Using ChIP in Combination with Bisulfite Sequencing

The evidence is building up for the existence of solid links between methylated DNA and the modification of chromatin. This article deals with a few of the latest techniques that are employed to allow researchers to understand how these phenomena are interrelated.

At first these were considered to be separate occurrences, and the only common feature was that both participated in regulating epigenetic modification of DNA. Thus it was common laboratory practice to specialize in one or the other of these fields. While this kind of demarcation did develop greater depth of technical skill in both fields, the weight of research now indicates that both of these mechanisms are intricately interwoven, and crosstalk is often detected (Fischle, 2008; Cedar & Bergman, 2009; Lee et al., 2010).

Recent studies have reported the presence of solid relationships between these events, but they are mostly correlative in nature or else experiments on various epigenetic marks conducted in parallel. This shows the lack of techniques specifically designed and powerful enough for parallel research on DNA methylation as well as the modification of chromatin.

Two groups working independently of each other came out with some unusual techniques in 2012. These combine some established protocols for epigenetic analysis to allow simultaneous research on both these events. The reports came out in the same issue of the journal Genome Research. Both used the same combination, namely, chromatin immunoprecipitation (ChIP) with bisulfite sequencing (BS), with the difference being in the order in which they are used.

Thus the same basic technology was used to formulate a new method successfully, allowing DNA methylation as well as chromatin modification to be studied in combination at the same moment.

ChIP Bisulfite Sequencing (ChIP-BS-seq)

One group comprising of researchers from Radboud University in the Netherlands and the Broad Institute in Boston developed the ChIP-BS-seq (Brinkman et al., 2012). This is a platform which uses bisulfite to treat a sample which has undergone chromatin immunoprecipitation. The samples are then subjected to sequencing.

By using this method the researchers were able to find out much more about how DNA methylation and chromatin features are linked to each other:

H3K27me3 (a repressive histone mark) and DNA methylation are found to occur in combination in almost all areas of the DNA except for CpG islands.

Embryonic stem cells lack DNA methyltransferase, which causes genomic alterations to occur throughout in H3K27me3. When there were identifiable marks of DNA methylation , H3K27me3 was found to be enriched locally in a broad fashion.

High Throughput Sequencing of Bisulfite Treated ChIP DNA (BisChIP-seq)

Another group of scientists from the Garvan Institute for Medical Research in Australia built up a tool to detect cross talk between DNA methylation and chromatin. This is called BisChIP-seq, and operates in the reverse of the previous technique. It uses bisulfite treatment of DNA first, to be followed by ChIP and sample sequencing (Statham et al., 2012).

The results of this study using BisChIP-seq assessment were intriguing:

Some genes that are silenced in prostate cancer show enrichment of CpG islands and transcription start sites (TSS) for H3K27me3 and DNA methylation.

On the other hand, there were low levels of DNA methylation wherever there was an intergenic region presenting H3K27me3.

Thirdly, alleles which showed methylation as well as nonmethylated alleles were found to express H3K27 simultaneously. This could mean that the polycomb chromatin status of any region is not a factor in the occurrence of DNA methylation in that region.

Both ChIP-BS-seq and BisChIP-seq are techniques that allow the direct analysis of the correlation between DNA methylation and chromatin modification. By combining these two events during analysis, the data obtained is superior to that arrived at by the older correlative methods. This is eagerly expected to provide means to explore epigenetic mechanisms in greater detail.


  • 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.

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Last updated: Jun 6, 2019 at 11:56 AM


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