T-cell acute lymphocytic leukemia protein 1 (TAL1) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). Its deregulation can occur through diverse cis-alterations, including SIL-TAL1 microdeletions, translocations with T-cell Receptor loci, and more recently described upstream intergenic non-coding mutations. These mutations consist of recurrent focal microinsertions that create an oncogenic neo-enhancer accompanied by activating epigenetic marks. This observation laid the groundwork for an innovative paradigm concerning the activation of proto-oncogenes via genomic alterations of non-coding intergenic regions. However, for the majority of T-ALL expressing TAL1 (TAL1+), the deregulation mechanism remains 'unresolved'. We took advantage of H3K27ac and H3K4me3 chromatin immunoprecipitation sequencing data of eight cases of T-ALL, including five TAL1+ cases. We identified a putative novel oncogenic neo-enhancer downstream of TAL1 in an unresolved monoallelic TAL1+ case. A rare but recurrent somatic heterozygous microinsertion within this region creates a de novo binding site for MYB transcription factor. Here we demonstrate that this mutation leads to increased enhancer activity, gain of active epigenetic marks, and TAL1 activation via recruitment of MYB. These results highlight the diversity of non-coding mutations that can drive oncogene activation.
T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive hematologic malignancy that accounts for 15% of pediatric and 25% of adult ALL.1,2 The use of intensified chemotherapy regimes has led to recent advances in overall survival.3 However, 30-35% of patients still relapse and face a dismal prognosis with an overall survival of less than 20% at 5 years.4,5 T-ALL is caused by the clonal expansion of immature T-cell precursors that are blocked in their thymic differentiation. Several genetic alterations engendering the ectopic expression of key oncogenic transcription factors have been reported, including the HOX gene family members (HOXA,6,7 TLX1,8 TLX3,9,10 NKX1-1 and NXK1-2,11 basic helix-loop-helix family members (TAL1/2,12-16 L Y L 1,17 BHLHB1,18 the LMO family members (LMO1, LMO219,20), and MYB.21 Of these transcription factors, the TAL1 oncogene is one of the most frequently deregulated in T-ALL. Indeed, increased TAL1 transcripts are found in up to 60% of patients.1 Under physiological conditions, TAL1 is an essential transcription factor (TF) for the development of the vascular system and for primary and definitive hematopoiesis. During definitive hematopoiesis, TAL1 is required for erythroid differentiation, yet it is epigenetically repressed during human thymopoiesis.14,22,23 Oncogenic events leading to the ectopic expression of TAL1 in the T-cell lineage are considered strong drivers of T-cell leukemogenesis. Such deregulation mechanisms occur in cis and involve the ‘hijacking’ of enhancer elements leading to oncogene activation. The SIL-TAL1 microdeletion is the predominant deregulation mechanism found in 25% of TALL. The interstitial deletion places the TAL1 gene under the regulatory control of the adjacent STIL gene promoter.13,24 In a minority of cases (<5%), the TAL1 gene is translocated into either the T-cell receptor b (TRB) locus or, more often, the T-cell receptor δ (TRD) locus and under the control of strong T-cell receptor (TCR) cis-regulatory elements.24-26
We and others have identified a third deregulation mechanism consisting of microinsertions 7kb upstream of the TAL1 gene in a non-coding region. These microinsertions create a super-enhancer identifiable by its enrichment in transcriptionally activating epigenetic marks14,15 and de novo MYB TF binding sites.15 The recruitment of MYB to the mutated sequence and subsequent super-enhancer formation leads to ectopic TAL1 expression.
Lastly, a rare, recurrent intronic point mutation responsible for monoallelic TAL1 expression was recently discovered in pediatric T-ALL. The mutation creates a de novo YY1 TF binding site, is associated with active epigenetic marks, and demonstrates enhancer activity.27 Nonetheless, these diverse deregulation mechanisms do not account for all TAL1 positive (TAL1+) patients, suggesting 'unresolved' TAL1 deregulation mechanisms.28-30 We, therefore, hypothesized that the identification of aberrantly formed intergenic histone marks in the vicinity of the TAL1 locus could uncover new TAL1 abnormalities in T-ALL. We discovered a novel recurrent microinsertion downstream of the TAL1 gene leading to the formation of an MYB-dependent neo-enhancer responsible for TAL1 activation.
