DNA methylation regulates the expression of the negative transcriptional regulators ID2 and ID4 during OPC differentiation

The differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is the prerequisite for remyelination in demyelinated disorders such as multiple sclerosis (MS). Epigenetic mechanisms, such as DNA methylation, have been suggested to control the intricate network of transcription factors involved in OPC differentiation. Yet, the exact mechanism remains undisclosed. Here, we are the first to identify the DNA-binding protein inhibitors, Id2 and Id4, as targets of DNA methylation during OPC differentiation. Using state-of-the-art epigenetic editing via CRISPR/dCas9-DNMT3a, we confirm that targeted methylation of Id2/Id4 drives OPC differentiation. Moreover, we show that in the pathological context of MS, methylation and gene expression levels of both ID2 and ID4 are altered compared to control human brain samples. We conclude that DNA methylation is crucial to suppress ID2 and ID4 during OPC differentiation, a process that appears to be dysregulated during MS. Our data do not only reveal new insights into oligodendrocyte biology, but could also lead to a better understanding of CNS myelin disorders.

Durable CRISPR-Based Epigenetic Silencing

Development of CRISPR-based epigenome editing tools is important for the study and engineering of biological behavior. Here, we describe the design of a reporter system for quantifying the ability of CRISPR epigenome editors to produce a stable gene repression. We characterize the dynamics of durable gene silencing and reactivation, as well as the induced epigenetic changes of this system. We report the creation of single-protein CRISPR constructs bearing combinations of three epigenetic editing domains, termed KAL, that can stably repress the gene expression. This system should allow for the development of novel epigenome editing tools which will be useful in a wide array of biological research and engineering applications.

Epigenetic Inheritance is Gated by Naive Pluripotency and Dppa2

Environmental factors can trigger cellular responses that propagate across mitosis or even generations. Perturbations to the epigenome could underpin such acquired changes, however, the extent and contexts in which modified chromatin states confer heritable memory in mammals is unclear. Here we exploit a modular epigenetic editing strategy to establish de novo heterochromatin domains (epialleles) at endogenous loci and track their inheritance in a developmental model. We find that naive pluripotent phases systematically erase ectopic domains of heterochromatin via active mechanisms, which acts as an intergenerational safeguard against transmission of epialleles. Upon lineage specification however, acquired chromatin states can be probabilistically inherited under selectively favourable conditions, including propagation of p53 silencing through in vivo development. Using genome-wide CRISPR screening, we identify the mechanisms that block heritable silencing memory in pluripotent cells, and demonstrate removal of Dppa2 unlocks the potential for epigenetic inheritance uncoupled from DNA sequence. Our study outlines a mechanistic basis for how epigenetic inheritance is restricted in mammals, and reveals genomic- and developmental- contexts in which heritable memory is feasible.