Population Variability Generated during Rescue Process and Passaging of Recombinant Mumps Viruses

Recombinant mumps viruses (MuVs) based on established vaccine strains represent attractive vector candidates as they have known track records for high efficacy and the viral genome does not integrate in the host cells. We developed a rescue system based on the consensus sequence of the L-Zagreb vaccine and generated seven different recombinant MuVs by (a) insertion of one or two additional transcription units (ATUs), (b) lengthening of a noncoding region to the extent that the longest noncoding region in MuV genome is created, or (c) replacement of original L-Zagreb sequences with sequences rich in CG and AT dinucleotides. All viruses were successfully rescued and faithfully matched sequences of input plasmids. In primary rescued stocks, low percentages of heterogeneous positions were found (maximum 0.12%) and substitutions were predominantly obtained in minor variants, with maximally four substitutions seen in consensus. ATUs did not accumulate more mutations than the natural MuV genes. Six substitutions characteristic for recombinant viruses generated in our system were defined, as they repetitively occurred during rescue processes. In subsequent passaging of primary rescue stocks in Vero cells, different inconsistencies within quasispecies structures were observed. In order to assure that unwanted mutations did not emerge and accumulate, sub-consensus variability should be closely monitored. As we show for Pro408Leu mutation in L gene and a stop codon in one of ATUs, positively selected variants can rise to frequencies over 85% in only few passages.

A predominant enhancer co-amplified with the SOX2 oncogene is necessary and sufficient for its expression in squamous cancer

Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3’ noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.

A variant noncoding region regulates Prrx1 and predisposes to atrial arrhythmias

Rationale Atrial Fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1, encoding the Paired Related Homeobox 1 transcription factor. Objective To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and results The mouse orthologue of the noncoding variant genomic region (R1A) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region (Kifap3, Prrx1, Fmo2, Prrc2c), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program (Mhrt, Myh6, Rbm20, Tnnt2, Ttn, Ckm) was upregulated in R1A−/− atrial cardiomyocytes, and that Mef2 binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A−/− mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared to controls. Conclusion The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fondation Leducq

A Variant Noncoding Region Regulates Prrx1 and Predisposes to Atrial Arrhythmias

Rationale: Atrial Fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1, encoding the Paired Related Homeobox 1 transcription factor. Objective: To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and Results: The mouse orthologue of the noncoding variant genomic region (R1A) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region (Kifap3, Prrx1, Fmo2, Prrc2c), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program (Mhrt, Myh6, Rbm20, Tnnt2, Ttn, Ckm) was upregulated in R1A-/- atrial cardiomyocytes, and that Mef2 binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A-/- mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared to controls. Conclusions: The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes.

Genome-wide association experiments have uncovered a slew of cardiometabolic trait-associated variants. This information can be useful in the implementation of new diagnostic and treatment strategies.

A slew of cardiometabolic trait-associated variants have been discovered thanksto genome-wide association studies. The majority of imputed SNPs are locatedin the noncoding region, suggesting that noncoding genes are significant. In thissense, lncRNAs are gaining attention due to their biological functions, andrising data suggests that lncRNAs could be able to assist researchers in betterunderstanding the pathogenesis of cardiometabolic disorders. The existing stateof knowledge about lncRNAs in cardiometabolic disorders is still in its earlystages, and further research will help us better understand how the noncodingsector regulates us. This knowledge would be important in the development ofmodern diagnosis and treatment methods.

Enhancer Hijacking of BCL11B Defines a Subtype of Lineage Ambiguous Acute Leukemia

