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Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In T-ALL patients, we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5;14)(q35;q32.2) rearranged patients, and especially when CTCF bindings sites are preserved in between the BCL11B-enhancer and the TLX3oncogene. These solitary intervening sites insulate TLX3from the enhancer by inducing competitive looping to multiple binding sites near the TLX3promoter. Reduced CTCF levels, or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3promoter to BCL11B-enhancer looping that elevates oncogene expression levels and leukemia burden.

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded–targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. Here, the utility of FFPE-TLC in bone and soft tissue tumor diagnostics was evaluated. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n= 44) and control samples (n= 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.

During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.

The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gradually reestablished during reprogramming. Our data confirm that long-range chromatin interactions are primarily associated with the spatial segregation of open and closed chromatin, defining overall chromosome conformation. Additionally, we identified two further levels of genome organization in ESCs characterized by colocalization of regions with high pluripotency factor occupancy and strong enrichment for Polycomb proteins/H3K27me3, respectively. Underlining the independence of these networks and their functional relevance for genome organization, loss of the Polycomb protein Eed diminishes interactions between Polycomb-regulated regions without altering overarching chromosome conformation. Together, our data highlight a pluripotency-specific genome organization in which pluripotency factors such as Nanog and H3K27me3 occupy distinct nuclear spaces and reveal a role for cell-type-specific gene-regulatory networks in genome organization.

Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7–9 days.

Introduction.The CCCTC-binding factor (CTCF) regulates the 3D chromatin architecture by facilitating chromosomal loops and forming the boundaries of structural domains. In addition, CTCF is an important transcription factor and regulator of antigen receptor and T cell receptor recombination events. CTCFinactivating events have been found in various human cancers. Loss-of-heterozygosity (LOH) or inactivating missense mutations in specific zinc- fingers have been identified in many human cancers including sporadic breast cancer, prostate cancer, Wilms-tumors and acute lymphoblastic leukemia (ALL). Heterozygous deletions or point mutations have been identified in over half of the patients with breast cancer or uterine endometrial cancers, deregulating global gene expression by altering methylated genomic states and poor survival. Here, we investigated the functional significance and molecular-cytogenetic associations of CTCFaberrations in T-cell acute lymphoblastic leukemia patients.

Splinter: Cergentis BV: Current Employment. Van Eyndhoven: Agilent Technologies Netherland: Current Employment. Van Min: Cergentis BV: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

Background: Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell (MCL) and follicular (FL) lymphoma [Pott C., Blood 2010; Ladetto M, Blood 2013]. However, some technical limitations hamper its feasibility in daily clinical routine. First of all, current techniques allow the identification of a molecular marker only in 75-80% of MCL and 55-60% of FL patients. All the other “no marker” cases cannot be studied for MRD purposes. Next generation sequencing (NGS) tools might overcome these limitations. Among them, the recently developed targeted locus amplification (TLA) technology [de Vree P.J., Nat Biotechnol 2014] that selectively amplifies and sequences entire genes on the basis of the crosslinking of physically proximal DNA loci, was able to identify novel candidate oncogenes and gene fusions in acute lymphoblastic leukemia. [Kuiper R.P.; Vroegindeweij EM, 57thASH Communication, Blood 2015].

Splinter: Cergentis BV: Employment. van Min:Cergentis BV: Employment.

Simonis: Cergentis BV: Employment. Klous:Cergentis BV: Employment. Yilmaz:Cergentis BV: Employment. Splinter:Cergentis BV: Employment.

Background: T-cell acute lymphoblastic leukemia is characterized by clonal and mutual exclusive chromosomal rearrangements that recurrently activate TAL1, LMO2, TLX1, NKX2-1, TLX3, HOXAor MEF2Concogenes. Most of these translocations or chromosomal rearrangements occur as erroneous D-J or V-DJ rearrangement attempts of T-cell receptor beta (TCRB) or TCR alpha/delta (TCRAD) genes, mostly positioning oncogenes under the transcriptional control of TCR enhancer elements. Alternatively, oncogenes can also be activated as consequence of BCL11Bchromosomal rearrangements. Although many oncogenes are known in T-ALL, the driving oncogenic lesion in particular T-ALL cases remains unknown.

Current risk assessments for treatment of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are based on several clinical and biological criteria, including genomic alterations. Genomic profiling of BCP-ALL in the last few years has substantially extended the number of risk factors that can be used for risk stratification, including a novel entity known as BCR-ABL1-like or Ph-like ALL. This subgroup of BCR-ABL1-like cases is characterized by the high recurrence of a diverse repertoire of novel gene fusions and mutations which frequently result in enhanced tyrosine kinase and cytokine receptor signaling [Roberts et al., NEJM 2014;371:1005-15]. Leukemia's with these alterations could potentially be targeted with appropriate tyrosine kinase inhibitors. Clinical trials with newly-diagnosed patients carrying these alterations are therefore required, but the large genomic diversity within this group of patients currently provides a major bottleneck.