Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: Inducible RNase H1 transgene expression in vitro. A) Schematic of hiPSC differentiation from fibroblasts to NPCs and neurons. Left panel: Immunofluorescent staining of the neuronal marker MAP2 (green) in hiPSC-neurons (top) and the NPC markers SOX2 (green) and Nestin (red) in hiPSC-NPCs (bottom); scale bar: 100 mm. Right panel: Immunofluorescent staining of S9.6 (red) and DAPI (blue) in each respective cell type; scale bar: 100 mm. B) Schematic of the effect of RNase H1-mediated knockdown of R-loops, or D145N mutant that is binding- competent but catalytically inactive. C) Overview of the lentiviral constructs used in this study, including the rtTA transactivator, RH1ΔMLS, RH1ΔMLS D145N catalytically-inactive mutant, and BFP-expressing lentiviral backbone control. D) (Top) Representative Western Blot of hiPSC-neurons expressing either BFP, RH1ΔMLS, or RH1ΔMLS D145N lentiviruses. Gradient triangles indicate concentration of virus. RNase H1 protein is 5.1-fold increased in RH1ΔMLS cells relative to BFP controls (normalized to β-actin loading control). (Bottom) Immunocytochemistry for S9.6 (red) and RNase H1 (green) in hiPSC-neurons expressing BFP, RH1ΔMLS D145N , or RH1ΔMLS transgenes. RNase H1 protein is robustly increased in the cell nucleus (DAPI, blue) in RH1ΔMLS and RH1ΔMLS D145N cells relative to BFP controls, indicating successful nuclear expression of the transgene-encoded protein; scale bars = 20 mm. E) Experimental timeline. NPCs are transduced with RH1ΔMLS or a control (RH1ΔMLS D145N or BFP backbone) lentivirus. After 4 days in vitro , puromycin (1:1,000) is added to the culture media to select for transgene-expressing cells. After 6 days in culture, puromycin concentration is dropped to 1:5,000. On day 0 (indicating transition from NPC to neuron differentiation), cells are replated and fed every 2 days with neuron differentiation medium containing 1:1,000 doxycycline to activate the transgene. Every week from week 2, electrophysiological recordings are captured by multielectrode array (MEA). After 6 weeks in culture, cells are harvested for DRIP-seq, bulk- and scRNA-seq. F) (top to bottom) DRIP-seq tracks for hiPSC-derived neurons transduced with BFP (blue tracks, BFP Neuron cultures 1-3) or RH1ΔMLS (bright red tracks, cultures 1-3) constructs over a well-defined, conserved R-loop hotspot region , demonstrating robustness of our DRIP-seq experimental and computational processing. Note that y-axis scaling 0-90 for BFP Neuron DRIP-seq is wider than RH1 Neuron scale, 0-10. RNA alone processed by the DRIP-seq protocol (RNA-only DRIP, magenta track) shows no discernable peaks, indicating that our S9.6 immunoprecipitation is specific for DNA/RNA hybrids and does not represent single-stranded or double-stranded RNA. Orange track: in silico HindIII/EcoRI/XbaI/ SSPI restriction enzyme digest showing expected cutting sites of the restriction enzyme cocktail used for DRIP-seq. G) Left: Volcano plot displaying the 6,380 differential promoter-bound R-loop peaks (DRIP-seq) between hiPSC-differentiated cultures expressing RH1ΔMLS (n = 3) or BFP (n = 3) (FDR < 0.05, pink dots) after 6 weeks in culture. Of these, 4,805 DRIP-seq peaks were enriched in BFP controls (log 2 fold change > 1) and 474 DRIP-seq peaks were enriched RH1ΔMLS cells (log 2 fold change < -1), indicating successful knockdown of R-loops in the latter. Right: Volcano plot displaying the 1,753 differential promoter-bound R-loop peaks between RH1ΔMLS (n = 3) and RH1ΔMLS D145N (n = 2) (FDR < 0.1, pink dots) cells after 6 weeks in culture. Of these, 1,709 DRIP-seq peaks were enriched in RH1ΔMLS D145N controls (log 2 fold change > 1) and 35 DRIP-seq peaks were enriched in RH1ΔMLS cells (log 2 fold change < -1), again indicating successful knockdown of R-loops in the latter. H) Representative DRIP-seq tracks over two neuronal genes for BFP-, RH1ΔMLS D145N -, and RH1ΔMLS- expressing hiPSC-neurons after 6 weeks in culture. Pink highlighted region and inset displays magnified promoter region.

Article Snippet: The Axion Maestro Multielectrode array system and AXIS software (Axion Biosystems) were used to perform 10-minute extracellular recordings of spontaneous network activity once a week for 8 weeks.

Techniques: Expressing, In Vitro, Staining, Marker, Mutagenesis, Binding Assay, Construct, Western Blot, Concentration Assay, Immunocytochemistry, Transduction, Derivative Assay, Immunoprecipitation, In Silico

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: RNase H1-mediated R-loop loss during neuronal differentiation in vitro results in electrophysiological deficits. A) Top left: Electrophysiological recordings were taken with an Axion multielectrode array (MEA) system to assess population-wide neuronal activity. Schematic of the 48-well MEA plate used for electrophysiological recordings. Bottom Left: Schematic of an individual well within the MEA plate, containing 16 recording electrodes. Right: Screen shot of the MEA recording on week 8, with BFP-expressing hiPSC-neurons plated on the left half of the plate and RNase H1-overexpressing neurons on the right. B) Representative spike raster plots over the 10-minute recording at week 8 for BFP-expressing (left) and RNase H1-overexpressing (right) hiPSC-neurons. Each row denotes a single recording electrode within a single well of the 48-well MEA plate. C-E) The number of spontaneous spikes ( C ), weighted mean firing rate (wMFR) in Hertz ( D ), and number of bursts ( E ) were recorded weekly from BFP-controls and RNase H1 transgene expressing hiPSC-neurons from two donor lines. Data from the two donor lines were combined and a two-way ANOVA was performed with Sidak’s multiple comparisons test to test significant differences in each metric between weeks 2-8. Each condition contained at least 14-20 viable well replicates after removing wells that had less than 10 active recording electrodes. F) Statistics were additionally calculated for each electrophysiological metric at week 8 (unpaired two-tailed student’s t test) between RNase H1-transgene hiPSC-neurons and BFP controls. Individual donors represented with either filled in or hollow shapes.

Article Snippet: The Axion Maestro Multielectrode array system and AXIS software (Axion Biosystems) were used to perform 10-minute extracellular recordings of spontaneous network activity once a week for 8 weeks.

Techniques: In Vitro, Activity Assay, Expressing, Two Tailed Test