In haematopoietic malignancies the MLL gene, located on chromosome 11q23, is frequently disrupted by chromosome rearrangement, generally resulting in fusion to various partner genes. We have previously reported a t(11;15)(q23;q14) in a case of acute myeloblastic leukaemia. Here, we report the cloning of a novel MLL partner, AF15q14, at chromosome 15q14. In this translocation, the breakpoint occurred in exon 8 of MLL and exon 10 of AF15q14. The normal AF15q14 transcripts of approximately 8.5 kb in size, are expressed in different tumoral cell lines, in a variety of normal tissues, and in all the foetal tissues tested. Sequencing of AF15q14 cDNA revealed a putative open reading frame of 1833 amino acids that had no homology with any other known protein. The C-terminal end of the putative AF15q14 contained a bipartite nuclear localization site. The translocation t(11;15) preserved the open reading frame between MLL and the 3′ end of AF15q14. The contribution of AF15q14 to the fusion protein was only 85 amino acids. Immunofluorescence staining experiments with expression vectors encoding these 85 amino acids confirmed the functionality of the predicted nuclear localization site.
The MLL gene (also known as ALL1 and HTRX), located on chromosome 11q23, is found rearranged in approximately 10% of acute lymphoblastic leukaemias, and in more than 5% of acute myeloid leukaemias (AML), and is generally associated with a very poor prognosis. This gene encodes a large protein (3968 amino acids) which contains several features of transcription factors, AT-hooks, methyl-transferase domain, zinc fingers, a nuclear localization site and a highly conserved SET domain. The MLL gene could be the human homologue of Drosophila trithorax (reviewed in Dimartino and Cleary, 1999). Defects in yolk-sac haematopoiesis in MLL-null mouse embryos (Yu et al., 1995), and the differentiation block in MLL-null embryonic stem cells (Fidanza et al., 1996), suggest that MLL plays an important role in haematopoiesis through its ability to regulate the expression of Hox genes. Most rearrangements that disrupt the MLL gene, e.g. reciprocal translocation and self-fusion, occur within a clustered region of 8.3 kb (Gu et al., 1994). Translocation disrupts the MLL gene between the AT-hooks and the zinc finger domains to produce a fused gene encoding a chimeric protein. Molecular and cytogenetic evidences indicate that the crucial chimeric product is the 5′MLL3′-partner fusion transcript in MLL associated leukaemias (Rowley, 1992). To date, the cloning of the fusion partners has led to the discovery of 20 different genes encoding various kinds of proteins with no common features. The apparently-insignificant role of the partner genes in leukaemogenesis is suggested by the tandem duplication of the MLL gene, whose effects are similar to those of translocations, and by a recent study which showed that truncated MLL, when fused to the bacterial Lac Z gene, is sufficient for tumorigenesis (Dobson et al., 2000). However, the contribution of the partner gene to leukaemogenesis could still be critical, in view of a mouse knock-in study which has shown that MLL-AF9 chimeras develop AML, whereas MLL-myc chimeras do not (Corral et al., 1996). Moreover, the immortalization of murine haematopoietic progenitors by the retrovirus-mediated gene transfer of MLL-ENL requires the integrity of the transactivation activity of ENL (Schreiner et al., 1999). Thus it would seem that the other partners of MLL have to be identified before one can attempt to find out whether it is the functional gain or the production of a dominant-negative protein that dictates the tumoral phenotype.
In the present work, we report the cloning of AF15q14, the fusion partner of the MLL gene in t(11;15)(q23;q14). We previously reported the case of a 48-year-old man with no previous history of toxic exposure, suffering from AML-M4, which was cytogenetically characterized as 46,XY,-3, t(11;15)(q23;q14), +mar (Archimbaud et al., 1998; Harrison et al., 1998). The bone marrow was hypercellular, with 80% blast cells. The patient was treated by intensive chemotherapy, and died four months after diagnosis. MLL involvement in the reciprocal translocation t(11;15)(q23;q14) was demonstrated by Southern blot analysis of the leukaemia-cell DNA. Rearranged DNA fragments of the der(11) and der(15) chromosomes were observed with MLLa probe encompassing the breakpoint cluster region (Figure 1a). Restriction mapping by Southern blot analysis, performed with 5′MLL and 3′MLL probes (data not shown), allowed us to locate the breakpoint between intron 7 and intron 8 within the common breakpoint cluster region of MLL (Figure 1b).
The t(11;15) translocation has already been described several times (Hernandez et al., 1995; Hunger et al., 1993; Rubnitz et al., 1994), and it was the detection of a t(11;15) in a patient with AML, involving the MLL locus, that prompted us to study the resulting molecular rearrangement. Unfortunately, no mRNA from this patient was available so we used genomic phage libraries prepared from leukaemic cells and placental tissue to isolate the der(11) chromosome and the normal counterpart on chromosome 15 (Figure 1b). Southern blot analysis of the leukaemic cells and control DNA, performed with AF15a, AF15b and AF15c probes, revealed the expected germinal and rearranged bands (data not shown). Hybridization onto DNA from interspecies somatic hybrid (Clontech) showed that these probes were derived from chromosome 15 (data not shown). Fluorescence in situ hybridization to normal human metaphases, using an artificial bacterial chromosome isolated by PCR screening with AF15q14 primers (BAC No. B590H1, CEPH, Paris, France), confirmed the location of these probes on chromosome 15 at band 15q14 (data not shown). Nucleotide sequence analysis revealed that the breakpoint on the der(11) chromosome was located in exon 8 of MLL (Figure 1c). Unlike other MLL rearrangements previously studied, no extra nucleotides or heptamer-like signals were observed at the junction point, which probably rules out any involvement of VDJ recombinase. It has been shown that large numbers of ALU sequences are present at the MLL breakpoint (Marschalek et al., 1997), which may render this region particularly prone to recombination events. An ALU sequence was found on chromosome 15 660 bp downstream from the breakpoint, which could thus be predisposed to translocation events.
