Editing DNA Methylation in the Mammalian Genome

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.National Institutes of Health (U.S.) (Grant HD045022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant HG002668)National Institutes of Health (U.S.) (Grant GM114864

Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions

More than a thousand proteins are thought to contribute to mammalian chromatin and its regulation, but our understanding of the genomic occupancy and function of most of these proteins is limited. Here we describe an approach, which we call “chromatin proteomic profiling,” to identify proteins associated with genomic regions marked by specifically modified histones. We used ChIP-MS to identify proteins associated with genomic regions marked by histones modified at specific lysine residues, including H3K27ac, H3K4me3, H3K79me2, H3K36me3, H3K9me3, and H4K20me3, in ES cells. We identified 332 known and 114 novel proteins associated with these histone-marked genomic segments. Many of the novel candidates have been implicated in various diseases, and their chromatin association may provide clues to disease mechanisms. More than 100 histone modifications have been described, so similar chromatin proteomic profiling studies should prove to be valuable for identifying many additional chromatin-associated proteins in a broad spectrum of cell types.National Institutes of Health (U.S.) (Grant HG002668)National Institutes of Health (U.S.) (Grant HG006046)National Institutes of Health (U.S.) (Grant HD045022

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

TALEN-mediated editing of the mouse Y chromosome

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes—Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.National Institutes of Health (U.S.) (US NIH grant R01-HG000257)National Institutes of Health (U.S.) (US NIH grant R01-CA084198)National Institutes of Health (U.S.) (US NIH grant R37-HD045022)Croucher Foundation (Scholarship)Howard Hughes Medical Institute (Investigator

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.National Institutes of Health (U.S.) (Grant RO1-GM34277)National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant PO1-CA42063)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Director's Pioneer Award 1DP1-MH100706)Damon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramSimons Foundatio

Differential clearance rates of microbial phylotypes by four appendicularian species

Appendicularians are abundant planktonic filter feeders that play a significant role in the pelagic food web due to their high clearance rates. Their diet and feeding rates have typically been measured as bulk chlorophyll or cell removal, with some attention given to prey size but no differentiation between the microbial phylotypes. Using a combination ofin situand laboratory incubations with flow cytometry and next-generation sequencing, we found species-specific differences in clearance rates and diet compositions of 4 common species:Oikopleura albicans,O. fusiformis,O. longicauda, andO. dioica. WhileO. albicansmost efficiently removed nano-eukaryotic algae, the other smaller species preferentially removed micron-sized pico-eukaryotic algae. Pico- and nano-eukaryotic cells constituted the major food source of the studied appendicularians despite their occurrence in oligotrophic water dominated by prokaryotic cells. Across species, pico- and nano-planktonic microalgae biomass comprised 45 to 75% of the appendicularian diets. Although non-photosynthetic bacteria were removed at lower rates than all other prey groups, their total contribution to the appendicularian diet was not trivial, representing 5 to 19% of the planktonic carbon in the appendicularian diet; pico-cyanobacteria contributed an additional 9 to 18%. Removal rates and efficiencies of pico-eukaryotes were higher than those of prokaryotes of similar size. Strikingly different clearance rates were observed for different prokaryotic phylotypes, indicating that factors other than size are involved in determining the capturability of the cells. Collectively, our findings provide additional evidence for differential retention of microbial prey among mucous-mesh grazers and its substantial effect on the upper-ocean microbial community.</jats:p

Ventricular arrhythmias after atrial fibrillation electrical cardioversion: A multicenter study

