Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.

Aging is associated with an abnormal increase in DNA methylation (DNAm) in human gene promoters, including in bone marrow stem cells. DNAm patterns are further perturbed in hematological malignancies such as acute myeloid leukemia but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNAm spreading beyond the gRNA location. We find that despite a transient delivery system, DNAm is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNAm is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33 ⁺ ) and lymphoid (CD19 ⁺ ) cells is unaffected, monocyte (CD14 ⁺ ) populations decreased and granulocytes (CD66b ⁺ ) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNAm can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNAm on the p15 locus. As such, other aging-associated aberrant DNAm may impact hematopoiesis in vivo.

Consolidation of contextual memories after a stressful encounter is essential for the survival of an organism and in allowing a more appropriate response to be elicited should the perceived threat reoccur. Recent evidence has explored the complex role that epigenetic mechanisms play in the formation of such memories, and the underlying signaling pathways are becoming more apparent. The glucocorticoid receptor (GR) has been shown to play a key role in these events having both genomic and non-genomic actions in the brain. GR has been shown to interact with the extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) signaling pathway which, in concert, drives epigenetic modifications and chromatin remodeling, resulting in gene induction and memory consolidation. Evidence indicates that stressful events can have an effect on the offspring in utero, and that epigenetic marks altered early in life may persist into adulthood. A new and controversial area of research, however, suggests that epigenetic modifications could be inherited through the germline, a concept known as transgenerational epigenetics. This review explores the role that epigenetic processes play in the central nervous system, specifically in the consolidation of stress-induced memories, the concept of transgenerational epigenetic inheritance, and the potential role of epigenetics in revolutionizing the treatment of stress-related disorders through the emerging field of pharmacoepigenetics and personalized medical treatment.

The desire to analyse limited amounts of biological material, historic samples and rare cell populations has collectively driven the need for efficient methods for whole genome sequencing (WGS) of limited amounts of poor quality DNA. Most protocols are designed to recover double-stranded DNA (dsDNA) by ligating sequencing adaptors to dsDNA with or without subsequent polymerase chain reaction amplification of the library. While this is sufficient for many applications, limited DNA requires a method that can recover both single-stranded DNA (ssDNA) and dsDNA. Here, we present a WGS library preparation method, called ‘degraded DNA adaptor tagging’ (DDAT), adapted from a protocol designed for whole genome bisulfite sequencing. This method uses two rounds of random primer extension to recover both ssDNA and dsDNA. We show that by using DDAT we can generate WGS data from formalin-fixed paraffin-embedded (FFPE) samples using as little as 2 ng of highly degraded DNA input. Furthermore, DDAT WGS data quality was higher for all FFPE samples tested compared to data produced using a standard WGS library preparation method. Therefore, the DDAT method has potential to unlock WGS data from DNA previously considered impossible to sequence, broadening opportunities to understand the role of genetics in health and disease.