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.
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 approximately 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.
Nagel
Stefan
S
German Collection of Microorganisms and Cell Cultures, Department of Cell Cultures, Inhoffenstrasse 7B, 38124 Braunschweig, Germany. sna@dsmz.de
Scherr
Michaela
M
Kel
Alexander
A
Hornischer
Klaus
K
Crawford
Gregory E
GE
Kaufmann
Maren
M
Meyer
Corinna
C
Drexler
Hans G
HG
MacLeod
Roderick A F
RA
D000107:Acetylation; D019457:Chromosome Breakage; D002883:Chromosomes, Human, Pair 14; D002895:Chromosomes, Human, Pair 5; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015973:Gene Expression Regulation, Leukemic; D024003:HMGA Proteins / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics* / Q000378:metabolism; D005810:Multigene Family; D015530:Nuclear Matrix / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014178:Translocation, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D000099232:Proto-Oncogene Protein Spi-1
D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D024003:HMGA Proteins; D006657:Histones; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D009841:Oligonucleotides; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D000099232:Proto-Oncogene Protein Spi-1; C479740:BCL11B protein, human; C095552:NKX2-5 protein, human; C489494:TLX3 protein, human