citations

Site- and allele-specific polycomb dysregulation in T-cell leukaemia

Authors:

Navarro JM1, Touzart A2, Pradel LC1, Loosveld M3, Koubi M4, Fenouil R1, Le Noir S2, Maqbool MA1, Morgado E1, Gregoire C1, Jaeger S1, Mamessier E1, Pignon C1, Hacein-Bey-Abina S5, Malissen B1, Gut M6, Gut IG6, Dombret H7, Macintyre EA2, Howe SJ8, Gaspar HB8, Thrasher AJ8, Ifrah N9, Payet-Bornet D1, Duprez E10, Andrau JC1, Asnafi V2, Nadel B1

In:

Source: Nat Commun.
Publication Date: (2015)
Issue: 6: 6094

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Experiment

Genome editing in T-ALL cell line by type II CRISPR system PEER T-ALL cells line were cultured in RPMI medium (Life Technologies) containing 20% fetal calf serum, 1% L-glutamine, 1% sodium pyruvate and 100?U?ml-1 penicillin/streptomycin (Life Technologies) at 37?°C in the presence of 5% CO2. The day of transfection, 1 million cells were nucleofected according to the manufacturer’s instruction (Lonza), with 500?ng DNA donor sequence containing Neomycin-resistant gene and 2?µg of the Cas9/gRNA expression vector (Addgene #42230). The chimeric guide RNA targeted TAL1 insertion site, and was cloned according to Cong et al.41. One day after nucleofection, cells were plated in 96-wells plate at 104 cells per well and incubated in presence of 1,200?µg?ml-1 geneticin G418 (Life Technologies) for 2 weeks. After selection and growing, a PCR was conducted to amplify the targeted region with genomic DNA derived from the surviving clones, and amplicons were separated on a 1% agarose gel then extracted with GEL/PCR clean up wizard (Promega) and sequenced (MWG-Biotech). CRISPR guide RNA: 5'-GAAAGACGTAACCCTACTTCC-3'.

Abstract

T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation.

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