A Novel Chromosomal Translocation T(1;14)(Q25;Q32) in Pre-B Acute Lymphoblastic Leukemia Involves the LIM Homeodomain Protein Gene, Lhx4

A novel chromosomal translocation t(1;14)(q25;q32) in pre-B acute lymphoblastic leukemia involves the LIM homeodomain protein gene, Lhx4

Norihiko Kawamata*,1, Sakura Sakajiri1, Kei-ji Sugimoto1, Yasushi Isobe1, Hirofumi Kobayashi1,2 and Kazuo Oshimi1

1Division of Hematology, Department of Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan; 2Division of Hematology, Department of Medicine, Saitama Cancer Center, 818 Komuro, Ina, Saitama 362-0806, Japan

Chromosome 1q21-25 is one of the hotspots of Introduction chromosomal abnormalities including translocations and duplications in hematological malignancies. This would In acute lymphoblastic leukemia (ALL), several genetic suggest that oncogene(s) reside in this region. We have abnormalities associated with leukemogenesis have cloned the junctional sequence of t(1;14)(q25;q32) in pre- been reported (Look, 1997). In adult ALL of B cell B acute lymphoblastic leukemia cells by an inverse PCR lineage, t(9;22)(q32;q11) resulting in the generation of method. A novel sequence was fused to the joining region an oncogenic fusion gene, BCR – ABL, is most of the immunoglobulin heavy chain gene. We confirmed frequently detected. In addition to BCR – ABL, this rearrangement by Southern blot analysis, genomic ETV6 – AML1, E2A – Pbx, IgH – IL3, E2A – HLF and PCR and fluorescence in situ hybridization. We found a rearrangements involving the MLL gene are detected in coding sequence which is homologous to the mouse Lhx4 ALL of B-cell lineage (Look, 1997; Willis and Dyer, cDNA sequence 17 kb from the breakpoint. The human 2000). However, many oncogenes involved in trans- Lhx4 gene encodes 390 amino-acids, including one locations in ALL remain to be analysed. tandem pair of LIM domains and one homeodomain. In human cancer cells, oncogenes are frequently The human Lhx4 gene consists of six exons. Lhx4 overexpessed by transcriptional deregulation (Lengauer protein is very homologous to human Lhx3 protein et al., 1998; Nowell, 1997; Popescu and Zimonjic, except in the N-terminal region. The transcripts of the 1997). Gene amplification of oncogenes induces Lhx4 gene were not detected in adult multiple tissues overexpression. This amplification is usually detected analysed by Northern blotting, but were detected in the as double minutes or homogeneously stained regions leukemic cells carrying t(1;14)(q25;q32) by reverse- on karyotyping (DeVita et al., 2001). Translocation transcription PCR. The protein expression of Lhx4 in also results in the overexpression of oncogenes, in these leukemic cells was confirmed by Western blot which the regulatory region of the oncogenes is analysis. Lhx4 activated the reporter gene carrying the replaced by the regulatory region, including enhancer mouse a-glycoprotein subunit promoter region, which is elements, of other genes (Willis and Dyer, 2000). In regulated by Lhx3. LIM protein and homeodomain hematological malignancies derived from B lympho- protein genes are frequently involved in translocations of cytes, rearrangements between the immunoglobulin hematological malignancies. The Lhx4 gene is deregu- heavy chain (IgH) gene, which resides on chromo- lated in the leukemic cells and Lhx4 protein may play an some 14q32, and oncogenes are frequently detected important role, possibly as an activator, in leukemogen- (Willis and Dyer, 2000). In these rearrangements, the esis. regulatory region of the oncogenes is replaced by the Oncogene (2002) 21, 4983 – 4991. doi:10.1038/sj.onc. enhancer region of the IgH gene, which induces 1205628 overexpression of the oncogene and is associated with tumorigenesis of the B cell lymphoid malignancies Keywords: inverse PCR; transcriptional factor; immu- (Willis and Dyer, 2000). noglobulin heavy chain gene; RT – PCR; reporter gene Dup(1q21-25) is frequently detected in ALL of B-cell assay lineage (Mitelman et al., 1997). Similarly, lymphomas and myelogenous malignancies often have genetic abnormalities involving this region (Mitelman et al., 1997). 1q21-25 is one of the hotspots, areas of chromosomal alteration including duplications and translocations, frequently detected in hematological malignancies (Mitelman et al., 1997). Although it is not clear whether duplications result in the gene *Correspondence: N Kawamata; E-mail: [email protected] amplification at present, this structural change in Received 6 November 2001; revised 18 April 2002; accepted 26 chromosomes might deregulate the expression pattern April 2002 of the genes. Novel oncogene(s) might reside in this IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4984 region and overexpression of the gene(s) might be using one primer specific to IgH (JH4AS) and one associated with leukemogenesis. novel sequence (WR1). PCR products were obtained T(1;14)(q25,q32) is a novel chromosomal abnorm- from the leukemic cells but not from control DNA ality detected in ALL of B-cell