Antibody-guided chromatin tagmentation
Genome-wide targeting of histone modifications and MYB were performed by antibody-guided chromatin tagmentation (ACT-seq), according to Carter et al.31 pA-Tn5ase protein was isolated from E. coli (C3013, New England Biolabs; Ipswich, MA, USA) and transformed with plasmid pET15bpATnp (#121137, Addgene; Watertown, MA, USA). The pA-Tn5 transposome (pA-Tn5ome) was generated by mixing pA-Tn5ase (final concentration either 1.9 mM or 3.3 mM, depending on the pA-Tn5ase preparation) and Tn5ME-A+B load adaptor mix (final concentration 3.3 mM) in complex formation buffer (CB31). The pA-Tn5ome-antibody complexes were formed by mixing 1 mL pA-Tn5ome with 0.8 µL CB and 0.8 mL antibody solution. We used antibodies against histone H3K27ac (#4729, Abcam; Berlin, Germany), H3K4me1 (#8895, Abcam; Berlin, Germany), H3K4me3 (#39915 active motif), IgG (#PP64B, Millipore; Burlington, MA, USA), H2B (#M30930, Hölzel Dianostika; Köln, Germany), and MYB (#45150, Abcam); 100,000 cells were used for the pA-Tn5ome-ab complex binding and tagmentation. Tagmented DNA was purified using MinElute kit (#28004, Qiagen; Venlo, The Netherlands) and eluted with 20 mL elution buffer (EB). Sequencing libraries were generated under real-time conditions with a LightCycler 480 (Roche Professional Diagnostics; Indianapolis, IN, USA) in 50 mL reaction mixes consisting of 20 mL tagmented DNA eluate, 25 mL NEBNext High Fidelity 2X Mix (#M0541, New England Biolabs; Ipswich, MA, USA), 0.5 mL 100x SYBRGreen, 2.5 mL primer Tn5McP1n and 2.5 mL barcode primer. Reaction conditions were 72°C, 5 minutes (min) (gap repair); 98°C, 30 seconds (sec) (initial melting); followed by cycles of: 98°C (10 sec), 63°C (10 sec), 72°C (10 sec). Cycling was stopped when fluorescence units (FU) had increased by 5. Libraries were purified with HighPrep magnetic beads (#220001, Biozym; Hessisch Oldendorf, Germany) with a bead:DNA ratio of 1.4:1 and eluted with 12 mL EB. Quantity and fragment size of the libraries were determined with a Qubit dsDNA HS assay kit (#Q32854, Invitrogen, ThermoFisher; Waltham, MA, USA) and a TapeStation 4150 with D1000 High Sensitivity Assay (#5067-5585, Agilent; SantaClara, CA, USA), respectively. Six to eight differently barcoded libraries were multiplexed and sequenced on a single lane of a NextSeq™ 550 system (paired-end, 75 bp, Illumina; San Diego, CA, USA) with mid-output at the Genome and Proteome Core Facility of DKFZ. ACT-seq data were analyzed as previously described.32
Alpha-cas phasing methods and sgRNA sequences
To phase the engineered heterozygous 3’ mutation with the SNP (Hg19: chr1: 47 684 223) in J-3’NE#1 cells, gRNA targeting the region downstream of the SNP (TAL1 SNP DS crRNA) and upstream of the mutation (TAL1 3’NE DS crRNA) were designed. Corresponding Cas9 RNP were prepared as described above and electroporated into J-3’NE cells. Forty-eight hours (h) post electroporation gDNA was isolated and amplified by polymerase chain reaction (PCR). Similarly, Jurkat 5’super-enhancer was phased using Cas9 RNP corresponding to TAL1 SNP DS crRNA and TAL1 5’SE US crRNA followed by PCR. The LMO1 activating point mutation in Jurkat cells was phased to an SNP in LMO1 exon 1 (Hg19: chr11: 8 285 124) using LMO1 SNP DS crRNA and LMO1 MUT US crRNA followed by PCR. The LMO2 activating microinsertion in MOLT4 cells was phased to an SNP in LMO2 exon 6 (Hg19: chr11: 33 881 016) by electroporating Cas9 RNP corresponding to LMO2 SNP DS crRNA and LMO2 MUT US crRNA (100,000 MOLT4 cells were electroporated using the following settings: 1350V, 10ms, 3 pulses), followed by PCR. In all cases the PCR product was cloned into a pCR4 TOPO TA vector using a TOPO TA Cloning Kit (Cat. # 450030, Thermo Scientific; Waltham, MA, USA) and transformed into electrocompetent Stbl3 cells. Ampicillin-resistant colonies were further inoculated for plasmid miniprep. Plasmids were Sanger sequenced using the M13 reverse primer.
