Genome Mining Reveals High Topological Diversity of ω-Ester-Containing Peptides and Divergent Evolution of ATP-Grasp Macrocyclases.

ω-Ester-containing peptides (OEPs) are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) containing intramolecular ω-ester or ω-amide bonds. Although their distinct side-to-side connections may create considerable topological diversity of multicyclic peptides, it is largely unknown how diverse ring patterns have been developed in nature. Here, using genome mining of biosynthetic enzymes of OEPs, we identified genes encoding nine new groups of putative OEPs with novel core consensus sequences, disclosing a total of ∼1500 candidate OEPs in 12 groups. Connectivity analysis revealed that OEPs from three different groups contain novel tricyclic structures, one of which has a distinct biosynthetic pathway where a single ATP-grasp enzyme produces both ω-ester and ω-amide linkages. Analysis of the enzyme cross-reactivity showed that, while enzymes are promiscuous to nonconserved regions of the core peptide, they have high specificity to the cognate core consensus sequence, suggesting that the enzyme-core pair has coevolved to create a unique ring topology within the same group and has sufficiently diversified across different groups. Collectively, our results demonstrate that the diverse ring topologies, in addition to diverse sequences, have been developed in nature with multiple ω-ester or ω-amide linkages in the OEP family of RiPPs.

A Topologically Distinct Modified Peptide with Multiple Bicyclic Core Motifs Expands the Diversity of Microviridin-Like Peptides.

Microviridins are ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain multiple intramolecular ω-ester or ω-amide crosslinks between two side chains in peptides. This type of the side-to-side macrocyclization may generate diverse structures with distinct topology and ring sizes, but the majority of the microviridin-like RiPPs present only a single consensus sequence with a tricyclic architecture. Here, we expanded the natural diversity of the microviridin-like modified peptides by determining the crosslinking connectivity of a new modified peptide, mTgnA and its homologous RiPPs, which we named the thuringinin group. Members of the thuringinin group have core motifs with a distinct consensus sequence, which is transformed to a novel hairpin-like bicyclic structure by the cognate ATP-grasp enzyme. We suggest that the microviridin-like RiPPs naturally have novel sequences and architectures beyond those found in microviridins and comprise a larger RiPP family, termed omega-ester containing peptides (OEPs).

Efficient prime editing in mouse brain, liver and heart with dual AAVs.

Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer's disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component.

Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery.

The genome can be divided into two spatially segregated compartments, A and B, which partition active and inactive chromatin states. While constitutive heterochromatin is predominantly located within the B compartment near the nuclear lamina, facultative heterochromatin marked by H3K27me3 spans both compartments. How epigenetic modifications, compartmentalization, and lamina association collectively maintain heterochromatin architecture remains unclear. Here we develop Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) to jointly measure chromosome conformation, DNA methylation, and lamina positioning. Through LIMe-Hi-C, we identify topologically distinct sub-compartments with high levels of H3K27me3 and differing degrees of lamina association. Inhibition of Polycomb repressive complex 2 (PRC2) reveals that H3K27me3 is essential for sub-compartment segregation. Unexpectedly, PRC2 inhibition promotes lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with this repositioning, genes originally marked with H3K27me3 in the B compartment, but not the A compartment, remain largely repressed, suggesting that constitutive heterochromatin spreading can compensate for H3K27me3 loss at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with lamina association are fundamental features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how compartmentalization and lamina association represent distinct but interdependent modes of heterochromatin regulation.