Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs

da | Set 10, 2020 | Biologia Molecolare

Abstract

In a recent work Song and collaborators [1].  used an allelic reporter approach to show how super-enhancers allelic DNA methylation dynamics underlies locus-specific heterogeneity, which functionally impacts transcription and cellular states of mouse embryonic stem cells.

Locus-specific DNA methylation heterogeneity across cells has been shown by recent scWGBS as a potential explanation for the variable low-to-intermediate levels of methylation at active enhancers in bulk measurements. The present work was based on an experimental paradigm that overcomes some of the limitations of single-cell sequencing approaches using an allele specific reporter system [1]. The authors have recently developed Reporter of Genome Methylation (RGM) allows tracing of locus-specific DNA methylation based on the on-and-off of a fluorescent signal in single cells in real time, and has been shown to faithfully reflect the endogenous DNA methylation states at multiple genomic loci [2][3]. They utilized this system at two pluripotency super-enhancers (SEs), Sox2 and Mir290 SEs, in ESCs.

To track DNA methylation heterogeneity on each allele, they targeted the Mir290 and the Sox2 SE independently in 129xCastaneus F1 hybrid ESCs with allele-specific RGM reporters and generated two cell lines, Sox2-129SE-RGM-tdTomato/Sox2 CASTSE-RGM-eGFP (abbreviated below as SOX2 SE-TG) and Mir290-129SE-RGM-tdTomato/ Mir290-CAST SE-RGM-eGFP (abbreviated below as MIR290-SE-TG) allowing to visualize the SE locus-specific DNA methylation state at allelic and single cell resolution. The FACS analysis detected a small fraction of single positive (T+G–, T–G+) as well as double-negative (T–G–) cells in both cell lines, though the majority of cells were double positive (T+G+), consistent with the heterogeneity reported in scWGBS data. The authors demonstrated that changes in DNA-methyltransferase activities modulate the dynamics of methylation. They compared RGM activities in Dnmt3a or Dnmt3b single-knockout and Dnmt3a/3b double-knockout (DKO) cells. Although the number of RGM negative cells was reduced in Dnmt3a or Dnmt3b single-knockout cells, cells with methylated SEs were eliminated only in the absence of both de novo methyltransferases in DKO cells preventing any de novo methylation. These results suggest that both DNMT3A and DNMT3B have redundant functions and independently contribute to de novo methylation of SE DMRs.

Figure 1. Highlights

 

To assess whether demethylation of the SEs involved active or passive mechanisms, they analyzed whether DNA demethylation would be affected in cells upon delaying cell-cycle progression using thymidine block. In all three populations carrying at least one methylated allele, the kinetics of demethylation upon thymidine block was significantly decreased upon 3 days in culture. This suggests that cell proliferation-driven passive demethylation is responsible for SE demethylation. The steady-state of such dynamic heterogeneity reflects a balance between de novo methylation dependent on both DNMT3A and DNMT3B and passive demethylation during rapid cell proliferation.

MED1 has been shown to be dynamically involved in increasing SE concentration for transcription of key cell identity genes. The authors wondered if different allelic methylation states influence the association of MED1 condensates with the SE Mir290. As the RGM reporter allowed isolation of cells with DNA methylation states in allele-specific SEs, they were able to demonstrate that dynamic changes in DNA methylation in SEs are closely related to promoter levels of the promoter- enhancers H3K27ac. This is due to the interruption of enhancers-promoter interactions consistent with the condensates of the Mediator complex which show a reduced association to the methylated SE Mir290.

By removing DNA methylation at the Mir290 SE through Dnmt1/Uhrf1 deletion, they showed that changes in SE DNA methylation is a dynamic process actively regulating its transcriptional activity. While Sox2 and Mir290 SE methylation affect target gene expression similarly, they detected some differences on cellular growth and differentiation. Cells with biallelically methylated Sox2 SE revealed impaired growth and upregulation of differentiation-related pathways. In contrast, Mir290 SE methylation had little effects on cell state. They identified additional differences of how DNA methylation suppresses activity of the two SEs. Mir290-295 expression was independently suppressed by methylation at either Mir290 SE DMR allele consistent with the observation that individual DMR constituents have independent activities [4]. In contrast, monoallelic Sox2 SE methylation did not significantly affect the overall Sox2 expression, suggesting additional regulatory mechanisms.

Finally, the authors wondered if DNA methylation may be present in pre-implantation embryos. To study the changes in DNA methylation of the two SEs in the single cell stage and allelic level, the authors generated homozygous transgenic mice for the 129SE-RGM-tdTomato allele or for the CASTSE-RGM-eGFP allele and thus obtained embryos of 2-4 cells presenting a 129SE-RGM-tdTomato allele and a CASTSE-RGM-eGFP allele by crossing homozygous animals for RGM-eGFP or RGM-tdTomato.

The two SEs present heterogeneity of methylation of allelic DNA at different times: the reporter’s activity became evident already in the 4-cell stage for the SE Mir290 while for the SE Sox2 only at the level of the morula. At the blastocyst stage, the expression of Sox2 was limited to the internal cell mass (ICM), while Mir290-295 showed wide expression in both ICM and trophectoderma (TE) [5] [6] [7]. The data therefore indicate that dynamic DNA methylation exists in active SEs in early pre-implantation embryos creating locus-specific epigenetic heterogeneity, recapitulating and extending our ESC observations in vitro.

This study provides a path towards the mechanistic understanding of dynamic T-DMR regulation in heterogeneous tissues and complex biological processes, such as development and diseases [8][9].

References

  1. Song et al. Dynamic enhancer DNA methylation as basis for transcriptional and cellular heterogeneity of ESCs. Molecular Cell, https://doi.org/10.1016/j.molcel.2019.06.045.
  2. Stelzer, Y., Shivalila, C.S., Soldner, F., Markoulaki, S., and Jaenisch, R. (2015). Tracing dynamic changes of DNA methylation at single-cell resolution. Cell 163, 218–229.
  3. Stelzer, Y., Wu, H., Song, Y., Shivalila, C.S., Markoulaki, S., and Jaenisch, R. (2016). Parent-of Origin DNA Methylation Dynamics during Mouse Development. Cell Rep. 16, 3167–3180.
  4. Suzuki, H.I., Young, R.A., and Sharp, P.A. (2017). Super-Enhancer-Mediated RNA Processing Revealed by Integrative MicroRNA Network Analysis. Cell 168, 1000–1014.
  5. Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487–492.
  6. Paikari, A., D Belair, C., Saw, D., and Blelloch, R. (2017). The eutheria-specific miR-290 cluster modulates placental growth and maternal-fetal transport. Development 144, 3731–3743.
  7. Wicklow, E., Blij, S., Frum, T., Hirate, Y., Lang, R.A., Sasaki, H., and Ralston, A. (2014). HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLoS Genet. 10, e1004618.
  8. Heyn, H., and Esteller, M. (2012). DNA methylation profiling in the clinic: applications and challenges. Nat. Rev. Genet. 13, 679–692.
  9. Robertson, K.D. (2005). DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610.

Simone D'Aietti

Master Industrial Biotechnology student