Lindsey Montefiori, Sonja Seliger, and Zhaohui Gu contributed equally. Acute leukemias of ambiguous lineage (ALAL), including those that express combinations of myeloid, T-lineage and stem cell markers such as T/myeloid mixed phenotype acute leukemia (MPAL) and early T cell precursor acute lymphoblastic leukemia (ETP-ALL), remain challenging to diagnose, classify and treat. To define the genomic basis of these leukemias, we conducted a large pan-acute leukemia analysis of 2,573 samples, including 774 T-ALL, 126 MPAL, 262 AML, and 1,411 B-ALL cases, with transcriptomic sequencing of all cases and whole genome sequencing of a subset. tSNE and hierarchical clustering analyses of RNA-seq data identified a new subtype of 60 samples with a distinct gene expression profile (Figure 1A) and immunophenotype (typically cCD3+ CD7+ CD1a- CD2+ CD5- CD8- cMPO+/- and myeloid/stem cell marker positive); of 55 cases with data, 25 (45.5%) were T/myeloid MPAL, 20 (36.4%) ETP-ALL, 8 (14.5%) AML and 2 (3.6%) undifferentiated leukemia; 80% of cases harbored FLT3 alterations. These cases exhibited monoallelic expression of BCL11B which encodes a T-lineage transcription factor that is repressed in hematopoietic stem and progenitor cells (HSPCs), a putative cell of origin for ALAL. WGS/RNA-seq identified recurrent BCL11B-deregulating structural variants (SVs) in 56/60 (93.3%) cases, including BCL11B fusion to RUNX1 or ZEB2 in 6 (10%) cases. Most SVs were noncoding and included rearrangement of BCL11B to a gene desert upstream of ARID1B on chromosome 6 (23 cases; 41%); rearrangement to the BENC enhancer at CCDC26, distal to MYC on chromosome 8 (9; 16%); rearrangement to an intronic region of CDK6 on chromosome 7 (4; 7%); a novel high-copy (~20x) tandem amplification of a 2.5 kb noncoding region 700 kb downstream of BCL11B on chromosome 14 which we term BCL11B Enhancer Tandem Amplification (BETA) in 12 cases (21%); and rearrangement to noncoding regions at the SATB1 and ETV6 loci, each observed in a single case (Figure 1B). BCL11B-deregulating SVs were otherwise not identified in WGS analysis of 5,550 pediatric and adult hematological malignancies, 344 pediatric brain tumors and 797 pediatric solid tumors. We hypothesized that these SVs result in enhancer hijacking and ectopic activation of BCL11B in a CD34+ HSPC. Accordingly, the ARID1B, CCDC26, CDK6, and ETV6 loci all harbor CD34+ super enhancers which are absent (ARID1B, CCDC26, ETV6) or diminished (CDK6) in committed T cell precursors (Figure 1C,D). The BETA region is nominally active in HSPCs; however, tandem amplification generates a ~50 kb chromatin domain which may transform this region into a potent transcriptional activator. To investigate this, we performed histone H3 lysine 27 acetyl (H3K27ac) chromatin conformation capture followed by high-throughput sequencing (HiChIP) on 5 primary samples (1 ARID1B, 1 CCDC26, 1 CDK6 and 2 BETA cases), normal cord blood CD34+ cells, and 2 T-ALL cell lines (Figure 1E,F). In each primary sample, HiChIP confirmed that the rearranged CD34+ enhancers are active and interact with BCL11B, supporting an enhancer hijacking mechanism. Moreover, in addition to looping to BCL11B, BETA also activates the T cell-specific ThymoD enhancer 1 Mb distal of BCL11B (Figure 1E). Thus, the tandem amplification of a short, inconspicuous noncoding region generates a powerful de novo enhancer that ectopically activates BCL11B, 700kb downstream, and co-opts a dormant T cell enhancer 300 kb in the opposite direction. These activation events likely collaborate to drive oncogenic BCL11B expression in HSPCs. In conclusion, this large-scale analysis has not only identified a new subtype-defining lesion in leukemia and a new mechanism of enhancer generation in cancer (BETA) but has also resolved two controversies. First, genotypic alterations transcend immunophenotype in the classification of lineage ambiguous leukemias, with BCL11B rearrangements unifying a subgroup of T/myeloid MPAL, ETP-ALL and poorly differentiated AML that often differ only by cMPO expression. This recapitulates prior observation of ZNF384-rearrangement defining a subtype of B-ALL and B/myeloid MPAL. Second, chromatin topology analysis demonstrates enhancer hijacking of BCL11B in a primitive stem/progenitor cell, and thus, at least for a subset of cases, a hematopoietic stem cell is the cell of origin for T/myeloid antigen-expressing lineage ambiguous leukemias. Disclosures Iacobucci: Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; AbbVie, Inc.: Research Funding.

