Combined ChIP and bisulfite sequencing

There is mounting evidence for substantial interaction between methylated DNA and chromatin modifications. We highlight current techniques that allow analysis of DNA methylation-chromatin crosstalk.

DNA methylation and chromatin modifications were once generally considered to be distinct mechanisms that were only grouped together based on their roles as epigenetic regulators. Many labs specialized in either chromatin or DNA methylation research.

While this approach certainly led to deeper technical expertise for studying each type of mark, mounting evidence suggests that these epigenetic mechanisms do not operate independently and often exhibit crosstalk (Fischle, 2008; Cedar & Bergman, 2009; Lee et al., 2010).

Several recent reports have indicated that there are substantial interactions between DNA methylation and chomatin modifications. However, those findings could only be derived from correlative studies or parallel experiments on various epigenetic marks, due to a lack of adequate techniques to allow simultaneous analysis of DNA methylation and chromatin modification.

In 2012, two groups, working separately, bridged that technology gap with unique approaches based on a combination of familiar epigenetic analysis protocols. The work was published in the same issue of Genome Research, and both applied chromatin immunoprecipitation (ChIP) and bisulfite sequencing (BS) together, but used them in a different sequence.

Each of the new methods combined the same basic tools to allow successful combinatorial profiling of DNA methylation and chromatin modifications at the same time.

ChIP bisulfite sequencing (ChIP-BS-seq)

Scientists at Radboud University in the Netherlands and the Broad Institute in Boston developed ChIP-BS-seq (Brinkman et al., 2012). This is a system where they bisulfite treat previously chromatin immunoprecipitated material and then sequence those samples.

Their approach allowed them to determine relationships between DNA methylation and chromatin features: 

  • H3K27me3 and DNA methylation exist together throughout most of the genome, with the exception of CpG Islands.
  • Lack of DNA methyltransferase in embryonic stem cells led to widespread genomic changes in H3K27me3, and broad local enrichments of H3K27me3 emerged where DNA methylation marks were previously found.
  • DNA methylation appears to prevent H3K27  deposition.

High throughput sequencing of bisulfite treated ChIP DNA (BisChIP-seq)

Researchers from the Garvan Institute for Medical Research in Australia, constructed their DNA methylation-chromatin crosstalk tool, BisChIP-seq, in the opposite way. This technique bisulfite treats the DNA first, then proceeds to ChIP and sequence samples (Statham et al., 2012).

Their BisChIP-seq assessment yielded several interesting results:

  • CpG islands and transcription start sites (TSS) of genes that are silenced in prostate cancer displayed enrichment for both the repressive histone mark H3K27me3 and DNA methlation.
  • Intergenic regions where H3K27me3 is present had less DNA methylation.
  • Both methylated and unmethylated alleles showed association with H3K27me3 histones simultaneously. This suggested that DNA methylation is not dependent on the polycomb chromatin status of that region.

Both ChIP-BS-seq and BisChIP-seq offer a means to directly investigate the relationship between chromatin modifications and DNA methylation. This combinatorial analysis of chromatin and DNA methylation represents a big advancement from previous correlative studies and promises to provide a platform towards a deeper understanding of epigenetic mechanisms.


  • Brinkman AB, Gu H, Bartels SJ, Zhang Y, Matarese F, Simmer F, Marks H, Bock C, Gnirke A, Meissner A and Stunnenberg HG (2012). Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res. 22, 1128–38.
  • Cedar H and Bergman Y (2009). Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet. 10, 295–304. 
  • Fischle W (2008). Talk is cheap–cross-talk in establishment, maintenance, and readout of chromatin modifications. Genes Dev. 22, 3375–82. 
  • Lee JS, Smith E and Shilatifard A (2010). The language of histone crosstalk. Cell 3, 682–5. 
  • Statham AL, Robinson MD, Song JZ, Coolen MW, Stirzaker C and Clark SJ (2012). Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA. Genome Res. 22, 1120–7.