See Online Supplementary Table S1 for primer sequences and Online Supplementary Table S2 for gRNA sequences. For ACT-seq oligonucleotide sequences see Online Supplementary Table S3.
Studies were conducted with informed consent from all patients and in accordance with the principles of the Declaration of Helsinki, and approved by local and multicenter research ethical committees.
Identification of a putative novel oncogenic neoenhancer associated with an intergenic 3’TAL1 microinsertion
To investigate TAL1 deregulation mechanisms, we took advantage of the Blueprint Consortium T-ALL chromatin immunoprecipitation sequencing (ChIP-seq) series.33,34 We focused on H3K4me3 and H3K27ac enrichment over the TAL1 locus in eight primary T-ALL samples and the normal thymus (Figure 1A). TAL1 expression was analyzed using available RNA-seq data (n=6) and/or real-time quantitative (RQ) PCR (n=8). Five patient samples displayed high TAL1 expression and were considered TAL1 positive (TAL1+) and three were considered TAL1 negative (TAL1-) (Online Supplementary Figure S1A and B). Allelic expression analysis by RNA-seq and Sanger sequencing revealed that all five TAL1+ samples had monoallelic TAL1 expression, indicating cis-deregulation mechanisms (Figure 1C-E, Online Supplementary Figure S1C). We screened TAL1+ patients for the known recurrent mechanisms of TAL1 deregulation. Of these five TAL1+ patients, one (UPNT-760) had a SIL-TAL1 microdeletion, one (UPNT-885) had the 5’ super-enhancer mutation (5’SE), and one (UPNT-820) had a translocation involving TAL1 and the TRB locus. Two patients (UPNT-753 and UPNT-802) had unidentified TAL1 deregulation mechanisms and were considered unresolved. ChIP-seq data for H3K27ac and H3K4me3 marks showed enrichment over the TAL1 gene body in all five monoallelically expressing TAL1+ patients (Figure 1A). As expected, H3K27ac enrichment encompassing the 5’super-enhancer was observed in the UPNT-885 sample. In addition, we noticed a unique dual monoallelic enrichment over an intergenic region downstream of the TAL1 gene in one unresolved monoallelic TA L1 + patient (UPNT-802) that was absent in all other TA L1 + patients, suggesting the presence of a novel regulatory element (Figure 1B, Online Supplementary Figure S2). We performed whole genome sequencing (WGS) on both tumoral and non-tumoral DNA (bone marrow remission sample with undetectable minimal residual disease [MRD]) from UPNT-802 and identified a heterozygous 21bp microinsertion (variant allele frequency [VAF] 36%) about 4 kb downstream of the TAL1 gene (Hg 19 chr1: 47 677 744) within the genomic region enriched in H3K27ac and H3K4me3 marks (Figure 1B). This variant was somatic and absent in the non-tumoral sample (Figure 1F). We also confirmed the presence of the mutation by Sanger sequencing. Notably, this mutation was stable in leukemic cells expanded in a patient-derived xeno-graft (PDX) (Figure 1G). As a final verification, we designed mutation specific primers to amplify the microinsertion in the diagnostic sample (Online Supplementary Figure S1D). Collectively, these results suggest the identification of a novel oncogenic neo-enhancer.
