Targeted epigenome editing of an endogenous locus with chromatin modifiers is not stably maintained

Abstract

BackgroundDNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter.ResultsExpression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment.ConclusionsThe clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0002-z) contains supplementary material, which is available to authorized users.

Generalized nucleation and looping model for epigenetic memory of histone modifications

“…Here, we show that a generalized nucleation-and-looping model explains key experimental findings on engineered H3K9 methylation domains in fission yeast (15,16,18) and epigenome editing experiments in human cells (19,24,25). Several features specifically arise from looping interactions beyond the next nucleosome and are therefore not observed in linear spreading simulations.…”

“…Therefore, intrinsically confined modification domains cannot be stably maintained. For the case of H3K9 methylation, Clr4/Suv39h-mediated feedback is indeed too weak to induce stable memory at engineered domains (15,16,19). How can cells selectively enable stable epigenetic memory at endogenous heterochromatin but exhibit only shortterm memory at engineered domains?…”

“…The enzymes that are responsible for heterochromatic H3K9me2/3 are Clr4 in fission yeast and Suv39h in metazoans, and the PRC2 complex catalyzes H3K27me2/3 (12). By stably tethering these enzymes to chromatin, extended engineered domains enriched for the respective modification can be formed (13)(14)(15)(16)(17)(18)(19)(20). Furthermore, both modifications can confer epigenetic memory at least across several cell divisions (14)(15)(16)(17)21).…”

Genome-wide repressive capacity of promoter DNA methylation is revealed through epigenomic manipulation

“…Although it has not been shown systematically that DNA methylation acts as a primary and causal signal genome-wide, it has been shown to be sufficient for transcriptional repression of individual genes (Razin and Cedar, 1991;Jackson-Grusby et al, 2001;Yang et al, 2012;Bintu et al, 2016;Amabile et al, 2016;Kungulovski et al, 2015), and even necessary for some mechanisms of long term promoter silencing such as X-inactivation and imprinting (Li et al, 1993;Jaenisch and Bird, 2003;Bestor et al, 2015). Transcriptional silencing is also stimulated by a complex network of epigenetic marks, including methylation and deacetylation of certain histone subunits of nucleosomes near the transcription start site (Kungulovski et al, 2015), which are thought to be regulated in part by the recruitment of methylated DNA-binding proteins (Klose and Bird, 2006).…”

Analysis of DNA modifications in aging research

“…Recent reviews provide a good overview of this potential paradigm shift (Voigt and Reinberg 2013;Thakore et al 2016;Stricker et al 2017;Willyard 2017). Briefly, the concept is to use tools like CRISPR (Vojta et al 2016), zinc fingers (Kungulovski et al 2015) or transcription activatorlike effector (TALES) (Maeder et al 2013) to drive fusion proteins with DNMTs or TETs to a specific location or locations in the genome to alter modification status. This offers great promise to move epigenomic patterns to more mechanistic investigations (Stricker et al 2017).…”