lineage. This (Figure 3a). The sequence of the PCR product was translocation might involve the IgH gene and novel confirmed (data not shown). Further, we performed genes. Overexpression of a novel gene residing on 1q25 Southern blot analysis using one probe specific to IgH and fusion to the IgH gene could be associated with and a novel sequence (w360 probe). Using the IgH leukemogenesis. probe, two rearranged bands were detected on HindIII In this study we cloned a junctional sequence of digestion and one rearranged band on EcoRI digestion, t(1;14)(q25;q32) and found a fusion of the IgH gene and using the w360 probe, a single rearranged band and a LIM homeodomain gene. was detected in the EcoRI and HindIII digest, respectively (Figure 3b). On HindIII digestion, one of the two rearranged bands detected by the IgH probe Results and the rearranged band detected by the W360 probe co-migrated (asterisks in Figure 3b). On EcoRI Clinical sample digestion, the rearranged band detected by the w360 A 53-year-old female was admitted to our hospital probe co-migrated with the germ line band detected by because of fever. She was diagnosed as having acute the IgH probe. This result might suggest that one of lymphoblastic leukemia of pre-B cell type. She was the rearranged bands detected by the IgH probe co- treated with multiple reagent chemotherapy. A migrated with the germ line band on EcoRI digestion, complete remission was achieved after induction and that the rearranged bands detected by the IgH and therapy, and consolidation and maintenance therapy w360 probes also co-migrated on EcoRI digestion. were performed. During the maintenance therapy, the Then we screened the phage genomic library using patient was transferred to another hospital to be the w360 probe and obtained overlapping clones treated by allogeneic bone marrow transplantation. (Figure 4). Using one of the phage clones (lambda Q-banded karyotyping of her leukemic cells were 1212) as a probe, we performed FISH to analyse the performed as previously reported (Sandberg, 1990). chromosomal localization of the novel sequence. The The karyotyping of her leukemic cells at admission signals were detected at 1q23-25 (Figure 5a). These revealed t(9;22)(q23;q11) and t(1;14)(q25;q32) (Figure results confirmed that the junctional sequence we 1a). After complete remission, these chromosomal cloned is derived from t(1;14)(q25;q32). Further, we abnormalities disappeared. Thus, she was diagnosed detected fusion signals between the IgH gene and the with de novo ALL, not blastic crisis from chronic novel sequence in these leukemic cells by the dual color myelogenous leukemia. FISH analysis (Figure 5b) (see below). T(1;14)(q25;q32) was detected in acute lymphoblastic We determined the genomic sequence around the leukemia of pre-B cell type. We employed inverse PCR breakpoint in the novel sequence and compared it with for the DNA from the leukemic cells. We obtained the rearranged sequence. The N-segment of 24 nucleo- several bands (Figure 1b) then we cloned the products tides was inserted between JH4 and the novel sequences into plasmids and sequenced them. Two bands revealed (Figure 2). Further, we sequenced the phage clones and a novel sequence fused to the IgH gene (Figure 2). The searched for a gene around the breakpoint. We found a breakpoint resided in the JH4 region of the IgH gene sequence highly homologous to mouse Lhx4 gene clones (Ravetch et al., 1981). To exclude artifacts during the about 25 kb from the break point (Figure 4). inverse PCR procedure, we performed a genomic PCR To obtain the coding sequence of this putative gene, we performed anchor PCR on HEK293 cells. Then, we assembled the sequences and obtained a 1243-nucleotide cDNA sequence which had a single open reading frame of 1170 nucleotides (Figure 6a). The deduced amino-acid sequence was highly homologous to that of mouse Lhx4 (Figure 6b), so we concluded that this gene is a human homolog for mouse Lhx4 and named it the human Lhx4 gene. After our submission of this manuscript, two other groups have reported the cDNA sequence of the human Lhx4 gene (Liu et al., 2002; Machinis et al., 2001). In the sequence reported by Machinis et al. (2001) the nucleotide sequence encoding Figure 1 Chromosomal abnormality and inverse PCR. (a) N-terminal region is missing. The cDNA sequence of T(1;14)(q25;q32) was detected in the leukemic cells. Numbers indi- the human Lhx4 gene reported by Liu et al. (2002) is cate chromosome numbers. Arrows indicate the translocated chro- the same as that reported in this study. mosomes. (b) Inverse PCR was performed on XbaI digested (Xb) The human Lhx4 gene encodes a product of 390 and BglII (Bg) digested DNA (see Materials and methods). Several PCR bands were obtained and all bands were sequenced. Arrow amino-acids, which is homologous to the Lhx family heads indicate the bands having the IgH gene fused to a novel se- proteins (Dawid et al., 1998). There is one tandem pair quence of LIM domains and one homeodomain, both of which