Background: Ventricular arrhythmias (VAs) after atrial fibrillation (AF) electrical cardioversion (ECV) have been reported. Objective: We sought to assess incidence, timing, and clinical characteristics of patients with post-AF ECV-related VAs. Methods: Multicenter observational retrospective study including 13 centers, incorporating patients with VAs or sudden cardiac death within 10 days of ECV. The total number of ECVs performed during the collecting period was provided. Patients with pre-ECV VAs were excluded. Results: Twenty-three patients with VAs were identified out of 11,897 AF ECVs performed in 13 centers during a median 2-year period, suggesting post-ECV VA incidence of 0.2%. The patients’ mean age was 71 ± 11 years, and 13 (56.5%) were female. AF duration prior to ECV was 71 ± 54 days. Congestive heart failure and hypertension were both found in 17 (74%) patients. QT-prolonging drugs were used by 17 (74%). Index VA occurred 28.5 (interquartile range 5.5–72) hours post-ECV, including torsades de pointes, nonsustained polymorphic ventricular tachycardia, and sudden cardiac death in 17 (74%), 5 (22%), and 1 (4%) patient, respectively. Post-ECV heart rate was slower and QT duration longer compared with pre-ECV (57 ± 11 beats/min vs 113 ± 270 beats/min; P &lt; .001; QT duration 482 ± 61 ms vs 390 ± 60 ms; P &lt; .001). VAs reoccurred in 9 (39%) patients, 11 (interquartile range 3–13.5) hours post–index VA. Two patients had an arrhythmic death within 72 hours post-ECV. Conclusion: VAs post-AF ECV are rare, occur within 3 to 72 hours post-ECV, and are potentially fatal. Our study gives a signal of caution favoring prolonged monitoring in small subset of patients as congestive heart failure patients treated with class III antiarrhythmic drugs, with post-ECV bradycardia, especially (but not exclusively) when QT prolongation noted

Securing wider EU commitment to the elimination of hepatitis C virus

In 2016, the Hepatitis B and C Public Policy Association (HepBCPPA), gathered all the main stakeholders in the field of hepatitis C virus (HCV) to launch the now landmark HCV Elimination Manifesto, calling for the elimination of HCV in the EU by 2030. Since then, many European countries have made progress towards HCV elimination. Multiple programs - from the municipality level to the EU level - were launched, resulting in an overall decrease of viremic HCV infections and liver-related mortality. However, as of 2021, most countries are not on track to reach the 2030 HCV elimination targets set by the WHO. Moreover, the COVID-19 pandemic has resulted in a decrease in HCV diagnoses and fewer direct acting antiviral treatment initiations in 2020. Diagnostic and therapeutic tools to easily diagnose and treat chronic HCV infection are now well established. Treating all patients with chronic HCV infection is more cost-saving than treating and caring for patients with liver-related complications, decompensated cirrhosis or hepatocellular carcinoma. It is more important than ever to reinforce and scale-up action towards HCV elimination. Yet, efforts urgently need the dedicated commitment of policymakers at all governmental and policy levels. Therefore, the 3rd EU Policy Summit, held in March 2021, featured EU parliamentarians and other key decision makers to promote dialogue and take strides towards securing wider EU commitment to advance and achieve HCV elimination by 2030. We have summarized the key action points and report the 'Call-to-Action' statement supported by all the major relevant European associations in the field

Securing wider EU commitment to the elimination of hepatitis C virus

In 2016, the Hepatitis B and C Public Policy Association (HepBCPPA), gathered all the main stakeholders in the field of hepatitis C virus (HCV) to launch the now landmark HCV Elimination Manifesto, calling for the elimination of HCV in the EU by 2030. Since then, many European countries have made progress towards HCV elimination. Multiple programmes—from the municipality level to the EU level—were launched, resulting in an overall decrease in viremic HCV infections and liver-related mortality. However, as of 2021, most countries are not on track to reach the 2030 HCV elimination targets set by the WHO. Moreover, the COVID-19 pandemic has resulted in a decrease in HCV diagnoses and fewer direct-acting antiviral treatment initiations in 2020. Diagnostic and therapeutic tools to easily diagnose and treat chronic HCV infection are now well established. Treating all patients with chronic HCV infection is more cost-saving than treating and caring for patients with liver-related complications, decompensated cirrhosis or hepatocellular carcinoma. It is more important than ever to reinforce and scale-up action towards HCV elimination. Yet, efforts urgently need the dedicated commitment of policymakers at all governmental and policy levels. Therefore, the third EU Policy Summit, held in March 2021, featured EU parliamentarians and other key decision makers to promote dialogue and take strides towards securing wider EU commitment to advance and achieve HCV elimination by 2030. We have summarized the key action points and reported the ‘Call-to-Action’ statement supported by all the major relevant European associations in the field.info:eu-repo/semantics/publishedVersio