of cells and sometimes don’t include proper controls.
So, Melanie Eckersley-Maslin’s team (Peter MacCallum Cancer Centre, University of Melbourne) optimized the sequential re-ChIP technique. The three-day protocol requires a little time, but it is specific and only needs about two million cells. Here are the different ChIP types:
- Control ChIPs include IgG-IgG, and H3K4me3-only and H3K27me3-only ChIPs
- Sequential ChIPs include H3K4me3, then H3K27me3 antibody beads in one set of tubes, as well as H3K27me3, then H3K4me3 in another set
Here’s the protocol:
- Cross-link chromatin with formaldehyde, lyse cells and treat with MNAse to obtain mononucleosomes
- To increase the signal-to-noise ratio and remove non-specific binding, pre-clear the chromatin with pre-washed polystyrene beads
- Form antibody-bead complexes for control IgG, H3K4me3, and H3K27me3 ChIPs
- Conduct the first overnight ChIP, wash the complexes and elute them with an SDS buffer
- Perform the second ChIP overnight—if antibodies against H3K4me3 were used in the first IP, then use antibodies against H3K27me3 this time, and vice versa
- Elute chromatin and reverse crosslinks
- Degrade RNA and proteins, and perform qPCR and/or high-throughput sequencing with the DNA
The hard-working team applied the method to mouse embryonic stem cells (mESCs) and identified bivalent sites consistent with previous studies. Almost 9,000 peaks were shared between the two sequential ChIPs, and about 5,300 of those overlapped with the independent single ChIPs, which were high-confidence sites. Similar to previous studies, the team found peaks mostly at promoters or transcription start sites with CpG islands. The sites were enriched for developmental regulation motifs. Other peaks overlapped possible enhancer elements. Here are some additional data:
- Looking at promoters, the method detected 77% of previously known bivalent genes
- 23% of the bivalent promoters were novel
- Bivalent genes were expressed at low levels, as expected, and dynamically expressed as differentiation took place—either to an active or repressed condition
- DPPA2 and DPPA4 are required for bivalency at some genes, and a double-knockout lost bivalent peaks at those genes
- Genes not dependent on DPPA2/4 for priming did not lose peaks in the double-knockout
- Some novel DPPA2/4-dependent genes were identified
In the end, the researchers say that the method could be used in different tissues and cell lines—not just in mESCs—to pinpoint true bivalent sites.
Double down on bivalent chromatin with reChIP over at Epigenetics & Chromatin, February 2024.