Oncogene IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4985

Figure 2 The junctional sequence of t(1;14)(q25;q32). IgH: the germinal IgH sequence. Fusion: the fusion sequence obtained from the leukemic cells by the inverse PCR. 1q25: the normal sequence of the novel sequence from 1q25. Bars indicate matching nucleotides. J4: J4 exon of the IgH gene. XbaI: the restriction enzyme XbaI site. A novel sequence is fused to the J4 sequence. There is 24 nucleotides of a N-segment between these two sequences

Lhx4 is homologous to human Lhx3. However, the N- terminal sequence is completely different (Figure 6b). Two isoforms (Lhx3A and Lhx3B) of human Lhx3 have been reported, in which the N-terminal regions are completely different (Sloop et al., 1999). The transcriptional activity of these two proteins is different; Lhx3A induces expression of a reporter gene carrying the promoter region of the aGSU gene, which had a Lhx3 binding sequence. In contrast to Lhx3A, Lhx3B can not induce expression of the reporter gene (Sloop et al., 1999). These results suggested that the N- terminal sequence of Lhx3 and Lhx4 might influence the functions of these proteins, especially transcriptional activity. Comparing the human Lhx4 cDNA sequence with genomic sequences of the phage clones and genomic sequences from the human genome project data base, Figure 3 Cloning of the breakpoint region from the leukemic we found that the human Lhx4 gene consists of six cells. (a) Genomic PCR. To exclude cloning artifacts, we exons (Figure 4 and 6c) and all exon-intron performed PCR using an IgH-specific primer (JH4) and a novel sequence, we cloned, the specific primer WR1. Lane1: 100bp DNA boundaries are compatible with the GT-AG exon- ladder (New England Biolabs). Lane 2: control DNA from a T-cell intron boundary rule (Breathnach and Chambon, line, Jurkat. Lane3: DNA from the leukemic cells. The PCR pro- 1981). In this translocation, the enhancer region of duct was obtained from the leukemic cells. (b) Southern blot ana- the IgH gene is fused to the 5’ regulatory region of lysis. The DNA from the leukemic cells was digested with EcoRI (lane 1) or HindIII (lane 2). The blots were hybridized with IgH the Lhx4 gene in a head-to-head configuration. The probe (right panel) or a novel sequence we cloned, the exon encoding the initiation site of the Lhx4 resides specific probe w360 (left panel). Asterisks and diamonds indicate about 17 kb from the breakpoint (Figure 4). Further, rearranged bands. The asterisks indicate the co-migrating bands we performed two color FISH on the leukemic cells detected by the IgH and w360 probes on HindIII digestion using the IgH gene and the PAC clone containing the Lhx4 gene as probes. We found fusion signals between the IgH and Lhx4 genes in most of the are conserved in Lhx proteins, including human Lhx2 leukemic cells carrying t(1;14)(q25;q32) (97%) (Figure (Figure 6b). The highest homology was observed for 5b). mouse Lhx4 except in the N-terminal region. The We found the Lhx4 gene to be expressed in sequence of the N-terminal end of the mouse Lhx4 HEK293 cells. In mouse, it is reported that Lhx4 is gene has not yet been reported. In not only LIM and important for organogenesis of pituitary. We used the homeodomain regions but also C-terminal regions, human cell line, HP75, established from human