The intergenic 3’-TAL1 microinsertion is responsible for TAL1 deregulation in cell-line models
To study the function of the new putative neo-enhancer, we introduced the heterozygous 3´ microinsertion in Jurkat (TAL1+) and Peer (TAL1-) T-ALL cell lines using CRISPR-Cas9 technology. In the Jurkat cell line, the 3’ microinsertion was introduced and the original 5’super-enhancer subsequently deleted (J-3’NE #1 and J-3’NE #2 derivative cell lines). Likewise, we introduced the 3’ mutation into the Peer cell line (P-3’NE #1, P-3’NE #2, P-3’NE #3 derivative cell lines), and also engineered a Jurkat cell line that was deleted for the original 5’ super-enhancer (J-del derivative cell line) (Figure 2A). Genotyping by Sanger sequencing confirmed successful genomic editing in the derivative cell lines with the insertion of the heterozygous microinsertion (Online Supplementary Figure S3A and B). As expected, a strong decrease in TAL1 expression was observed in J-del cells, which was rescued upon introduction of the 3’micro-insertion in J-3’NE #1 and J-3’NE #2 derivative cell lines (Figure 2B and G). Whereas cell proliferation was affected in J-del cells, J-3’NE #1 cells displayed unaffected proliferation (similar to normal unedited Jurkat) (Figure 2C). The introduction of this mutation also led to a strong TAL1 activation in the Peer derivative cell lines (P-3’NE #1, P-3’NE #2, P-3’NE #3) (Figure 2D and G). Importantly, whereas wild-type (WT) Peer showed low and biallelic TAL1 expression, the three Peer derivative cell lines displayed monoallelic TAL1 expression in line with cis-activation (Figure 2E). The mutated sequence was cloned in a PGL4.23 plasmid (containing the luciferase gene and a minimal promoter) and demonstrated significantly increased enhancer activity (P=0.004) compared to the WT sequence in the luciferase reporter assay (Figure 2F), confirming the enhancer function of this variant. Taken together, these results suggest that the novel 3´ microinsertion leads to the creation of a new regulatory element able to activate TAL1 expression and sustain cell proliferation.
Aberrant TAL1 expression from the mutated allele
In order to associate the 3’ microinsertion with the observed aberrant monoallelic TAL1 expression in J-3’NE #1 cells (Figure 3A), and to prove that the expressed allele carried the mutation, we phased the mutation and the heterozygous 3´-UTR SNP used to study the allelic expression in this cell line. Since the mutation and the SNP are approximately 6.5 kb apart, this region was difficult to clone. To circumvent this problem, we used CRISPRCas9 to delete a large DNA fragment between the SNP and the mutation to reduce the distance between the two variants. The region was then PCR amplified. The small PCR product was either cloned into a plasmid and Sanger sequenced, or barcoded and directly sequenced using Mi-seq (Illumina; San Diego, CA, USA). We called this allele-phasing method “Alpha-Cas” (see Figure 3B). After deletion of the intervening DNA and cloning into the plasmid, 9 clones were sequenced. Sequences of two clones contained the unexpressed SNP G and the WT sequence; conversely, six clones contained the expressed SNP A and the mutated sequence, demonstrating the phasing of the mutation on the expressed allele. In addition, we found one clone with the unexpressed SNP G and the microinsertion, which likely resulted from a low rate of allelic exchange during the CRISPR-Cas9 cutting and recombination process (technical artefact) (Figure 3C). Mi-seq produced 56,054 aligned sequenced reads with 81% of reads having