Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29a∼29b-1, mir-100∼let-7a-2∼125b-1 and miR-221∼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci.

Progression of malignancy to overt disease requires multiple genetic hits. Activation-induced deaminase (AID) can drive lymphomagenesis by generating off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription. The first active transcriptional profiles from acute lymphoblastic leukemia (ALL) patients acquired here reveal striking similarity at structural variation (SV) sites. Specific transcriptional features, namely convergent transcription and Pol2 stalling, were detected at breakpoints. The overlap was most prominent at SV with recognition motifs for the recombination activating genes (RAG). We present signal feature analysis to detect vulnerable regions and quantified from human cells how convergent transcription contributes to R-loop generation and RNA polymerase stalling. Wide stalling regions were characterized by high DNAse hypersensitivity and unusually broad H3K4me3 signal. Based on 1382 pre-B-ALL patients, the ETV6-RUNX1 fusion positive patients had over ten-fold elevation in RAG1 while high expression of AID marked pre-B-ALL lacking common cytogenetic changes.DOI: http://dx.doi.org/10.7554/eLife.13087.001

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Progression of malignancy to overt disease requires multiple genetic hits. Activationinduced deaminase (AID) can drive lymphomagenesis by generating off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription. The first active transcriptional profiles from acute lymphoblastic leukemia (ALL) patients acquired here reveal striking similarity at structural variation (SV) sites. Specific transcriptional features, namely convergent transcription and Pol2 stalling, were detected at breakpoints. The overlap was most prominent at SV with recognition motifs for the recombination activating genes (RAG). We present signal feature analysis to detect vulnerable regions and quantified from human cells how convergent transcription contributes to R-loop generation and RNA polymerase stalling. Wide stalling regions were characterized by high DNAse hypersensitivity and unusually broad H3K4me3 signal. Based on 1382 pre-B-ALL patients, the ETV6-RUNX1 fusion positive patients had over tenfold elevation in RAG1 while high expression of AID marked pre-B-ALL lacking common cytogenetic changes.

Acute lymphoblastic leukemia (ALL) is the most common pediatric leukemia. It comprises of multiple subtypes with combinations of chromosomal rearrangements, indels and copy number variations. Furthermore, there is a significant number of somatic mutations located outside of protein coding regions of the genome. These DNA alterations can be classified based on whether they 1) introduce novel transcription factor binding sites, 2) create aberrant (super-)enhancers, or 3) lead to the loss of regulatory boundaries at oncogene loci upon disruption of CTCF binding sites. We performed whole genome sequencing (WGS) on two T-ALL and 16 pre-B ALL patients from diagnostic and remission samples, and measured transcriptional activity using Global Nuclear Run-on sequencing (GRO-seq). These results were integrated with chromatin topology information and transcript annotations to yield comprehensive characterization of the gene regulatory landscape. The effect on gene expression was further analyzed based on monoallelic expression. Altogether, we found 49 translocations, 153 deletions, 9 duplications and 22 inversions across the pre-B ALL samples. Majority of the changes were located within the annotated gene regions. At 25 of these 233 sites, we found evidence that active enhancers resulted in transcription-coupled genetic instability at DNA breakpoints. In a pre-B ALL case, our analysis successfully captured complex translocation event involving ETV6 (ETV6-MUTYH, ETV6-chr7, ETV6-chr21, ETV6-TXNRD2), and deletion at chr22 that hit to active promoters of PATZ1 and DRG1 genes leading to monoallelic expression of these genes. In T-ALLs, we found 13 translocations (8 intergenic) and 9 deletions. In a T-ALL case, we found that translocation between TCR-beta locus and chr9 brought an active promoter to the proximity of TMEM38B resulting in increased gene expression. We also found a deletion in active promoter at chr9 leading to decreased expression of MTAP and CDKN2A genes. Moreover, a large deletion in chr11 brought oncogenic transcription factor LMO2 and RAG1 close to each other. In summary, we present a genome-wide approach to link structural variation events with gene regulatory features in ALL cells. This extends previous approaches that have mainly focused on genetic changes at coding regions. Citation Format: Laura Oksa, Miikka Vouttilainen, Tapio Vuorenmaa, Saara Laukkanen, Mari Lahnalampi, Olli Lohi, Merja Heinäniemi. Analysis of DNA structural variations in context of the gene regulatory landscape of pediatric ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2078.