Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1.

Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.

Scarless Genomic Point Mutation to Construct a Bacillus subtilis Strain Displaying Increased Antibiotic Plipastatin Production.

Bacillus strains produce various types of antibiotics, and random mutagenesis has traditionally been used to overproduce these natural metabolites. However, this method leads to the accumulation of unwanted mutations in the genome. Here, we rationally designed a single nucleotide substitution in the degU gene to generate a B. subtilis strain displaying increased plipastatin production in a foreign DNA-free manner. The mutant strain (BS1028u) showed improved antifungal activity against Pythium ultimum. Notably, pps operon deletion in BS1028u resulted in complete loss of antifungal activity, suggesting that the antifungal activity strongly depends on the expression of the pps operon. Quantitative real-time PCR and lacZ assays showed that the point mutation resulted in 2-fold increased pps operon expression, which caused the increase in antifungal activity. Likewise, commercial Bacillus strains can be improved to display higher antifungal activity by rationally designed simple modifications of their genome, rendering them more efficient biocontrol agents.

Random knock-in expression system for high yield production of heterologous protein in Bacillus subtilis.

Chromosome-integrated recombinant protein expression in bacteria has advantages for the stable maintenance of genes without any use of antibiotics during large-scale fermentation. Even though different levels of gene expression were reported, depending upon their chromosomal position in bacterial species, only a limited number of integration sites have been used in B. subtilis. In this study, we randomly integrated the GFP and AprE expression cassettes into the B. subtilis genome to determine integration sites that can produce a high yield of heterologous protein expression. Our mariner transposon-based expression cassette integration system was able to find integration sites, which can produce up to 2.9-fold and 1.5-fold increased expression of intracellular GFP and extracellular AprE, respectively, compared to the common integration site amyE. By analyzing the location of integration sites, we observed an adjacent promoter effect, gene dosage effect, and gene knock-out effect all complexly contributing to the increased level of integrated gene expression. Besides obtaining a high yield of heterologous protein expression, our system can also provide a wide-range of expression to expand the systematic application for steady-state metabolic protein production.

A Highly Efficient CRISPR-Cas9-Mediated Large Genomic Deletion in Bacillus subtilis.

In Bacillus subtilis, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the eco-friendliness of B. subtilis, it is critical that engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in B. subtilis without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing pps operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered Streptococcus pyogenes-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in spo0A (∼100%), point mutation (∼68%) and GFP gene insertion (∼97%) in sigE and demonstrated its broad applicability for various types of site-directed mutagenesis in B. subtilis.