the expected allele phasing (47.9% of reads contained the expressed SNP A and microinsertion, 33.1% of reads had the unexpressed SNP G and WT sequence), and a minority of reads (19%) demonstrating allelic exchange (Figure 3D). We and others have previously described the TAL1 monoallelic expression associated with the 5’ super-enhancer mutation;14,28 however, allele phasing was not performed at the time of its discovery. To validate the Alpha-Cas method, we used it to phase the 5’ mutation and the expressed allele in the Jurkat cell line. We deleted the 20 kb intervening region between the SNP and 5’ mutation before phasing. Alpha-Cas found the expected SNP and mutation phasing by both cloning and Mi-seq methods: 86.8% of sequencing reads had the expected phased alleles (51.4% of reads with the expressed SNP A and the micro-insertion, 35.4% of reads with unexpressed SNP G and the WT sequence). Similarly, we observed a low rate of allelic exchange (13.1%) (Online Supplementary Figure S4A-D). This method was also suitable to phase intergenic mutations associated with other T-ALL oncogenes. The LMO1 gene is also activated and monoallelically expressed by the creation of a neo-enhancer bound by MYB transcription factor in the Jurkat cell line.35 The mutation and the informative SNP are 4.3 kb apart (Online Supplementary Figure S5A and B). We compared the direct cloning of the entire 4.3 kb region (Online Supplementary Figure S5C) to the Alpha-Cas method for allele-phasing (Online Supplementary Figure S5D and E). Direct cloning and Alpha-Cas produced comparable results with the expected phasing observed in 91% of the Mi-seq sequencing reads, thus validating this method. In the Molt-4 cell line, LMO2 is activated via a distal microinsertion, creating once again a de novo MYB binding motif which leads to its ectopic monoallelic expression36,37 (Online Supplementary Figure S6A and B). This mutation is situated at a long distance from the informative SNP (75.7 kb) making direct cloning impossible, and the Alpha-Cas method proved, therefore, very useful. Alpha-Cas revealed the expected phasing in 93.8% of reads (Online Supplementary Figure S6C). Despite a low rate of allelic exchange occurring during Cas9 recombination, Alpha-Cas is a simple, efficient, and reliable method for allele-phasing, especially in the case of very distant variants.
3’ neo-enhancer: epigenetic profiling
Using the recently described ACT-seq method as an alternative to conventional ChIP-sequencing (see Methods), we observed the creation of the active 3’ neo-enhancer by the increased H3K27ac and H3K4me1 enrichment at the microinsertion in J-3’NE #1, J-3’NE #2 and P-3’NE #1 derivative cell lines (Figure 4A and B). The corresponding sequencing reads contained uniquely the mutated sequence confirming the monoallelic epigenetic activation of the mutated allele (Figure 4C). As control, we also observed an enrichment at the native 5’ super-enhancer in the unedited Jurkat cell line, which was lost in the J-3’NE #1, J-3’NE #2 cell lines (Figure 4A). Using primers spanning the 3’ microinsertion site, we performed H3K27ac ChIP-qPCR and observed an increased enrichment in J-3’NE #1 cells compared to Jurkat cells (Figure 4D). We also carried out an allele specific H3K27ac ChIP-qPCR in J-3’NE #1 cells and observed an increased enrichment of this histone mark at the mutated allele compared to the WT allele (Figure 4E).
Microinsertions downstream of the TAL1 gene are recurrent in T-cell acute lymphoblastic leukemia and neo-enhancer activity depends on MYB transcription factor binding.