Oncogene IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4986

Figure 4 Schema of genomic structure around the breakpoint on chromosome 1q25. Upper: overlapping lambda phage clones. Low- er: genetic map around the breakpoint at 1q25. An arrow indicates the breakpoint. Boxes indicate exons. Numbered boxes indicate the exons of the Lhx4 gene. The numbers of exons are provisional. A black bold bar (IgH enhancer) shows the enhancer region of the IgH gene. A circle indicates the CpG island. A small black box indicates the position of the probe used for Southern blot analysis

Figure 5 Chromosomal localization of the novel sequence and fusion signals between the IgH and Lhx4 genes. (a) Lambda 1212 was used as a probe for ﬂuorescence in situ hybridization. The signals were detected at 1q23-25 (arrow heads). (b) Fusion signals between the IgH and Lhx4 genes were detected in the leukemic cells. A green signal indicates the IgH gene, a red signal the Lhx4 gene and a yellow signal the fusion signal between the IgH and Lhx4 gene (arrow)

pituitary adenoma (Jin et al., 1998), for Northern blot Lhx4 gene and expression of the transcript and protein analysis and detected the transcript to be about 4.3 kb of Lhx4 in the leukemic cells. long (Figure 7a). To analyse the expression pattern of Further, we performed a reporter gene assay to the Lhx4 gene in normal tissues, we performed define the transcriptional activity of Lhx4. Lhx4 has a Northern blotting and found no expression in any homeodomain identical to that of the human Lhx3, so adult tissue examined (Figure 7b). We detected we used the aGSU reporter gene which is controlled by expression of the Lhx4 gene in the original case with Lhx3 (Bach et al., 1995). As a positive control, we t(1;14)(q25;q32) by RT – PCR (Figure 7c). Then, we transfected the Lhx3A gene, resulting in induction of performed Western blot analysis to confirm the reporter gene expression (Figure 8 upper). Lhx4 also protein expression of Lhx4 in leukemic cells. The size strongly induced expression of the reporter gene of the protein was 47 kDa and leukemic cells carrying (Figure 8 lower). Nuclear LIM interactor (NLI) (Jurata t(1;14)(q25;q32) expressed Lhx4 (Figure 7d). Recently, et al., 1996), also called Ldb (LIM-domain binding) Sloop et al. (2001) have reported that a functional (Agulnick et al., 1996) or CLIM (cofactor of LIM alternate translation form are generated from Lhx3 homeodomain protein) (Bach et al., 1997), binds the transcripts even in transfection analysis using the full- LIM domains of LIM homeodomian proteins and length Lhx3 cDNA expression vector. In our experi- influences their transcriptional activity (Agulnick et al., ments, we could not detect an alternate translation 1996; Bach et al., 1997; Jurata et al., 1996). Co- form of Lhx4. transfection of the NLI genes with the Lhx3A or Lhx4 These results suggested that the rearrangement of the gene decreased the induction of expression of the IgH and Lhx4 genes led to the deregulation of the reporter genes (Figure 8).

Oncogene IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4987

Figure 6 Structure of human Lhx4. (a) cDNA sequence and deduced amino-acid sequence of the Lhx4 gene. Lines indicate LIM domains (tandem pair of LIM domain) and a bold line indicates the homeodomain. (b) Structure and homology of human Lhx4. Bold lines indicate LIM domains and dots in the LIM domain indicate the residues conserved between Lhx4, Lhx3 and Lhx2. A box indicates the homeodomain. The sequence between the LIM domain and homeodomain of Lhx2 (444) was partially deleted to compare the structures of the four proteins. Lhx3 has two isoforms (Lhx3A and Lhx3B) and amino-acid sequences of these two proteins are identical except in the N-terminal region. So, the N-terminal sequence of the Lhx3B is shown. (c) Exon-intron boundaries of the Lhx4 genes. Capital letters indicate coding regions, small letters indicate intron sequences. All exon-intron junctional sequences are compatible with the GT-AG rule. Numbers of introns are provisional

