Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3′Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

SUMMARYDuring class switch recombination (CSR), B cells replace the Igh Cμ or δ exons with another down-stream constant region exon (CH), altering the anti-body isotype. CSR occurs through the introduction of AID-mediated double-strand breaks (DSBs) in switch regions and subsequent ligation of broken ends. Here, we developed an assay to investigate the dynamics of DSB formation in individual cells. We demonstrate that the upstream switch region Sμ is first targeted during recombination and that the mechanism underlying this control relies on 53BP1. Surprisingly, regulation of break order occurs through residual binding of 53BP1 to chromatin before the introduction of damage and independent of its established role in DNA repair. Using chromosome conformation capture, we show that 53BP1 mediates changes in chromatin architecture that affect break order. Finally, our results explain how changes in Igh architecture in the absence of 53BP1 could promote inversional rearrangements that compromise CSR.

Abstract4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes. PLOS Computational Biology |

Recent studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL), identifying several oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic lesions in cancer are located in intergenic regions, whose role remains poorly understood. In this context, 20% of T-ALL patients show loss of expression of the tumor suppressor PTEN, but not all can be explained by mutations/deletions of its coding region, defective splicing or promoter hypermethylation. Thus, we hypothesized that alterations of yet unidentified enhancers might play a critical role in the regulation of PTEN expression in T-ALL. To test this, we first performed 4C-seq of the PTEN promoter in human T-ALL cells. Integration of our results with epigenetic profiling revealed a distal non-coding region (named PE for PTEN enhancer) that shows concomitant interaction with the PTEN promoter and bona fide enhancer marks. Moreover, reciprocal 4C-seq assays using PE as viewpoint confirmed its interaction with the PTEN promoter. Next, multispecies DNA sequence alignment revealed notable conservation of PE in mammals and detailed analysis of H3K27ac across 64 cell lines and tissues revealed that PE is specifically active in T-cells and other hematopoietic cells, but is largely repressed in non-hematopoietic cells. Moreover, luciferase assays showed strong, orientation-independent activation of reporter constructs containing either the mouse or human PE sequence in Jurkat cells, and CRISPR/Cas9-induced deletion of PE in human T-ALL cells led to reduced PTEN levels, increased phospho-AKT and enhanced cellular proliferation. To formally test the functional relevance of PE in T-cell development and transformation, we generated PE knockout and conditional knockout mice. PE-null animals are viable and show a marked increase in thymus size and cellularity as their only developmental alteration. Mechanistically, this phenotype is associated with a marked reduction in Pten expression. Next, we analyzed the role of PE in the induction of NOTCH1-driven T-ALLs by transplanting mice with PE wild-type or knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1. In this context, mice transplanted with NOTCH-infected PE-null cells showed 100% penetrance and developed T-ALL ~50 days after transplant. However, mice transplanted with NOTCH-infected PE wild-type cells showed delayed T-ALL kinetics and reduced penetrance. In addition, secondary loss of PE in already established NOTCH1-induced T-ALLs from PE conditional knockout mice led to accelerated T-ALL progression and a gene expression signature driven by Pten loss. Finally, analyses of human T-ALL patient samples uncovered recurrent deletions encompassing PE without concomitant deletion of the PTEN coding sequence. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in the pathogenesis of human leukemia. Citation Format: Luca Tottone, Olga Lancho, Maria Victoria da Silva-Diz, Shirley Luo, Shreeta Chakraborty, Pedro P. Rocha, Daniel Herranz. A tumor suppressor enhancer of PTEN in T-cell development and transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3434.

