Journal: Nature Communications

Article Title: MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia

doi: 10.1038/s41467-020-15733-8

Figure Lengend Snippet: a RT-PCR demonstrating the predicted increase in intron retention at alternative splice sites in DOT1L and SETD1A transcripts in MOLM13 cells after MBNL1 knockdown. For DOT1L , Actin serves as a loading control, and primer product (“Intron Inclusion Band”) only occurs when the intron is retained. Representative gel images shown, two biological replicates performed. b qRT-PCR results from RNA-immunoprecipitation assay assessing for enrichment of DOT1L and SETD1A transcripts in MBNL1-precipitated RNA. Error bars show mean ± SD. Data is from three biological replicates for MOLM-13 and two biological replicates for RS4;11. c Western blots of MOLM13 and MV4;11 cells following knockdown of MBNL1, demonstrating changes in DOT1L and SETD1A protein levels. Representative western blots shown, two biological replicates performed. SETD1A image represents same samples run and processed in parallel on separate blot. d Volcano plots representing differentially expressed genes in MOLM13 cells at 4 days (left) and 12 days (right) after knockdown. X -axis thresholds represent 1.5-fold change in expression; Y -axis threshold represents p < 0.01. Differentially expressed genes determined by empirical Bayes two-tailed moderated t -test. e Differentially expressed genes were significantly enriched positively and negatively by GSEA for corresponding gene sets related to perturbation of essential MLL leukemogenesis processes (i.e., HOXA9, upper GSEA plots; GSK3, lower plots.) Enrichment scores, p values, and false-discovery rate q values calculated as previously published .

Article Snippet: After RNA-immunoprecipitation, RNA was extracted and analyzed via qRT-PCR through the process described in the “RT- and Quantitative RT-PCR” section, with the exception that the qRT-PCR was run using Taqman Probes (Thermo Fisher Scientific, Hs_00986924 for SETD1A and Hs_05017433 for DOT1L ) instead of SYBR Green.

Techniques: Reverse Transcription Polymerase Chain Reaction, Knockdown, Control, Quantitative RT-PCR, RNA Immunoprecipitation, Western Blot, Expressing, Two Tailed Test

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Permutation tests (n=20,000, randomization method: circularRandomizeRegions) indicate there is significant overlaps between conserved and chromatin accessible Setd1a ChIP-Seq peaks (P = 0.0055, Z-score = 2.9) and established SCZ-GWAS loci identified. X-axis is the number of overlaps, Y-axis is the density of expected number of overlaps determined by permutation. EVperm, Expected average number of overlaps by permutation, EVobs, actual observed number of overlaps. Permutation P value threshold = 0.05.

Article Snippet: We transfected 5ng of pRL Renilla vector, 50 ng of 3X Mef2-Luciferase vector (Addgene #32967), 75 ng of Mef2c CDS (Addgene #32515) and/or Setd1a CDS (Origene #MR215352), Lsd1 CDS (Origene #MR210741), pcDNA3.1 vector.

Techniques: ChIP-sequencing

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Representative ChIP-Seq locus (Kmt2a gene). Normalized reads per genomic coverage from PFC ChIP-Seq for Setd1a (black) and chromatin marks (H3K4me1, red; H3K4me2, orange; H3K4me3, blue; H3K27ac, green) are shown. Black boxes below the Setd1a track depict significant peaks that passed all quality checks (see Methods).

Article Snippet: We transfected 5ng of pRL Renilla vector, 50 ng of 3X Mef2-Luciferase vector (Addgene #32967), 75 ng of Mef2c CDS (Addgene #32515) and/or Setd1a CDS (Origene #MR215352), Lsd1 CDS (Origene #MR210741), pcDNA3.1 vector.

Techniques: ChIP-sequencing

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A-B) Volcano plots of differentially expressed genes in whole-PFC RNA-Seq of Setd1a+/− mice are depicted in (A). 342 differentially expressed genes (FDR<5%) are shown in red, while non-significant changes are shown in grey (left panel). Of the 342 differentially expressed genes, 271 are Setd1a targets (orange and blue, right panel). We partitioned these genes in 4 categories based on the binding regions of Setd1a in the PFC. We defined ‘Setd1a bound promoters up-regulated genes’ and ‘Setd1a bound promoters down-regulated genes’ as genes in which promoters are bound by Setd1a. We defined ‘Setd1a bound enhancers up-regulated genes’ and ‘Setd1a bound enhancers down-regulated genes’ as genes in which enhancers, but not promoters, are bound by Setd1a. Genes with Setd1a binding at both promoters and enhancers, were partitioned into the “Setd1a-promoter bound” classes. A summary of the number of genes in each category is shown in (B).

Article Snippet: We transfected 5ng of pRL Renilla vector, 50 ng of 3X Mef2-Luciferase vector (Addgene #32967), 75 ng of Mef2c CDS (Addgene #32515) and/or Setd1a CDS (Origene #MR215352), Lsd1 CDS (Origene #MR210741), pcDNA3.1 vector.

Techniques: RNA Sequencing Assay, Binding Assay

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Restoration of knockout-first allele after TAM treatment. For the rescue experiments, Setd1a+/− mice were crossed to R26FlpoER mice. FLP (Flpo) induced globally by TAM converts the “knockout-first” allele to a conditional allele, restoring gene activity.

Article Snippet: We transfected 5ng of pRL Renilla vector, 50 ng of 3X Mef2-Luciferase vector (Addgene #32967), 75 ng of Mef2c CDS (Addgene #32515) and/or Setd1a CDS (Origene #MR215352), Lsd1 CDS (Origene #MR210741), pcDNA3.1 vector.

Techniques: Knock-Out, Activity Assay

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Neurod6 locus. ChIP-Seq of LSD1 (grey), Setd1a (black), Mef2 (dark blue), H3K27ac (green), H3K4me3 (light blue) is shown. Grey, black and dark blue boxes below Lsd1, Setd1a and Mef2 tracks respectively represent significant peaks that passed all quality checks (see methods).

Article Snippet: We transfected 5ng of pRL Renilla vector, 50 ng of 3X Mef2-Luciferase vector (Addgene #32967), 75 ng of Mef2c CDS (Addgene #32515) and/or Setd1a CDS (Origene #MR215352), Lsd1 CDS (Origene #MR210741), pcDNA3.1 vector.

Techniques: ChIP-sequencing

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: We transfected 5ng of pRL Renilla vector, 50 ng of 3X Mef2-Luciferase vector (Addgene #32967), 75 ng of Mef2c CDS (Addgene #32515) and/or Setd1a CDS (Origene #MR215352), Lsd1 CDS (Origene #MR210741), pcDNA3.1 vector.

Techniques: Recombinant, Luciferase, Plasmid Preparation, Software