Oncogene IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4988

Figure 7 Northern blot, RT – PCR and Western blot analysis of Lhx4. (a) Northern blot analysis of HEK293 cells. Twenty micrograms of total RNA extracted from HEK293 was electrophoresed. The membrane was hybridized with the Lhx4 probe. The size of the Lhx4 transcript is about 4.3 kb. (b) Northern blot analysis of multiple organ tissues. Two micrograms of mRNA of normal tissues was electrophoresed. Upper panel: Lhx4 expression. Lower panel: beta- microglobulin expression as an internal control. 1: brain, 2: heart, 3: skeletal muscle, 4: colon, 5: thymus, 6: spleen, 7: kidney, 8: liver, Figure 8 Reporter gene assay. Upper: Lhx3A transfection. Low- 9: small intestine,10: placenta, 11: lung, 12: peripheral blood leuko- er: Lhx4 transfection. Results are expressed as fold-activation, cyte. (c) RT – PCR analysis. Total RNA extracted from cell line mean+standard deviation of the mean, compared to the activity and the clinical sample was subjected to RT – PCR. Multiplex of the aGSU promoter in the presence of an empty vector. The PCR was performed using the primers for the Lhx4 and beta 2 mi- combinations of co-transfected expression vectors and aGSU re- croglobulin (b2MG) genes. Upper band: RT – PCR for the Lhx4 porter vectors are indicated gene. Lower bands: RT – PCR for the b2MG gene as an internal control. The PCR products of Lhx4 were detected in the leukemic cells with t(1;14)(q25:q32). 1: marker DNA, 2: A549 (negative control), 3: the leukemic cells with t(1;14). (d) Western blot analysis. hematopoietic cells (Buske and Humphries, 2000; Upper: Lhx4 protein expression. An arrow head indicates the Tenen et al., 1997). Lhx4 proteins. Lower: beta-actin expression for an internal control. Chromosomal translocations in leukemias involve 1: A549 cells transiently transfected with a empty vector (negative several homeobox genes (Buske and Humphries, 2000; control). 2: A549 cells transiently transfected with Lhx4 expression vector (positive control). 3: leukemic cells with t(1;14)(q25; q32) Look, 1997). In T cell ALL with t(10;14)(q24;q11), the Hox11 gene (10q24) is translocated to the TCR gene (14q11), resulting in overexpression of Hox11. Pbx, which is frequently involved in E2A-Pbx rearrangement of pre-B ALL with t(1;19)(q23; p13), is also one of the Discussion homeodomain proteins. The MLL gene, which is involved in 11q23 translocations and has many We found a rearrangement of the IgH and Lhx4 translocation partner genes, also encodes a homeo- genes, in which the 5’ regulatory region of the Lhx4 domain protein. Alteration of these homeodomain gene was replaced by the enhancer region of the IgH protein genes influences the normal development of gene. This could lead to the overexpression of the hematopoietic cells and could lead to leukemogenesis. Lhx4 gene in leukemic cells. Lhx4 has a home- LIM homeodomain proteins differ from other odomain which is one of the DNA binding domains homeobox proteins in several structural features and binds to a specific DNA sequence (Buske and (Dawid et al., 1998). They have one tandem pair of Humphries, 2000). Genes encoding proteins having LIM domains and one homeodomain, and are also homeodomains are called homeobox genes, and transcriptional factors for neurogenesis (Sharma et al., include Hox and Pbx genes. These genes play 1998; Sheng et al., 1997; Thor et al., 1999). The LIM important roles in organogenesis and morphorogen- domain is a cysteine-rich sequence recognized in the esis (Buske and Humphries, 2000; Tenen et al., 1997). three homeobox gene products, Lin-1, Iyl-1, and Mec-3 Additionally, some homeobox proteins function in (Dawid et al., 1998; Dawid and Chitnis, 2001; Freyd et the normal differentiation and proliferation of al., 1990). The LIM domains contributes to protein –

