DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner

Formation of γH2Ax serves as a checkpoint for double-strand break (DSB) repair pathways. Here the authors reveal via integrated chromatin analysis that γH2Ax domains are established by chromosomal contacts with the DSB site

Antigen glycosylation regulates efficacy of CAR T cells targeting CD19

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy

Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts

Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While “exitrons” are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed “falsitrons,” that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation

A Cyfip2-Dependent Excitatory Interneuron Pathway Establishes the Innate Startle Threshold

Summary: Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process. : Using forward genetics, electrophysiology, and combined behavior and Ca2+ imaging in zebrafish, Marsden et al. show that cyfip2 regulates the acoustic startle threshold by controlling the activity of excitatory spiral fiber interneurons. Keywords: zebrafish, acoustic startle response, Mauthner cell, Cyfip2, spiral fiber neurons, behavior threshold, GCaM

Simulation-based comprehensive benchmarking of RNA-seq aligners

none6simixedBaruzzo, Giacomo; Hayer, Katharina E; Kim, Eun Ji; DI Camillo, Barbara; Fitzgerald, Garret A; Grant, Gregory RBaruzzo, Giacomo; Hayer, Katharina E; Kim, Eun Ji; DI CAMILLO, Barbara; Fitzgerald, Garret A; Grant, Gregory R

Novel fusion transcript between E4orf6 and DBP is expressed, translated, and conserved.

(A) Enlarged transcriptome map of Ad5 E4 and E2A transcriptional units. Promoter transcription start sites are indicated with left-facing arrows, and cleavage and polyadenylation sites (pA) labeled with downward facing arrows. Novel E4-derived transcripts that terminate in E2A are highlighted in red. Ad5 mutant viruses dl1004 and dl355 contain deletions (indicated by hashed boxes) that remove most of the E4 region and splice donors (ΔE4) or a 14-base deletion inside E4orf6 that only abrogates E4orf6 expression (ΔE4orf6). (B) Reverse-transcriptase PCR on cDNA derived from Ad5-infection of A549 cells reveals characteristic bands of both E4orf6/Unk and E4orf6/DBP. L denotes DNA ladder and triangle indicates increasing cDNA concentration. (C) A549 or W162 cells were uninfected (mock) or infected with WT Ad5, ΔE4orf6, or ΔE4 viruses for 40 hours and proteins detected by immunoblot analysis. When blotting with antisera raised against the N-terminus of E4orf6, a prominent band is detected at the predicted size of E4orf6/DBP. This band is absent during ΔE4 infection and not observed in W162 cells where only the E4 region is provided in trans. Kilodalton size markers are shown to the left of each blot. (D) Infections and immunoblot analysis were performed as described in panel C. Two independently derived anti-N-terminal E4orf6 antibodies (RSA3 and M45) detect E4orf6/DBP. (E) Proteins expressed over a time-course of Ad5 infection in A549 were detected by immunoblot analysis at indicated hpi. (F) A549 cells were infected with adenoviruses from four different serotypes in a time-course. All tested adenovirus serotypes express a protein corresponding to E4orf6/DBP. (G) Quantitative reverse-transcriptase PCR was performed to demonstrate mRNA accumulation of Ad5 E1A (a representative early transcript), Fiber (a representative late transcript), and three E4orf6 containing transcript isoforms. Transcripts were normalized to expression level at 48 hpi and internal HPRT1 housekeeping gene.</p

Direct RNA Sequencing (dRNA-seq) unambiguously distinguishes early and late transcription.

(A) dRNA-seq was performed on polyadenylated RNA from Ad5-infected A549 cells extracted at 12 hours post-infection (hpi). Sequence reads were aligned to the re-annotated transcriptome and filtered to retain only unambiguous primary alignments. Normalized read count indicates the number of RNAs for a particular transcript once normalized to the total number of mappable reads (human plus adenovirus) for the entire sequencing reaction. For all panels, grey bars indicate early genes, black bars indicate late genes, and red bars indicate novel isoforms discovered in this study. Undetectable transcripts (nd) or those with fewer than 10 counts of a particular isoform detected ((B) Same as in Panel A, but with RNA harvested at 24 hpi.</p