Here at EpiGenie we like to think of ourselves as reporters of genomic methylation, but two new studies from the lab of Rudolf Jaenisch at MIT give us a run for our money and demonstrate the utility of their reporter of genomic methylation (RGM). Using RGM, the talented group brings forth a deeper understanding of the heterogeneity of genomic imprinting and also new insight into manipulating DNA methylation by epigenome editing.
RGM utilizes the minimal promoter from the imprinted Snrpn gene. This promoter not only drives methylation-sensitive expression, but it also takes on the methylation profile of any regulatory sequence that lies next to it. In RGM, the Snrpn promoter is coupled to green fluorescent protein (GFP) and produces fluorescence only when not methylated. Therefore, with some clever CRISPR/Cas9 mediated insertion, RGM can be placed next to a regulatory element of interest and give a fluorescent readout of its DNA methylation profile.
Cellular Dynamics of Genomic Imprinting
Stelzer et al. made use of the cellular resolution of their RGM system to understand the parent-of-origin specific dynamics of genomic imprinting during mouse development and in adulthood. They targeted RGM to the intergenic differentially methylated region (IG-DMR) of the Dlk1–Dio3 locus, which is involved in growth and neurodevelopment. At the IG-DMR, the paternal allele is hypermethylated, while the maternal allele is hypomethylated.
In this approach to study imprinting, they crossed two mouse strains, which allowed for allele specific insertion, via CRISPR/Cas9, into the mouse embryonic stem cells (mESCs) of the hybrid offspring. To enable allele specific readout, they used two different constructs containing one of two fluorescent proteins: tdTomato (Tom) produces red fluorescence and was inserted next to the maternal allele, while GFP was inserted alongside the paternal allele.
As expected, bisulfite sequencing revealed that DNA methylation from the paternal IG-DMR spread to the Snrpn promoter and silenced GFP, while the lack of DNA methylation at the maternal IG-DMR allowed the Snrpn promoter to drive the expression of Tom.
Now, with that complex construct validated, the insights shined through. Here’s what they found:
- There are some atypical profiles of bialleic hypo- or hypermethylation, which increase with cell passage in mESCs.
- In newly derived mESCs, there is a sex-specific effect where female, but not male, cells exhibit rapid demethylation of the paternal IG-DMR.
- The methylation status of the RGM system correlates with the expected natural parent-of-origin specific gene expression.
- In order to assess developmental potency, they created chimeric embryos that incorporated cells with funky fluorescence patterns, which indicated bialleic hypo- or hypermethylation at the lG-DMR. They observed embryonic death and birth defects in these chimeras, as well as differences in how the cells were incorporated in the brain.
Switching back to their original RGM approach, they targeted the Snrpn-GFP RGM construct to the maternal IG-DMR to investigate tissue and cell-type specific differences that occur during development. They discovered that:
- While the embryos examined all expressed GFP, a closer look showed this was not true in all tissues, revealing that the maternal IG-DMR was hypermethylated in some tissues.
- The tissues that repressed the expression of GFP in embryos were the exact same ones that repressed it in adulthood.
- In the adult brain, methylation of the maternal IG-DMR had cell-type specific heterogeneity.
Overall, the study provides novel insights into the dynamics of imprinting across development by profiling the heterogeneity at a locus critical for growth and neurodevelopment.
In Vitro and In Vivo DNA Methylation and Demethylation with dCas9
In a second study, by Liu et al. and done in collaboration with the lab of Richard Young, RGM enabled validation of effectors from the deactivated Cas9 (dCas9) epigenome editing toolbox, specifically those that edit DNA methylation. By fusing dCas9 with the catalytic domains of a methylation writer (dCas9-Dnmt3a) or eraser (dCas9-Tet1), the precision targeting of sgRNA can be taken advantage of and enable designer epigenetic modification.
The team made use RGM to validate successful epigenetic editing in mESCs, where they inserted their construct at the desired location and used fluorescence readouts alongside bisulfite sequencing:
- First, they inserted Snrpn-GFP into Dazl, a germline specific gene that isn’t active in mESCs, making it a perfect target for the DNA demethylation powers of dCas9-Tet1, which activated GFP expression.
- Reciprocally, they added Snrpn-GFP to Gapdh, a gene widely expressed in mESCs and an ideal site for some de novo DNA methylation by dCas9-Dnmt3a, which silenced GFP expression.
The team then targeted endogenous sequences in vitro. They confirmed gene expression alterations with qPCR and differential methylation with bisulfite sequencing. Here’s what went down:
- Given that neuronal activity induces the expression of brain-derived neurotrophic factor (BDNF) and demethylation of its promoter IV, they utilized dCas9-Tet1 in post-mitotic mouse cortical neurons to recreate the natural biological effect.
- MyoD is a master regulator of muscle development, and targeting its enhancer with dCas9-TET1 in fibroblasts improved their conversion to myoblasts.
- The team then played with chromatin looping by targeting CTCF binding sites with dCas9-Dnmt3a in mESCs, interfering with DNA looping and altering the expression of genes at a neighboring loop.
Finally, they went in vivo with mice and closed the loop on the two papers by inserting the Snrpn-GFP cassette into the paternal allele of the IG-DMR belonging to Dlk1-Dio3. By targeting the locus with dCas9-Tet1, they were able to activate GFP expression in skin and the brain. Overall, this paper reveals how editing DNA methylation can be used for stem cell programming and for gaining a functional understanding of regulatory sequence.
Taken together, these two papers demonstrate the utility of the RGM system for studies examining methylation at the single-cell level across development, while furthering our understanding of the heterogeneity of genomic imprinting and validating the powers of epigenome editing.