Benoit Molinie

Genome-wide Translocation Sequencing Reveals Mechanisms of Chromosome Breaks and Rearrangements in B Cells

Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.

Transcription factor trapping by RNA in gene regulatory elements.

Noncoding RNA helps protein binding Besides reading the coding regions of genes, RNA polymerase generates RNA at promoter-proximal and -distal DNA elements, but the function of these molecules is largely unknown. Sigova et al. show that these RNAs facilitate interactions between gene regulators and the regulatory elements they occupy. Nascent RNA associates with the transcription factor YY1 and increases its ability to bind DNA. Thus, transcription at active regulatory elements may provide a positive feedback loop that reinforces regulatory elements contributing to the stability of gene expression programs. Science, this issue p. 978 Nascent RNAs facilitate interactions between gene regulators and regulatory elements. Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs.

Enhancing GTEx by bridging the gaps between genotype, gene expression, and disease

Genetic variants have been associated with myriad molecular phenotypes that provide new insight into the range of mechanisms underlying genetic traits and diseases. Identifying any particular genetic variants cascade of effects, from molecule to individual, requires assaying multiple layers of molecular complexity. We introduce the Enhancing GTEx (eGTEx) project that extends the GTEx project to combine gene expression with additional intermediate molecular measurements on the same tissues to provide a resource for studying how genetic differences cascade through molecular phenotypes to impact human health.

Genome-wide Analysis of Immune System Genes by Expressed Sequence Tag Profiling

Profiling studies of mRNA and microRNA, particularly microarray-based studies, have been extensively used to create compendia of genes that are preferentially expressed in the immune system. In some instances, functional studies have been subsequently pursued. Recent efforts such as the Encyclopedia of DNA Elements have demonstrated the benefit of coupling RNA sequencing analysis with information from expressed sequence tags (ESTs) for transcriptomic analysis. However, the full characterization and identification of transcripts that function as modulators of human immune responses remains incomplete. In this study, we demonstrate that an integrated analysis of human ESTs provides a robust platform to identify the immune transcriptome. Beyond recovering a reference set of immune-enriched genes and providing large-scale cross-validation of previous microarray studies, we discovered hundreds of novel genes preferentially expressed in the immune system, including noncoding RNAs. As a result, we have established the Immunogene database, representing an integrated EST road map of gene expression in human immune cells, which can be used to further investigate the function of coding and noncoding genes in the immune system. Using this approach, we have uncovered a unique metabolic gene signature of human macrophages and identified PRDM15 as a novel overexpressed gene in human lymphomas. Thus, we demonstrate the utility of EST profiling as a basis for further deconstruction of physiologic and pathologic immune processes.

Identification and characterization of m6A circular RNA epitranscriptomes

This study brings together the expanding fields of RNA modifications and circular (circ) RNAs. We find that cells express thousands of m6A methylated circRNAs, with cell-type specificity observed between human embryonic stem cells and HeLa cells. m6A-circRNAs were identified by RNA sequencing of total RNA following ribosome depletion and m6A immunoprecipitation. The presence of m6A-circRNAs is corroborated by the identification of complexes between circRNAs and YTHDF1 and YTHDF2, proteins that “read” m6A sites in mRNAs. Furthermore, m6A modifications on non-linear RNAs depend on METTL3 and METTL14, the known m6A methyltransferase “writer” complex components, suggesting that circRNAs are methylated by the same complexes responsible for m6A modification of linear RNAs. Despite sharing m6A readers and writers, m6A-circRNAs are frequently derived from exons not methylated in mRNAs. Nevertheless, m6A-mRNAs that are methylated on the same exons as those composing m6A-circRNAs exhibit less stability than other m6A-mRNA, and this circRNA-mRNA cross-talk is regulated by YTHDF2. Thus, our results expand the m6A regulatory code through identification of the first circRNA epitranscriptome.

Genome-Wide Location Analyses of N6-Methyladenosine Modifications (m 6 A-Seq)

N6-methyladenosine-sequencing (m6A-seq) is a critical tool to obtain an unbiased genome-wide picture of m6A sites of modification at high resolution. It allows the study of the impact of various perturbations on m6A modification distribution and the study of m6A functions. Herein, we describe the m6A-seq protocol, which entails RNA immunoprecipitation (RIP) performed on fragmented poly(A) RNA utilizing anti-m6A antibodies. The captured/enriched m6A positive RNA fragments are subsequently sequenced by RNA-seq in parallel with background control non-immunoprecipitated input RNA fragments. Analyses reveal peaks of m6A enrichment containing sites of modifications analogous to chromatin modification immunoprecipitation experiments.