Oncogene IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4989 protein interaction and is bound by NLI/CLIM2/Ldb1, of Lhx4 might be associated with anti-apoptosis rather leading to an increase in the transcriptional activity of than cellular proliferation. Nevertheless, identifying the LIM homeodomain proteins (Agulnick et al., 1996; target genes controlled by Lhx4 could be a key to Bach et al., 1997; Jurata et al., 1996). However, in our elucidating the contribution of Lhx4 to leukemogen- system, co-transfection of NLI with Lhx4 or Lhx3A esis. suppressed the transcriptional activity of Lhx4 or We screened for the rearrangements of 10 cases with Lhx3A. Some feature of the cell line, A549, we used pre B ALL using the w360 probe and found none might cause this difference in the function of NLI. (Sakajiri et al. unpublished data). IgH/Lhx4 rearrange- Nevertheless, the Lhx4 gene is deregulated in leukemic ments might be rare in ALL, or the breakpoints might cells and the Lhx4 protein may play an important role, exist outside the regions detected using the w360 probe. possibly as an activator, in leukemogenesis. Unfortunately, we could not perform FISH analysis LMOs, including LMO1 (Rhombotin 1, ttg1) and and RT – PCR on these clinical samples. We examined LMO2 (Rhombotin 2, ttg2), also members of the LIM the levels of Lhx4 in human leukemic and cancer cell family, were cloned from translocations of lines by RT – PCR and found expression in HEK293 t(11;14)(p15;q11) and t(11;14)(p13;q11) of T-cell ALL. (embryonic kidney) and SY5Y (neuroblastoma) cell Although LMOs have one tandem pair of LIM lines (data not shown). Recently, overexpression of domains, they lack a homeodomain and do not directly Lhx2 has been reported in chronic myelogenous bind to DNA (Rabbitts, 1998). A complex of LMO, leukemias and acute lymphoblastic leukemias (Wu et SCL and GATA1 proteins is detected in premature al., 1996; Wu and Minden, 1997). Demethylation of the erythroid cells. Mice deficient in LMO1 cannot promoter region of the Lhx2 gene could be associated generate hematopoiesis, which suggests that LMOs with deregulation of this gene. Although no functional contribute to hematopoiesis (Rabbitts, 1998). LMO contribution of Lhx2 to leukemogenesis was reported, transgenic mice develop leukemia after a long period, Lhx family proteins might play important roles in the which suggests that the overexpression of LMOs is not cellular transformation of B-lymphocytes. enough for leukemogenesis and a ‘second hit’ promotes the development of leukemia in these mice (Rabbitts, 1998). The structure of the tandem pair of LIM Materials and methods domains in LMOs and in Lhx4 are homologous. The LIM domains of Lhx4 might also function as LMOs DNA extraction and Southern blotting and be associated with cellular transformation in B-cell Genomic DNA was extracted from the leukemic cells by the lineage ALL. standard phenol-chloroform method as previously described Some of the target genes of Lhx3, which is very (Sambrook and Russell, 2001) with informed consent. The homologous to Lhx4, were pituitary hormone genes DNA was digested with restriction enzymes, EcoRI or HindIII (Bach et al., 1995). However, no target genes for (Takara, Kyoto, Japan), and electrophoresed in 0.8% agarose proliferation or anti-apoptosis have been reported for gel containing Tris acetate EDTA (TAE) buffer. It was then transferred to nylon membrane (Hybond N+, Amersham- Lhx proteins. In organogenesis, it is not clear which pharmacia, Uppsala, Sweden) as recommended by the genes are the target of Lhx4. Mice deficient in Lhx4 manufacturer. The membrane was hybridized with radio- had an impaired respiratory function and died (Li et labeled DNA probes. A probe specific to the JH region of the al., 1994). Because of this early death, no study was immunoglobulin gene (Miki et al., 1994), and the w360 probe performed on the function of the lymphocytes of these specific to the 1q25 region was generated by PCR using a pair of mice. Recently, Machinis et al. (2001) have reported primers, W1:CAG GCC GGA GTC CAG TAG TA and mutations of the human Lhx4 gene which cause the WR1:GCA CTT TAG GGA GCT TTG AG, from normal short stature syndrome. They speculated that the human DNA. The PCR products were ligated into pGEM-T mutations of the Lhx4 gene lead the defect of pituitary vector (Promega, Madison, WI, USA) and the nucleotide and lack of hormones, including growth hormone, sequence was confirmed. The radio-labeled probes were generated using a Ready-to- which could result in the cause of short stature go DNA labeling kit and 32P-dCTP (Amershampharmacia) (Machinis et al., 2001). There is no report about the according to the manufacturer’s protocols. Pre-hybridization, impairment of hematopoiesis in Lhx4-deficient mice hybridization and washing were performed as recommended and human (Li et al., 1994; Machinis et al., 2001). The by the manufacturer. The membranes were exposed to exact function of Lhx4 in hematopoiesis remains to be imaging plates and analysed by an imaging analyser BAS disclosed. At present, it is unclear how Lhx4 is 2500 (Fuji Film, Tokyo, Japan). associated with leukemogenesis. We performed a cellular transformation assay using rat fibroblast RNA extraction, Northern blotting and reverse transcription (Rat1) and mouse fibroblast (NIH3T3) cells, however PCR (RT – PCR) we could not define the transformation activity in the Total RNA from clinical samples and cell lines was extracted Lhx4 gene (Sakajiri et al. unpublished data). The using Isogen solution (Nippon gene, Tokyo, Japan). A functions of Lhx4 in tumorigenesis might not be human pituitary adenoma cell line, HP75 was provided from defined by these systems. Recently, Liu et al. (2002) Dr RV Lloyd (Jin et al., 1998). Human leukemic cell lines, reported the Lhx4 are predominantly expressed in fetal HL60 (myeloid), U937 (monocyte), K562 (erythroid), Jurkat and adult central nervous system. Adult neuronal cells (T-cell), Daudi, Raji (B-cell), HEK293 (embryoic kidney), do not usually proliferate rapidly. So, overexpression A549 (Lung cancer), MCF7 (breast cancer) and SY5Y