Long range enhancers play critical roles in the control of gene expression during development and have emerged as key regulators of lineage commitment and oncogenic programs in hematopoiesis and leukemia. The MYC oncogene is dynamically regulated in the hematopoietic system under the control of a network of clustered distal enhancers, which provide modular regulation of MYC expression during lymphoid and myeloid development. In thymocyte development MYC transcription critically depends on the activity of N-Me, a distinct T-cell specific enhancer controlled by NOTCH1 signaling and located 1.4 Mb telomeric to the MYC transcription start site. Yet, the specific mechanisms governing N-Me enhancer activity and lineage specific control of MYC expression remain rudimentarily understood. Analysis of chromatin looping by 4C and chromatin accessibility by ATACseq revealed an unanticipated high density of chromatin contacts between N-Me and additional regulatory elements in the Myc locus and showed a distinct pattern of N-Me chromatin accessibility -opening as progenitors mature into T cell committed CD4 CD8 double negative (DN) 2b cells and returning to a closed configuration in CD4 CD8 double positive (DP) thymocytes-. To explore potential regulators of N-Me activity we performed Mass Spectrometry proteomic profiling of N-Me binding proteins and ChIPseq analyses identifying numerous factors involved in hematopoietic and lymphoid development (ERG, ETS1, GATA3, RUNX1, TCF3 and TCF12) and transcription factor oncogenes with prominent roles in the pathogenesis of T-ALL (HOXA9, MYB, MYC, LMO1, LMO2, TAL1 and TLX1). Moreover, phylogenetic footprinting analyses across vertebrate species identified two ultraconserved elements matching GATA factor binding motifs (GS1 and GS2). To test the functionality of these elements we introduced targeted mutations in the N-Me sequence at these sites using CRISPR/CAS9 directed mutagenesis. Mice homozygous for combined N-Me GS1 and GS2 mutations (GS1+2mut) revealed a marked defect in thymus cellularity with characteristic accumulation of DN and intermediate single positive (ISP) thymocytes and decreased numbers of more mature populations. Mechanistically, immunohistochemical, flow cytometry and single cell RNaseq analyses revealed decreased Myc protein levels in thymocyte poulations of GS1+2 mutant animals. In this context, we hypothesized that GATA3, a prominent N-Me binding transcription factor in our ChIP and proteomic analyses critically implicated in T-cell commitment, could play a major role in N-Me regulation via interaction with the GS1 and GS2 N-Me GATA sites. Consistent with this hypothesis analysis of Gata3 ChIPs from heterozygous GS1+2 mutant mice recovered only the N-Me wild type sequence, formally demonstrating the strict requirement of these sites for N-Me Gata3 binding. Mechanistically, ATACseq analysis revealed a marked reduction in chromatin accessibility and nucleosome invasion in thymocytes from GS1+2 mutant mice in support of a critical pioneering activity for GATA3 in the control of N-Me activity. Finally, given the important role of NOTCH1 induced MYC upregulation in the pathogenesis of T-ALL, we hypothesized that disruption of N-Me activity via targeted mutation of N-Me GATA sites could effectively impair the development of NOTCH1-driven T-ALL in N-Me GS1+2 mutant mice. To test this possibility we infected hematopoietic progenitors from N-Me wild type and N-Me GS1+2 homozygous mice with retroviruses driving the expression of an oncogenic constitutively active form of NOTCH1 (DE-NOTCH1) and transplanted them into sublethally irradiated recipients. In these experiments, mice transplanted with DE-NOTCH1 infected N-Me wild type cells developed overt T-ALL 6 weeks postransplant with 100% penetrance. In contrast, mice transplanted with DE-NOTCH1-expressing N-Me GS1+2 homozygous cells showed complete protection from NOTCH1 induced T-ALL (P <0.001). In all these results identify GATA3 binding to the N-Me enhancer as a critical driver of nucleosome eviction and enhancer activation strictly required for thymocyte development and NOTCH1-induced T-cell transformation. Disclosures No relevant conflicts of interest to declare.

Class Switch Recombination (CSR) involves the introduction of double stranded breaks (DSBs) at the switch regions of the immunoglulin heavy chain (Igh) locus by the enzyme Activation Cytidine Deaminse (AID). AID can also act as a general mutator targeting other loci in the genome which can then either be repaired faithfully or in an error-prone fashion introducing mutations and potentially initiating B cell lymphoma. The factors contributing to the choice of repair pathway are not fully understood. Here we tested the hypothesis that repair pathway choice is influenced by differential accessibility and expression levels of target loci across cell cycle. More specifically in the context of CSR we tested whether differential regulation of gene accessibility across cell cycle is an important determinant for AID binding and subsequent repair pathway choice as different repair pathways predominate at different stages of cell cycle. Using 3D-FISH in conjunction with Immunofluorescence we observed that AID target genes that are faithfully repaired are more accessible (found in euchromatic regions) in the G2 phase of the cell cycle then genes that are frequently mutated. In contrast, those genes which are repaired in an error prone fashion are more accessible in the G1 phase of cell cycle. Since Homologous Recombination mediated repair (HR), which is a faithful repair mechanism, occurs in G2 we speculate that accessibility of these genes at this stage of cell cycle facilitates action by this repair pathway. Conversely, genes that are more accessible during the G1 phase of cell cycle will be repaired by the non-homologous end joining (NHEJ) repair pathway and therefore are more likely to be mutated. Thus, HR could be the pathway by which faithful repair is accomplished and use of the NHEJ pathway on the other hand could contribute to the introduction of dangerous DNA mutations that might lead to B cell transformation and cancer. To connect differences in accessibility with repair pathway usage, we used a mouse model carrying a hypomorphic mutation in BRCA2, a protein involved in HR. This is the first mouse model impaired in HR that eludes embryonic lethality and allows inspection of the role of this pathway in maintaining genomic stability in splenocytes undergoing CSR. Our preliminary investigations indicate that in Brca2 mutant B cells not only is the integrity of fathfully repaired loci compromised, but the Igh locus is also damaged. Taken together these results support our hypothesis and further indicate that the HR pathway is involved in repairing Igh. Given that approximately 95% of lymphomas are of B cell origin and many of these are associated with AID mediated breaks, it is crucial for us to understand the factors that influence targeting and repair. Disclosures No relevant conflicts of interest to declare.

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