Research Article 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 Stefan Nagel,1 Michaela Scherr,2 Alexander Kel,3 Klaus Hornischer,3 Gregory E. Crawford,4 Maren Kaufmann,1 Corinna Meyer,1 Hans G. Drexler,1 and Roderick A.F. MacLeod1 1Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures), Department of Cell Cultures, Braunschweig, Germany; 2Department of Hematology, Hemostasis and Oncology, Hanover Medical School, Hanover, Germany; 3BIOBASE GmbH, Wolfenbu¨ttel, Germany; and 4Duke University, Institute for Genome Sciences and Policy, Durham, North Carolina Abstract Introduction In T-cell acute lymphoblastic leukemia, alternative t(5;14)- BCL11B (also known as CTIP2/Rit1) is a Kru¨ppel familyzinc (q35;q32.2) forms effect dysregulation of either TLX3 or finger gene located at 14q32, which is expressed during thymocyte À À NKX2-5 homeobox genes at 5q35 by juxtaposition with maturation from CD4 /CD8 to CD4+/CD8+ stages (1). BCL11B 14q32.2 breakpoints dispersed across the BCL11B downstream mayact either as a tumor-suppressor gene, first detected via genomic desert.Leukemic gene dysregulation by t(5;14) was inactivating mutations or deletions in murine radiation-induced investigated by DNA inhibitory treatments with 26-mer lymphomas (2) and more recently in T-cell acute lymphoblastic double-stranded DNA oligonucleotides directed against can- leukemia (T-cell ALL; ref. 3); or, as an oncogenic activator, first didate enhancers at, or near, orphan T-cell DNase I hypersen- detected byits juxtaposition with the TLX3 (also known as sitive sites located between 3¶-BCL11B and VRK1. NKX2-5 HOX11L2) homeobox gene at 5q35 via the recurrent t(5;14) down-regulation in t(5;14) PEER cells was almost entirely (q35;q32) rearrangement also in T-cell ALL (4–6). A neighboring restricted to DNA inhibitory treatment targeting enhancers related homeobox gene, NKX2-5 (also known as CSX), is similarly within the distal breakpoint cluster region and was dose and activated in T-cell ALL bya variant t(5;14)(q35;q32) or by sequence dependent, whereas enhancers near 3¶-BCL11B t(5;14)(q35;q11.2) involving the T-cell receptor D locus at 14q11.2 regulated that gene only.Chromatin immunoprecipitation (7, 8). Recently, interleukin-2 has been identified as a transcrip- assays showed that the four most effectual NKX2-5 ectopic tional target of BCL11B in both developing and transformed T enhancers were hyperacetylated.These enhancers clustered cells (9). The ectopic expression of Nirenberg-Kim family f1 Mbp downstream of BCL11B, within a region displaying homeobox genes (e.g., TLX3) has been recentlyshown to drive multiple regulatory stigmata, including a TCRA enhancer proliferation of myeloid progenitor cells in a mouse model (10). motif, deep sequence conservation, and tight nuclear matrix Nevertheless, the mechanism underlying leukemic deregulation by attachment relaxed by trichostatin A treatment.Intriguingly, t(5;14) remains obscure, mainlybecause 14q32.2 breakpoints are although TLX3/NKX2-5 promoter/exon 1 regions were hypo- widelyscattered over the f1 Mbp downstream breakpoint cluster acetylated, their expression was trichostatin A sensitive, region. Analogies with T-cell receptor loci and the nature of the implying extrinsic regulation by factor(s) under acetylation ‘‘genomic desert’’ covering 3¶-BCL11B support the existence of PU.1 control.Knockdown of , known to be trichostatin A underlying interactions between TLX3/NKX2-5 and remote TLX3 NKX2-5 responsive and which potentially binds / pro- transcriptional enhancers. moters, effected down-regulation of both homeobox genes. Chromosomal mechanisms governing remote transcriptional Moreover, genomic analysis showed preferential enrichment regulation have latelyattracted increasing interest. In particular, near ectopic enhancers of binding sites for the PU.1 cofactor conserved noncoding elements/regions/sequences identified near NKX2-5 HMGA1, the knockdown of which also inhibited .We metazoan developmental genes show coordinate enhancer activity suggest that HMGA1 and PU.1 coregulate ectopic homeobox when tested in zebrafish embryos (11). Physical juxtaposition gene expression in t(5;14) T-cell acute lymphoblastic leukemia between regulators and their targets has now been shown (12). by interactions mediated at the nuclear matrix.Our data These interactions are believed to occur at chromatin loops tethered document homeobox gene dysregulation by a novel regulatory to nuclear scaffold matrix attachment regions (13–15). DNaseI ¶ BCL11B region at 3 - responsive to histone deacetylase inhibi- hypersensitive sites denote ‘‘active’’ chromatin and are believed to tion and highlight a novel class of