Reproduction Abstracts

DNA methylation is a broadly studied epigenetic modification that is essential for normal mammalian development. Over the years, numerous methodologies were developed trying to cope with the intrinsic challenge of reading the “second dimension” epigenetic code. The recent rapid expansion of sequencing technologies has made it possible to fully chart the methylation landscape of different cell types at single-base resolution. However, current methods provide only a static “snapshot” of DNA methylation, thus precluding the study of real-time methylation dynamics during cell fate changes. Therefore, a key challenge in the field is to generate tools that allow tracing real-time changes in DNA methylation. We have recently establish a Reporter of Genomic Methylation (RGM) that relies on a synthetic imprinted gene promoter driving a fluorescent protein. We showed that insertion of RGM proximal to promoter-associated CpG islands, or non-coding regulatory elements such as tissue-specific super-enhancer regions, allows faithful reporting on gain and loss of DNA methylation. Importantly, we demonstrated that RGM allows to trace real-time DNA methylation dynamics, at single-cell resolution, during cell fate changes. In placental mammals, differential DNA methylation at imprinting control regions (ICRs) regulates the parent-of-origin monoallelic expression of multiple imprinted genes in clusters. To study allele-specific methylation dynamics associated with ICRs during mouse development, we targeted RGM to the intergenic ICR located at the Dlk1-Dio3 locus. Targeted mouse embryonic stem cells allowed to isolate and expand rare cell population that exhibit aberrant methylation, and study the consequences of loss-of-imprinting on normal development. Furthermore, we show that RGM faithfully reflects parent-of-origin methylation inheritance throughout generations, thus facilitating a systematic analysis of methylation dynamics during embryonic development and adults. Taken together, locus-specific readout of endogenous methylation states holds great promise for mechanistic studies with potential broad implications for the field.