The 3´neo-enhancer is a rare but recurrent oncogenic event, as we identified another patient (UPNT-613) with a 6 bp microinsertion at the same genomic location from an independent series of 189 T-ALL samples screened for this mutation by Sanger sequencing. This mutation was also detected in its corresponding PDX model (Online Supplementary Figure S7A). Moreover, this patient displayed a high unresolved TA L1 expression (Figure 5A) and, like UPNT-802, this high TA L1 expression was stable in the PDX model (Figure 5B). Using Oxford Nanopore sequencing technology (Oxford Nanopore Technologies; Oxford, UK), we performed a targeted analysis of TAL1 expression in the two 3’NE samples (UPNT-802 and UPNT-613 PDX cells) and in two 5’SE samples (Jurkat cells and UPNT-525 PDX cells) (Figure 5C, Online Supplementary Figure S7B-D). Our results revealed differences in the transcription of the 5’region of the TAL1 gene between the 3’NE and 5’SE samples. Long amplicons were detected (S1/R2 primer pair) uniquely in 5’SE samples whereas 3’NE samples initiated transcription downstream of exon 1 without affecting the TA L1 open reading frame (ORF) (S3/R2 primer pair). We also detected transcription activated downstream of the TA L1 gene near the 3’NE. However, transcription initiated here was not specific to the 3’NE regulatory element but specific to TAL1+ samples, as it was detectable in both 3’NE and 5’SE samples and absent in the TAL1- sample (DND-41) (S9b/R5 primer pair). Similarly to the previously described 5’ super-enhancer mutation, both the 3´ microinsertions in UPNT-802 and UPNT-613 patient samples were predicted (JASPAR) to create a de novo binding site for MYB transcription factor and other known members of the TAL1 complex38 (Figure 5D, Online Supplementary Table S4) suggesting the creation of the 3’ neo-enhancer is dependent on MYB binding. Using primers spanning either the 5’ super-enhancer site in Jurkat cells (5’SE) or the 3’ microinsertion site (3’NE), we performed MYB ChIP qPCR and observed an increased MYB enrichment at the 3’NE in J-3’NE #1 and P-3’NE #1 cells. As controls, we verified MYB enrichment at the 5’SE in Jurkat WT cells and its absence in Peer cells lacking MYB-driven super-enhancers (Figure 6A). MYB binding at the 3’NE was also detected in UPNT-802 PDX cells (Online Supplementary Figure S8A). Furthermore, to prove that the observed MYB binding was specific to the 3’mutated sequence, we performed an allele specific MYB ChIP-qPCR in J-3’NE #1, P-3’NE #1 cells and UPNT-802 cells and observed an increased MYB enrichment at the mutated allele compared to the WT allele (Figure 6B, Online Supplementary Figure S8B). In order to definitively link the MYB-dependent epigenetic activation of TA L1, we repressed MYB expression in Jurkat cells using an inducible dCas9-KRAB-MECP2 system with two sgRNA targeting the MYB promoter (Online Supplementary Figure S8C) and measured enhancer activity using a luciferase reporter assay. MYB silencing significantly reduced the enhancer activity associated with the mutated sequence (Figure 6C). Using the same inducible MYB repression system in J’3-NE#1 cells (Figure 6D) resulted in significantly reduced transcriptional and protein TAL1 expression (Figure 6E), demonstrating the essential role of MYB TF for TAL1 expression in these cells. LMO1, whose expression is also driven by a MYB-dependent neo-enhancer in Jurkat cells, served as a positive control (Online Supplementary Figure S8D).