Oncogene IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4990 (neuroblastoma) were provided from American Type Culture (Promega), the PCR products were sequenced on a Genetic Collection (Manassas, VA, USA). Aliquots (20 mg) of the analyzer 310 (Applied biosystems, Foster City, CA, USA). RNA were electrophoresed in 1% agarose gel containing To confirm the fusion sequence, we performed PCR using 2.2 M formaldehyde as previously described (Sambrook and the JH-specific primer JH4-AS:GCA GGA GAG AGG TTG Russell, 2001). They were then transferred to nylon TGA GG and the WR1 primer, on DNA extracted from the membrane (Hybond N+) according to the instruction leukemic sample carrying t(1;14) and or a Tcell line, Jurkat, manual. A multiple tissue blot was purchased from Clontech as a control. (Palo Alto, CA, USA). Pre-hybridization, hybridization and To obtain the full length of the human Lhx4 cDNA washing were performed as recommended by the manufac- sequence, we performed anchor PCR on RNA extracted from turer. The coding region of the human Lhx4 gene was used human embryonic kidney (HEK) 293 cells using the Mara- for the radio-labeled probe. As an internal control, the beta 2 thon cDNA amplification kit (Clontech) according to the microglobulin (b2MG) gene was radio-labeled. The signals manufacturer’s protocol. The sequences were assembled and were exposed to imaging plates and analysed by the BAS2500 a single open reading frame was identified. (Fuji Film). RT – PCR was performed with the total RNA using Genomic library screening Ready-To-Go RT – PCR beads (Amershampharmacia) according to the manufacturer’s protocol. For detection of A Lambda phage genomic library was obtained from Health the Lhx4 transcripts, we used as primers, Det-Lhx4S: GGG Science Research Resources Bank (Tokyo, Japan). The AGC GTC TAC TGC AAG G and Det-Lhx4AS: GCT TAG library was screened using the w360 probe as previously CTC CAG CCT CTG A. As an internal control, we described (Sambrook and Russell, 2001). A PAC library was amplified the transcripts of the b2MG gene using, B2MG-S: obtained from Research Genetics (Huntsville, AL, USA) and ACC CCC ACT GAA AAA GAT GA and B2MG-R: ATC screened by PCR using as primers, Lhx4-HD-S: CAA GAA TTC AAA CCT CCA TGA TG. The PCR products were CTC CCC CAA GCC TG and Lhx4-BoAS: GGA AGC electrophoresed in 2% agarose gel containing TAE buffer, TCA GCT CAC TGT C, according to manufacturer’s then stained with ethidium bromide and photographed under recommendations. Then a PAC clone, RP5-1180C10, which UV light. contained the human Lhx4 gene, was obtained from Research Genetics. Fluorescence in situ hybridization (FISH) Reporter gene assay Phage and PAC DNAs were extracted as previously described (Sambrook and Russell, 2001). DNA probes were For the mouse a-glycoprotein subunit (aGSU) reporter gene, labeled with biotin-14-dATP (Gibco BRL, Rockville, MD, the fragment, 7440 to +1, containing the promoter region USA) and hybridized to metaphase spreads from normal was amplified by PCR using as primers, MaGSU-S: TTT human lymphocytes as previously described (Kobayashi et GGT ACC TTT ATT AAA TCC AGA GCC TG, and al., 1994). Hybridization signals and the corresponding MaGSU-AS: TTT CTC GAG TGC CTG CAG TTA ATA chromosomal bands were visualized with fluorescein isothio- AAG TCT (underlined sequences are additional sequences of cyanate (FITC)-conjugated avidin (Oncor, Gaitherburg, MD, KpnI and XhoI sites designed for subcloning) and ligated into USA) after staining with 4’,6-diamidino-2-phenylindole pGL3 basic vector (Promega) (Bach et al., 1995). The dihydrochloride. sequence of the ligated fragment was determined. Internal To confirm the rearrangements between the IgH and Lhx4 control vector, pRL was purchased from Promega. The full- genes, we performed two color FISH as previously reported length coding region of the Lhx4 gene was ligated into (Kobayashi et al., 1994). The DNA extracted from the PAC pcDNA3.1(+) vector (Invitrogen, Groningen, The Nether- clone containing the Lhx4 gene was labeled with rhodamine. lands). The NLI expression vector was provided by Dr GN The probes for IgH were labeled with FITC. The signals were Gill, and the Lhx3A and Lhx3B expression vectors were detected by fluorescence microscopy and photographed. provided by Dr SJ Rhodes. The mixtures of DNA were transfected into the A549 human lung cancer cell line using Effecten Transfection Reagent (Quagen, Hilden, Germany) Inverse PCR, genomic PCR and anchor PCR according to the manufacturer’s instructions. We used 6-well Inverse PCR was performed according to the protocol plates for transfection, and seeded 16105 cells per well 1 day reported by Willis et al. (1997). Briefly, high molecular before transfection. The cells were transfected with 0.1 mgof weight DNAs were digested with restriction enzymes (BglII pRL vector, 1 mg of pGL-aGSU or pGL3 vector, 1 mgof or XbaI ), then 0.4 micrograms of the digested DNAs were Lhx4 or Lhx3 or empty vector, and 1 mg of NLI or empty purified and ligated at 158C overnight to generate circular vector. The experiments were triplicated. The luciferase DNAs. The self-ligated circular DNAs were purified and activity was measured using a Dual-luciferase reporter assay subjected to PCR. For BglII-digested DNAs, the first round kit (Promega) on a luminometer, Lumat LB9507 (Berthold, of PCR was performed using the primers, JBE: GAA GCA Bad Wildbad, Germany). The activity was calibrated with GGT CAC CGC GAG AGT and J6E: CCC ACA GGC activity of internal control Renilla luciferases translated from AGT AGC AGA AAA CAA. The second round of nested pRL vectors. Results are expressed as fold-activation PCR was performed using JBI: CTT CTG GTT GTG AAG compared to the activity of the aGSU promoter in the AGG TGG TTT TG and J6I: TCT GGG CTC GAG TCG presence of an empty vector. ACG CAG AAA ACA AAG GCC CTA GAG GG. For XbaI-digested DNAs, the first-round PCR was performed Western blot analysis using JXE: CAC TGG CAT CGC CCT TTG TCT AA and J6E. The second round of nested PCR was performed using Western blot analysis was performed using an antibody JXI: CCC ATG CCT TCC AAA GCG ATT and J6I. specific to mouse Lhx4 proteins provided by Dr SL Pfaff. For The amplified PCR bands were cut out from the agarose an internal control, we used anti-beta-actin antibody (Sigma, gels and purified. After ligation into pGEM-3Zf(+) vectors St. Louis, MI, USA). As positive and negative controls for