potential therapeutic target heighten the accessibilityof nucleosome-depleted regulatoryDNA amid noncoding DNA. [Cancer Res 2007;67(4):1461–71] regions and facilitate loading of transcription factor binding sites. Chromatin relaxation is also associated with histone hyperacetyla- tion (16) and is antagonized byhistone deacetylases.A global survey of regulatoryregions in CD4 + T cells uncovered >5,000 DNaseI Note: Supplementarydata for this article are available at Cancer Research Online hypersensitive sites mostly bracketing active genes (17). A notable (http://cancerres.aacrjournals.org/). exception included an orphan DNaseI hypersensitive site cluster Requests for reprints: Roderick A.F. MacLeod or Stefan Nagel, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Department of Cell Cultures, lying inside the BCL11B gene desert amid a region subsequently Inhoffenstrasse 7B, 38124 Braunschweig, Germany. Phone: 49-531-261-6167; Fax: 49- shown to host an acetylation island in T cells (18). 531-261-6150; E-mail: [email protected] or [email protected]. ¶ I2007 American Association for Cancer Research. To investigate the role of 3 -BCL11B in leukemic cis-activation, doi:10.1158/0008-5472.CAN-06-2615 we adapted DNA inhibitorytreatments byusing matching www.aacrjournals.org 1461 Cancer Res2007; 67: (4). February 15, 2007 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Cancer Research oligonucleotides directed against specific noncoding sequences Chromatin immunoprecipitation assay. Chromatin immunoprecipi- based on previous work on the IgH enhancer (19, 20). Regulatory tation (ChIP) was done as recommended using the ChIP Assaykit (Upstate), 6 potential was further analyzed via nuclear matrix attachment in whereby10 cells were sonicated for 30 seconds, producing fragments A nuclear halo preparations, and bycharacterizing protein acetyla- ranging 100 to 1,000 bp, and precipitated with 10 g of the anti–acetyl- histone H3 antibody. For PCR analysis, 400 ng of DNA (ChIP or complete tion associated with both genes and noncoding regions. Binding genomic DNA) were used as templates (SupplementaryTable S1). site matrices for transcription factors PU.1 and HMGA1, identified PCR. Total RNA was isolated 6 or 24 h after trichostatin A treatment, in the promoter regions of TLX3 and near regulatoryDNaseI either 24 h after electroporation with the individual oligonucleotides, or 4 to hypersensitive sites, respectively, by using the TRANSFAC database 5 days after lentiviral transduction. RNA extraction, cDNA synthesis, and (21) were targeted for inhibition bysmall interfering RNA (siRNA) PCR were done as described previously(7). Real-time quantitative-PCR (RQ- treatment, and their effects on leukemic homeobox gene PCR) was done as described previously(28). Oligonucleotide sequences are expression in t(5;14) cells were measured. listed in SupplementaryTable S1. Our data support the existence of a remote regulatoryDNaseI Oligonucleotide inhibitory treatments. Transient inhibitorytreat- hypersensitive site cluster associated with leukemic homeobox ments were modified from that described byCutrona et al. (20), substituting gene activation in t(5;14) T-cell ALL. TRANSFAC data show this double-stranded 26-mer double-stranded oligonucleotide (DSO) phosphor- othioate modified at positions 1-2/25-26 (purchased from MWG) for peptide putative regulatoryenhancer region to be enriched in HMGA1 nucleic acid oligonucleotides. In 10 of 16 cases, DSO sequences (Table 1) binding sites juxtaposed bypartner PU.1 sites at promoter regions were uniquelylocated at, or near ( V1 kbp), orphan T-cell DNaseI of both TLX3 and NKX2-5. We propose that leukemic activation hypersensitive sites (17), whereas the remainder lay either offset by f1 overlays HMGA1-PU.1 interactions mediated cytogenetically by to 10 kbp (DSO.12-14), or remote by>10 kbp (DSO.3/5), from DNaseI transcription factor binding site juxtaposition, highlighting the hypersensitive sites. Target sequences were chosen using TRANSFAC role of noncoding DNA in neoplasia and as a potential therapeutic databanks listing paired transcription factor binding sites displaced by target therein. <10 bp (the so-called composite elements; ref. 21). The selection process is shown in SupplementaryFig. S1 A for DSO.6. DSOs were produced by pooling equimolar amounts of complementarysingle-stranded oligonucleo- tides dissolved in water, heating to 95jC for 5 min and incubating at room Materialsand Methods temperature for 30 min. For electroporation, 2 Ag pEGFP-N1 reporter Cells and culture. Detailed descriptions of cell lines and culture (Clontech, Heidelberg, Germany) or DSO (20 Amol/L) were used to treat 106 methods