Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.

Small RNA pathways are important players in posttranscriptional regulation of gene expression. These pathways play important roles in all aspects of cellular physiology from development to fertility to innate immunity. However, almost nothing is known about the regulation of the central genes in these pathways. The forkhead box O (FOXO) family of transcription factors is a conserved family of DNAbinding proteins that responds to a diverse set of cellular signals. FOXOs are crucial regulators of cellular homeostasis that have a conserved role in modulating organismal aging and fitness. Here, we show that Drosophila FOXO (dFOXO) regulates the expression of core small RNA pathway genes. In addition, we find increased dFOXO activity results in an increase in RNA interference (RNAi) efficacy, establishing a direct link between cellular physiology and RNAi. Consistent with these findings, dFOXO activity is stimulated by viral infection and is required for effective innate immune response to RNA virus infection. Our study reveals an unanticipated connection among dFOXO, stress responses, and the efficacy of small RNA-mediated gene silencing and suggests that organisms can tune their gene silencing in response to environmental and metabolic conditions.O rganisms must be able to respond to changing conditions to survive. Radical changes in environment or metabolism can induce cellular stress signaling pathways meant to help the cell return to a normal range of function. Whereas there are dedicated transcriptional response pathways to deal with specific individual stresses such as heat shock (HSF) (1) or heavy metals (MTF-1) (2), the forkhead box O (FOXO) pathway has emerged as a general stress responsive transcription factor. Although initially characterized as downstream targets of insulin signaling, it is now clear that the FOXOs respond to multiple cellular stress signaling pathways including nutrient deprivation, oxidative stress, mitochondrial dysfunction, DNA damage, bacterial infection, and other cellular stress signals (3, 4).The FOXO family of transcription factors is conserved from Caenorhabditis elegans to humans. Invertebrates have a single FOXO gene: daf-16 in worms and dFOXO in Drosophila (5, 6). In mammals, the four FOXO genes (FOXO1, FOXO3, FOXO4, and FOXO6) have diversified, with some factors having a stressspecific response (7-10). Therefore, invertebrates offer a particularly attractive system for understanding FOXO function because the single isoform is responsible for all of the roles of the four mammalian FOXOs.Small noncoding RNA pathways are capable of regulating gene expression in a sequence-specific manner (11). Effector complexes called RNA-induced silencing complexes (RISC) containing small single-stranded RNAs bound to Argonaute proteins find target mRNAs and posttranscriptionally prevent their expression. Three major small RNA pathways have been identified in animal cells, the microRNA (miRNA) pathway, the small interfering RNA pathway (siRNA), and PIWI RNA (piRNA) pathways, each contain uni...

Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5′ UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5′ UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions.DOI: http://dx.doi.org/10.7554/eLife.00542.001

Background We sought to compare the outcomes of patients treated with intravenous (IV)-only vs. oral transitional antimicrobial therapy for infective endocarditis (IE) after implementing a new Expected Practice within The Los Angeles County Department of Health Services (LAC DHS). Methods We conducted a multi-centered, retrospective cohort study of adults with definite or possible IE treated with IV-only vs. oral therapy at the three acute care, public hospitals in the LAC DHS system between December 2018 to June 2022. The primary outcome was clinical success at 90 days, defined as being alive, and without recurrence of bacteremia or treatment-emergent infectious complications. Results We identified 257 patients with IE treated with IV-only (n=211) or oral transitional (n=46) therapy who met study inclusion criteria. Study arms were similar for many demographics; however, the IV cohort was older, and had more aortic valve involvement, hemodialysis patients, and central venous catheters present. In contrast, the oral cohort had a higher percentage of IE caused by methicillin-resistant S. aureus. There was no significant difference between the groups in clinical success at 90 days or last follow-up. There was no difference in recurrence of bacteremia or readmission rates. However, patients treated with oral therapy had significantly fewer adverse events. Multivariable regression adjustments did not find significant associations between any selected variables and clinical success across treatment groups. Conclusions These results demonstrate similar outcomes of real-world use of oral vs. IV-only therapy for IE, in accord with prior randomized controlled trials and meta-analyses.

No abstract