Activation of TLX3 and NKX2-5 in t(5;14)(q35;q32) T-Cell Acute Lymphoblastic Leukemia by Remote 3′-BCL11B Enhancers and Coregulation by PU.1 and HMGA1

Abstract

In T-cell acute lymphoblastic leukemia, alternative t(5;14)-(q35;q32.2) forms effect dysregulation of either TLX3 or NKX2-5 homeobox genes at 5q35 by juxtaposition with 14q32.2 breakpoints dispersed across the BCL11B downstream genomic desert. Leukemic gene dysregulation by t(5;14) was investigated by DNA inhibitory treatments with 26-mer double-stranded DNA oligonucleotides directed against candidate enhancers at, or near, orphan T-cell DNase I hypersensitive sites located between 3 ¶-BCL11B and VRK1. NKX2-5 down-regulation in t(5;14) PEER cells was almost entirely restricted to DNA inhibitory treatment targeting enhancers within the distal breakpoint cluster region and was dose and sequence dependent, whereas enhancers near 3 ¶-BCL11B regulated that gene only. Chromatin immunoprecipitation assays showed that the four most effectual NKX2-5 ectopic enhancers were hyperacetylated. These enhancers clustered f1 Mbp downstream of BCL11B, within a region displaying multiple regulatory stigmata, including a TCRA enhancer motif, deep sequence conservation, and tight nuclear matrix attachment relaxed by trichostatin A treatment. Intriguingly, although TLX3/NKX2-5 promoter/exon 1 regions were hypoacetylated, their expression was trichostatin A sensitive, implying extrinsic regulation by factor(s) under acetylation control. Knockdown of PU.1, known to be trichostatin A responsive and which potentially binds TLX3/NKX2-5 promoters, effected down-regulation of both homeobox genes. Moreover, genomic analysis showed preferential enrichment near ectopic enhancers of binding sites for the PU.1 cofactor HMGA1, the knockdown of which also inhibited NKX2-5. We suggest that HMGA1 and PU

Disruption of ETV6 in intron 2 results in upregulatory and insertional events in childhood acute lymphoblastic leukaemia

“…Thus t(5;14) T-cell ALL provides a novel paradigm for oncogene dysregulation. 57,58 The T-ALL case which we have presented here showed, that as in the recurrent t(5;14)(q35;q32) translocation of T-ALL, the end result of the t(5;12)(q35;p13) was the ectopic expression of TLX3. We were able to demonstrate that TLX3 was as highly upregulated in our patient, as it was in the t(5;14) positive HPB-ALL cell line, when compared to a series of controls.…”

MEF2C is activated by multiple mechanisms in a subset of T-acute lymphoblastic leukemia cell lines

“…7 These elements co-localize to DNAse I hypersensitive sites, contain acetylated histones, bind to the nuclear matrix and are enriched in HMGA1-binding sites juxtaposed by t(5;14) rearrangements with PU.1 sites located in the promoter regions of both TLX3 and NKX2-5. 20 Treatment with oligonucleotides matching enhancer elements at 3 0 -BCL11B effected downregulation of NKX2-5 in T-ALL PEER cells where this gene is juxtaposed to BCL11B by t (5,14). 20 In the present study this enhancer inhibition assay was used to effect NKX2-5 knockdown in T-ALL cells therein to evaluate candidate downstream targets of this gene previously identified in other tissues, notably developing spleen and heart.…”

“…20 Treatment with oligonucleotides matching enhancer elements at 3 0 -BCL11B effected downregulation of NKX2-5 in T-ALL PEER cells where this gene is juxtaposed to BCL11B by t (5,14). 20 In the present study this enhancer inhibition assay was used to effect NKX2-5 knockdown in T-ALL cells therein to evaluate candidate downstream targets of this gene previously identified in other tissues, notably developing spleen and heart. 21 We report aberrant myocyte enhancer factor 2C (MEF2C) expression, which may implicate analogous activation mechanisms in T-ALL cell lines.…”

“…23 Electroporation of PEER was performed as previously described. 20 Transfection of HeLa with plasmid DNA and siRNA oligonucleotides was performed according to the manufacturers' instructions (Qiagen, Hilden, Germany). Trichostatin-A (TSA) and p38 inhibitor SB203580 were obtained from Sigma (Mü nchen, Germany), and the BCL2 inhibitor YC137 from Calbiochem (Darmstadt, Germany).…”

“…To examine the role of NKX2-5 in the expression of the 5 co-expressed genes, NKX2-5 knockdown experiments were performed in PEER by enhancer inhibition. 20 Here we used double stranded oligonucleotide DSO6 previously shown to inhibit NKX2-5 exclusively, while expression levels of neighboring genes, BCL11B and BC043585, remain unperturbed. 20 Reduction of NKX2-5 protein expression was confirmed by western blotting (Figure 1a).…”