Inhibitory antibodies identify unique sites of therapeutic vulnerability in rhinovirus and other enteroviruses

The existence of multiple serotypes renders vaccine development challenging for most viruses in theEnterovirusgenus. An alternative and potentially more viable strategy for control of these viruses is to develop broad-spectrum antivirals by targeting highly conserved proteins that are indispensable for the virus life cycle, such as the 3C protease. Previously, two single-chain antibody fragments, YDF and GGVV, were reported to effectively inhibit human rhinovirus 14 proliferation. Here, we found that both single-chain antibody fragments target sites on the 3C protease that are distinct from its known drug site (peptidase active site) and possess different mechanisms of inhibition. YDF does not block the active site but instead noncompetitively inhibits 3C peptidase activity through an allosteric effect that is rarely seen for antibody protease inhibitors. Meanwhile, GGVV antagonizes the less-explored regulatory function of 3C in genome replication. The interaction between 3C and the viral genome 5′ noncoding region has been reported to be important for enterovirus genome replication. Here, the interface between human rhinovirus 14 3C and its 5′ noncoding region was probed by hydrogen–deuterium exchange coupled mass spectrometry and found to partially overlap with the interface between GGVV and 3C. Consistently, prebinding of GGVV completely abolishes interaction between human rhinovirus 14 3C and its 5′ noncoding region. The epitopes of YDF and GGVV, therefore, represent two additional sites of therapeutic vulnerability in rhinovirus. Importantly, the GGVV epitope appears to be conserved across many enteroviruses, suggesting that it is a promising target for pan-enterovirus inhibitor screening and design.

Mitochondrial Genome Sequences of Diorhabda carinata and Diorhabda carinulata, Two Beetle Species Introduced to North America for Biological Control

We announce the complete circularized mitochondrial genome assemblies of Diorhabda carinata and Diorhabda carinulata, beetle species introduced to North America for the biological control of invasive shrubs of the genus Tamarix L. (Tamaricaceae). The assemblies (16,232 and 16,298 bp, respectively) each comprise 13 protein-coding genes, 22 tRNAs, two rRNAs, and a noncoding region.

EF-G–induced ribosome sliding along the noncoding mRNA

Translational bypassing is a recoding event during which ribosomes slide over a noncoding region of the messenger RNA (mRNA) to synthesize one protein from two discontinuous reading frames. Structures in the mRNA orchestrate forward movement of the ribosome, but what causes ribosomes to start sliding remains unclear. Here, we show that elongation factor G (EF-G) triggers ribosome take-off by a pseudotranslocation event using a small mRNA stem-loop as an A-site transfer RNA mimic and requires hydrolysis of about two molecules of guanosine 5′-triphosphate per nucleotide of the noncoding gap. Bypassing ribosomes adopt a hyper-rotated conformation, also observed with ribosomes stalled by the SecM sequence, suggesting common ribosome dynamics during translation stalling. Our results demonstrate a new function of EF-G in promoting ribosome sliding along the mRNA, in contrast to codon-wise ribosome movement during canonical translation, and suggest a mechanism by which ribosomes could traverse untranslated parts of mRNAs.

Characterizing Students’ Ideas about the Effects of a Mutation in a Noncoding Region of DNA

Understanding student ideas in large-enrollment biology courses can be challenging, because easy-to-administer multiple-choice questions frequently do not fully capture the diversity of student ideas. As part of the Automated Analysis of Constructed Responses (AACR) project, we designed a question prompting students to describe the possible effects of a mutation in a noncoding region of DNA. We characterized answers from 1127 students enrolled in eight different large-enrollment introductory biology courses at three different institutions over five semesters and generated an analytic scoring system containing three categories of correct ideas and five categories of incorrect ideas. We iteratively developed a computer model for scoring student answers and tested the model before and after implementing an instructional activity designed to help a new set of students explore this concept. After completing a targeted activity and re-answering the question, students showed improvement from preassessment, with 64% of students in incorrect and 67% of students in partially incorrect (mixed) categories shifting to correct ideas only. This question, computer-scoring model, and instructional activity can now be reliably used by other instructors to better understand and characterize student ideas on the effects of mutations outside a gene-coding region.