TAL1 is a major oncogene in both adult and pediatric TALL. Intriguingly, the mechanisms leading to TAL1 over-expression are extremely diverse. Genetic rearrangements of the TA L1 gene such as SIL-TAL1 microdeletions and TA L1 translocations with TCR loci (TRD or TRB) have long been described.12,13,24-26 More recently, we and others14,28 uncovered novel mutations in a non-coding region leading to oncogene activation via the creation of an oncogenic neo-enhancer upstream of TAL1. The discovery of these mutations represented a major conceptual advancement, highlighting the underestimated contribution of intergenic mutations in genomic alterations driving cancers. In this circumstance, somatic heterozygous microinsertions of variable size create de novo binding sites for MYB TF, leading to the recruitment of a multi-protein complex, epigenetic activation and TAL1 overexpression in leukemic cells. Since this discovery, similar 5’ non-coding mutations affecting two other major T-ALL oncogenes, LMO1 and LMO2, that are frequently co-deregulated with TAL1 were found.35,37
Intergenic non-coding mutations leading to oncogene deregulation are not limited to T-ALL. A pivotal example is the frequent TERT promoter mutations found in many aggressive solid cancers such as melanomas, glioblastomas, medulloblastomas, and hepatocellular carcinomas.39 These non-coding mutations occur in the promoter region of TERT and create de novo binding sites for ETS TF, increase chromatin accessibility of the mutant alleles, and cause an epigenetic switch. Finally, a rare, recurrent non-coding mutation in the promoter region of TAL1 was discovered in two pediatric T-ALL samples. The authors showed that this mutation creates a de novo binding site for YY1 TF and leads to TAL1 activation.27
In the present work, by combining histone ChIP-seq and gene expression data analysis we identified a rare but recurrent novel oncogenic neo-enhancer responsible for TAL1 activation in T-ALL with unknown deregulation mechanisms. By a mechanism analogous to TAL1 5’SE mutations, we demonstrated that 3’NE mutations create a de novo binding site for MYB TF, which drives the deposition of the observed epigenetic marks and aberrant expression of TAL1, probably via its recruitment of CBP/p300 and members of the TAL1 complex (or core regulatory circuit, CRC).38 Interestingly, these novel microinsertions are the first oncogenic neo-enhancers found downstream of the TAL1 gene. This characteristic is in line with the enhancers’ independent position. Enhancers can be located upstream or downstream of a regulated gene and interact with proximal regulatory elements in the vicinity of transcription start sites, sometimes via long-range chromatin loops.40,41 These types of long-range interactions may be a consequence of genetic alterations at more distant loci and could be pertinent deregulation mechanisms underlining outstanding unresolved monoallelic TA L1 expression in T-ALL. A systematic analysis of histone ChIP-seq data in such cases could be an efficient way to discover potential distant oncogenic neo-enhancers and should be investigated in further studies.
In addition to identifying 3’microinsertions, we demonstrated the mutation’s exclusive presence on the aberrantly-expressed allele using allele phasing. This is also true for TA L1 5’SE mutations and LMO1 and LMO2 intergenic neo-enhancer mutations. These results were expected but had not yet been proven. We circumvented the challenges of phasing distant variants in these cases by developing a new technique based on CRISPR-Cas9 genome editing technology, called Alpha-Cas, to bring these variants closer and facilitate allele phasing analysis. This method is easy to implement and could provide a useful tool to phase potentially very distant mutations and oncogenes.
- Received June 16, 2022
- Accepted December 28, 2022
No conflicts of interest to disclose.
VA, AT and CP conceived and designed the study. AT, CS, AG, DW, EA, AR, MM, AP and OM performed the experiments. AM, UT, ES and KB analyzed the sequencing data. All authors critically reviewed the manuscript.
Data generated during this study have been submitted to the NCBI Gene Expression Omnibus repository under the accession number GSE200860.
The study was supported in part by the Helmholtz-Foundation. AT was supported by a DKFZ postdoctoral fellowship. The work in the VA lab was supported by ARC-Labellisation and the associations “Force Hémato” and “Laurette Fugain.” This study was also supported in the lab by grants from INCA PLBIO18-031, PLBIO 2018-00252, and ITMO Cancer Epig-2015 (to VA and SS). SS was supported by the Ligue Contre le Cancer (labeling 2018) and the RNA-sequencing was funded by LIGUE. This work was also supported by La Fondation pour la Recherche Médicale with grant FDT202012010638 (to CS). The ONT sequencing was also supported by ANR funding KREM-AIF (ANR-21-CE17-0014-02) (to EA).
The authors would like to thank the Genomics and Proteomics Core facilities at DKFZ and the Transcriptomics and Genomics, Marseille-Luminy platform for sequencing the ChIP-seq samples. We would also like to thank Guillaume Charbonnier for his help processing the NGS data and Frederic Tores from the bioinformatics platform of Imagine Institute for the ONT analysis.
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