Epigenetic mechanisms in stem cells and disease

The development of more than 300 cell types present in our body requires the action of complex molecular mechanisms, involving signal transduction pathways that instruct gene transcription by impacting on transcription factor networks and the epigenetic landscape. Epigenetic mechanisms play a crucial role in installing and maintaining cell fate decisions. DNA methylation is among the best studied epigenetic modifications, but genome-wide analysis of DNA methylation remains technically challenging and costly. We recently developed the Methylated DNA-sequencing (MeD-seq) technology that is based on a DNA methylation-dependent restriction enzyme, LpnPI, and therefore restricts CpG methylation analysis to methylated regions of the genome only. This significantly reduces the sequencing depth required, and simplifies subsequent bioinformatics analysis. In contrast to other methyl dependent restriction enzymes, LpnPI activity is blocked by a fragment size smaller than 32 base pairs. This unique property prevents over-digestion of methylation-dense DNA, as observed for other methyl dependent restriction enzymes (such as MspJI and FspEI), and allows accurate genome-wide analysis of CpG methylation at single nucleotide resolution, at a sequencing depth <1/10th required for whole genome bisulfite sequencing (WGBS). In addition, MeD-seq can be applied on DNA retrieved from FFPE material, and requires a minimum of 1000 cells. At present we are applying the MeD-seq technology and other technologies to study the dynamics of epigenetic modifications in embryonic development, stem cell differentiation, and disease with an emphasis on cancer.

 

The MeD-seq wet lab (left) and bio-informatics pipelines (right).

DNA methylation profiles of ZFP42 and the HOXA genes in fibroblasts, iPSCs and ESCs showing loss of DNA methylation in pluripotent stem cells (left two panels).
Right, unsupervised clustering of fibroblast, ESC en iPSC lines based on DNA methylation differences observed in HOX gene loci.

Comparison of MeD-seq with several other technologies to examine DNA methylation genome wide (addapted from Stirzaker 2004).

 

Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI. 2018. Boers R, Boers J, de Hoon B, Kockx C, Ozgur Z, Molijn A, van IJcken W, Laven J, Gribnau J. Genome Res. 28:88-99.

Gunhanlar N, Shpak G, van der Kroeg M, Gouty-Colomer LA, Munshi ST, Lendemeijer B, Ghazvini M, Dupont C, Hoogendijk WJG, Gribnau J, de Vrij FMS, Kushner SA. 2017. A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells. Mol Psychiatry. 10.1038/mp.2017.56

Dupont C, Loos F, Kong ASJ, Gribnau J. 2017. FGF treatment of host embryos injected with ES cells increases rates of chimaerism. Transgenic Res 26: 237-246.

Fernandes TG, Duarte ST, Ghazvini M, Gaspar C, Santos DC, Porteira AR, Rodrigues GM, Haupt S, Rombo DM, Armstrong J et al. 2015. Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells. Biotechnol J 10: 1578-1588.

Barakat TS, Ghazvini M, de Hoon B, Li T, Eussen B, Douben H, van der Linden R, van der Stap N, Boter M, Laven JS et al. 2015. Stable X chromosome reactivation in female human induced pluripotent stem cells. Stem Cell Reports 4: 199-208.

de Esch CE, Ghazvini M, Loos F, Schelling-Kazaryan N, Widagdo W, Munshi ST, van der Wal E, Douben H, Gunhanlar N, Kushner SA et al. 2014. Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation. Stem Cell Reports 3: 548-555.

Group leader: Joost Gribnau

Group members: Ruben Boers, Joachim Boers, Beatrice Tan