In order to identify a transcriptional unit on 15q14, repeat-free genomic DNA fragments were used as probes against Northern blot containing poly(A)+ RNA from a variety of cell types. The AF15c probe hybridized to a transcript of about 8.5 kb which is relatively abundant in HeLa cells (Figure 2a). In adult tissue, AF15q14 mRNA was found to be almost wholly restricted to the thymus, testis and bone marrow, whereas it seemed to be ubiquitous in the foetal tissues tested (Figure 2b). In line with the nomenclature for MLL fusion partner genes, we designated this gene AF15q14 (ALL-1 fused gene from chromosome 15q14). In order to characterize the AF15q14 transcript, a HeLa cDNA library was screened with the AF15c probe. Among the nine cDNAs isolated, the longest ones (1.2 kbp, 0.8 kbp and 0.6 kbp) were sequenced. Most of the 5′ and 3′ AF15q14 sequences were found by PCR screening of a testis cDNA library and/or by the rapid amplification of 5′/3′cDNA ends obtained from HeLa-cell poly(A)+ RNA. Sequencing of the genomic counterpart confirmed the nucleotide sequences of the PCR-isolated fragments. Subsequent walking and compilation of sequence data from the different overlapping cDNA clones allowed us to sequence 5925 bp of the AF15q14 transcript. The AF15q14 sequence has been deposited at the Data Library of the National Center for Biotechnology Information (accession No. AF248041). Genomic sequencing showed that the AF15q14 gene spanned a region of more than 35 kb, and contained at least 10 exons (Figure 3a), with typical donor and acceptor sites at the intron-exon junctions (data not shown).
Analysis of AF15q14 cDNA showed an open reading frame of 5499 bp encoding a protein of 1833 amino acids, with a predicted molecular mass of 206 kDa. This open reading frame contained an initiation sequence consensus ATGG (Kozak, 1986) at nucleotide 146 in exon 2, and a stop codon at nucleotide 5645 in exon 10. We observed an imbalance in its nucleotide composition, with 63% of A/T. Sequence comparison of the AF15q14 cDNA and the deduced protein with those found in databases did not reveal any significant homologies. Computerized analysis of the AF15q14 revealed two closely-related motifs of 105 and 103 amino acids coded by exon 10 (Figure 3b). These motifs, which are probably due to a functionally-important duplicated region, do not themselves have any currently known functional features. A database search for functional domains revealed only the presence of a predicted Bipartite Nuclear Localization Site (NLS-BP) at the C-terminal end of the putative AF15q14 protein (amino acids 1761–1778).
As with all the other 11q23 translocations so far investigated, the t(11;15)(q23;q14) breakpoint resulted in frame fusion between exon 8 (nt 4253) of MLL and exon 10 (nt 5389) of AF15q14 cDNA. The fused transcript MLL-AF15q14 could encode a putative protein of 1503 amino acids, composed of the N-terminal part of MLL (1418 amino acids) and the C-terminal part of AF15q14 (85 amino acids), which includes the NLS-BP. As no mRNA from leukaemic cells was available to us, this chimeric transcript remains to be studied in other t(11;15) translocations. In an attempt to assess the functionality of the NLS-BP, expression vectors encoding the C-terminal part of AF15q14 (85 amino acids) were transfected into COS-7 cells. Nuclear immunofluorescence was observed in COS-7 cells transfected with the pSG5flagAF15-85aa expression vector (Figure 4). The wild-type pSG5-flag vector, used as a control, did not show any signal (data not shown). Moreover an expression vector encoding these 85 amino acids, fused to Green Fluorescent Protein (pGFP expression vector, Clontech) and transfected into COS-7 cells, showed a similar nuclear localization pattern (data not shown). Our results indicate that AF15q14 protein may be a nuclear protein. Similarly to other MLL-partners such as AF3p21, AF4, AF5α, AF9, ENL and ELL, the NLS of AF15q14 is fused to MLL. All the MLL fusion proteins studied so far have been found to localize in the nucleus and although some partners of MLL such as AF1p/eps15 and AF6 localize in the cytoplasm, they show a nuclear localization when they are fused to MLL (Rogaia et al., 1997; Joh et al., 1997).
Further functional analysis of translocation partner genes, as well as MLL, should provide new insights into the leukaemogenesis of 11q23-associated leukaemia. Molecular analysis of t(11;15)(q23;q14), of which only a few cases have so far been reported, allowed us to isolate a new gene, AF15q14, fused to MLL. It will be interesting to see if this gene is found to be involved in other cases of t(11;15)(q23;q14).
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The authors wish to thank Bénédicte Santa-Lucia and Annick Bertholin for their technical assistance. This work was supported by grants from INSERM, the Association pour la Recherche contre le Cancer and the Ligue Nationale contre le Cancer (Comités du Rhône et de la Saône et Loire).
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Hayette, S., Tigaud, I., Vanier, A. et al. AF15q14, a novel partner gene fused to the MLL gene in an acute myeloid leukaemia with a t(11;15)(q23;q14). Oncogene 19, 4446–4450 (2000). https://doi.org/10.1038/sj.onc.1203789
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