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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

¹ Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures), Department of Cell Cultures, Braunschweig, Germany; ² Department of Hematology, Hemostasis and Oncology, Hanover Medical School, Hanover, Germany; ³ BIOBASE GmbH, Wolfenbüttel, Germany; and ⁴ Duke University, Institute for Genome Sciences and Policy, Durham, North Carolina

Requests for reprints: Roderick A.F. MacLeod or Stefan Nagel, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Department of Cell Cultures, Inhoffenstrasse 7B, 38124 Braunschweig, Germany. Phone: 49-531-261-6167; Fax: 49-531-261-6150; E-mail: rml{at}dsmz.de or sna{at}dsmz.de.

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 1 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.1 coregulate ectopic homeobox gene expression in t(5;14) T-cell acute lymphoblastic leukemia by interactions mediated at the nuclear matrix. Our data document homeobox gene dysregulation by a novel regulatory region at 3'-BCL11B responsive to histone deacetylase inhibition and highlight a novel class of potential therapeutic target amid noncoding DNA. [Cancer Res 2007;67(4):1461–71]

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