Oncogene IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4991 Lhx4 protein expression, we transfected the Lhx4 expression Rockford, IL, USA). The signals were exposed to X-ray and empty vectors into the A549 cell line, which does not films according to the manufacturer’s instructions. endogenously express Lhx4 mRNA. Cells were lysed with RIPA lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 140 mM NaCl, 100 mM sodium Acknowledgements fluoride, 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluor- We thank Ms Koshimura, Ms Wakairo, Ms Hayashi and ide, 0.5% sodium deoxycholate, 0.1% SDS) and proteins Ms Ohyanagi for technical help. We also thank Dr GN were electrophoresed in SDS-polyacrylamide gel using Gill and Dr J Rhodes for the expression vectors, and Dr standard methods (Sambrook and Russell, 2001). Frac- SL Pfaff for antibody specific to mouse Lhx4. We also tionated proteins were transferred onto nitrocellulose filters, thank Dr RV Llyod for the pituitary adenoma cell line, Hybond ECL (Amershampharmacia). The secondary horse HP75. radish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin antibody was purchased from New England Biolab (Beverly, MA, USA). Blocking, the reaction and Accession numbers washing were performed according to the manufacturer’s The Genebank accession number of the junctional recommendations. The signals were detected using Super- sequence between IgH and Lhx4 is AF416729 and the signal West Pico chemiluminescent substrate (Pierce, cDNA sequence of human Lhx4 is AY053457.

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