Epigenome

Cancer is the second leading cause of death worldwide and disease burden is expected to increase globally throughout the next several decades, with the majority of cancer-related deaths occurring in metastatic disease. Cancers exhibit known hallmarks that endow them with increased survival and proliferative capacities, frequently as a result of de-stabilizing mutations. However, the genomic features that resolve metastatic clones from primary tumors are not yet well-characterized, as no mutational landscape has been identified as predictive of metastasis. Further, many cancers exhibit no known mutation signature. This suggests a larger role for non-mutational genome re-organization in promoting cancer evolution and dissemination. In this review, we highlight current critical needs for understanding cell state transitions and clonal selection advantages for metastatic cancer cells. We examine links between epigenetic states, genome structure, and misregulation of tumor suppressors and oncogenes, and discuss how recent technologies for understanding domain-scale regulation have been leveraged for a more complete picture of oncogenic and metastatic potential.

Permanent epigenetic silencing using programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases1-3. However, to unlock its full therapeutic potential, an experimental confirmation of durable epigenetic silencing after the delivery of transient delivery of editors in vivo is needed. To this end, here we targeted Pcsk9, a gene expressed in hepatocytes that is involved in cholesterol homeostasis. In vitro screening of different editor designs indicated that zinc-finger proteins were the best-performing DNA-binding platform for efficient silencing of mouse Pcsk9. A single administration of lipid nanoparticles loaded with the editors' mRNAs almost halved the circulating levels of PCSK9 for nearly one year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks also persisted after forced liver regeneration, further corroborating the heritability of the newly installed epigenetic state. Improvements in construct design resulted in the development of an all-in-one configuration that we term evolved engineered transcriptional repressor (EvoETR). This design, which is characterized by a high specificity profile, further reduced the circulating levels of PCSK9 in mice with an efficiency comparable with that obtained through conventional gene editing, but without causing DNA breaks. Our study lays the foundation for the development of in vivo therapeutics that are based on epigenetic silencing.

Although more studies are demonstrating that a father's environment can influence child health and disease, the molecular mechanisms underlying non-genetic inheritance remain unclear. It was previously thought that sperm exclusively contributed its genome to the egg. More recently, association studies have shown that various environmental exposures including poor diet, toxicants, and stress, perturbed epigenetic marks in sperm at important reproductive and developmental loci that were associated with offspring phenotypes. The molecular and cellular routes that underlie how epigenetic marks are transmitted at fertilization, to resist epigenetic reprogramming in the embryo, and drive phenotypic changes are only now beginning to be unraveled. Here, we provide an overview of the state of the field of intergenerational paternal epigenetic inheritance in mammals and present new insights into the relationship between embryo development and the three pillars of epigenetic inheritance: chromatin, DNA methylation, and non-coding RNAs. We evaluate compelling evidence of sperm-mediated transmission and retention of paternal epigenetic marks in the embryo. Using landmark examples, we discuss how sperm-inherited regions may escape reprogramming to impact development via mechanisms that implicate transcription factors, chromatin organization, and transposable elements. Finally, we link paternally transmitted epigenetic marks to functional changes in the pre- and post-implantation embryo. Understanding how sperm-inherited epigenetic factors influence embryo development will permit a greater understanding related to the developmental origins of health and disease.

Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is difficult to engineer variants with enhanced activity while maintaining specificity. Here we engineer the compact Obligate Mobile Element Guided Activity (OMEGA) RNA-guided endonuclease IscB and its guiding RNA (ωRNA) by combining ortholog screening, structure-guided protein domain design and RNA engineering, and deep learning-based structure prediction to generate an improved variant, NovaIscB. We show that the compact NovaIscB achieves up to 40% indel activity (~100-fold improvement over wild-type OgeuIscB) on the human genome with improved specificity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAoff, that is compact enough to be packaged in a single adeno-associated virus vector for persistent in vivo gene repression. This study highlights the power of combining natural diversity with protein engineering to design enhanced enzymes for molecular biology applications.