Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited
© Gué et al; licensee BioMed Central Ltd. 2006
Received: 26 July 2005
Accepted: 24 January 2006
Published: 24 January 2006
Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism.
Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines.
In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation.
The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.
The Mixed Lineage Leukaemia (MLL) gene (also called HRX, HTRX or ALL-1) on chromosome band 11q23 is a recurrent target of reciprocal translocation in human acute leukaemias. The 11q23 translocation occurs in 5–6% of patients with acute myelogenous leukaemia (AML) and 7–10% of patients with acute lymphoblastic leukemia (ALL). More than 40 partners are involved in MLL translocation [2, 3]; the most common partner being the AF4 gene, on band 4q21. Translocation t(4;11)(q21;q23) is involved in a third of all MLL translocation cases  and represents 95% of translocations involved in ALL, compared to only 3.3% in AML . Another frequent partner of translocation is the ENL gene located on band 19p13.3, but whilst translocation t(11;19)(q23;p13.3) is found in both ALL and AML, it represents only 6% of all MLL translocations.
It has been demonstrated that pairs of genes (and chromosomal domains) which are frequently involved in reciprocal chromosomal translocation are close to each other in interphase nuclei. These observations have established a direct correlation between the spatial proximity of loci in interphase nuclei and the frequency of their translocation. This implies that the translocation takes place where and when the chromatin fiber containing the partner genes are co-localized (static "contact first" model). However, recent investigations, using alpha-particles for targeting double-strand breaks (DSBs), have demonstrated some displacements of DNA breaks into DNA repair foci where chromosomal exchange can occur. In other words, breaks formed at distal locations can subsequently be brought together to induce translocation (dynamic "breakage first" model).
In the present work, we propose a new direct approach to study the correlation between the relative position of genes in interphase nuclei and the corresponding frequencies of translocation. We have applied our approach to genes involved in 11q23 translocations (MLL, AF4 and ENL genes) in differentiated haematological cell lineages by 3D-FISH and in-depth image data analysis.
Results and discussion
Cell Lineage [No of loci]
NALM-6 [n = 324]
IM-9 [n = 448]
PLB-985 [n = 432]
AML-193 [n = 444]
45.0 ± 1.1%
45.3 ± 1.0%
45.7 ± 0.9%
39.4 ± 0.8%
54.8 ± 1.1%
55.4 ± 1.0%
57.8 ± 1.0%
46.6 ± 0.9%
48.5 ± 1.1%
50.6 ± 0.9%
43.7 ± 0.8%
49.5 ± 1.0%
In all the studied cell lines, the average separation between MLL and ENL loci was significantly smaller than that observed for MLL and AF4 (P < 0.0001). The mean smallest intergenic distance (corresponding to the minimum value of each of the four inter-loci distances) follows the same tendancy (26% of nuclear diameter for MLL-ENL distance vs. 34% for MLL-AF4 distance in NALM-6 and IM-9 cell lines, 26% vs. 36% for PLB-985 cell lines and 24% vs. 29% for AML-193 cell lines).
For each nucleus, the two smallest intergenic distance – dMLL-AF4 and dMLL-ENL – are displayed in figure 2. Firstly, our results show that, for the majority of nuclei, the ENL gene is closer to the MLL gene than to the AF4 gene. Secondly, according to the gene proximity criterion, we have calculated the number of nuclei where only the AF4 gene should be translocated with MLL gene (i.e. dMLL-AF4 < 2 μm and dMLL-ENL > 2 μm, greyed area in figure 2). In the same way, we computed the number of cells in which only the ENL gene should be translocated with the MLL gene (i.e. dMLL-ENL < 2 μm and dMLL-AF4 > 2 μm, greyed area in figure 2). The ratio of these two values, respectively, indicates a greater probability of a translocation event between MLL and ENL. (0.33, 0.35, 0.30 and 0.68 for NALM-6, IM-9, PLB-985 and AML-193 cell lines respectively).
Published MLL translocation frequencies report a high level of translocation t(4;11) compared to translocation t(11;19). According to the static "contact first" model, AF4 loci should be closer to MLL than ENL loci. Clearly, this is not in accordance with our data. In the case of the 11q23 translocation, the dynamic of chromatin must be taken into account and our results could be the first observation of the dynamic breakage first model proposed by Kanaar and co-workers.
It should be noted that published translocation frequencies were established after manifestation of cancer. Taking into account that a second genetic event seems to be required to induce cancer , we cannot exclude that the chromatin architecture context after MLL-AF4 translocation may be more favourable to the induction of a second event compared to MLL-ENL translocation. This could explain the high level of translocation t(4;11) detected in acute leukaemia. This could be illustrated by the relatively small distances between the ENL gene and the AF4 gene which could be favorable to translocation while the corresponding translocation is never reported in literature.
This work, based on simultaneous multi-probe hybridization in 3D-FISH experiments describes a new approach to study translocation mechanisms in a chromatin context. This method offers the advantage of a direct comparison of several translocation frequencies with localization of corresponding loci in each nucleus. Furthermore, it allows the simultaneous measurement of multiple intergenic distances in a cell by cell manner in a single experiment, without the chromatin distortion common to existing procedures.
Our results are not in accordance with the static model of translocation mechanism and may represent the first observation of the dynamic "breakage first" model. We are currently extending our approach to study MLL and up to six partner genes to confirm these intriguing results.
The authors would like to thank Maïté Coppey and Christophe Chamot at Institut Jacques Monod (Paris, France) for providing access and assistance with confocal microcopy, Marriono Rocchi (University of Bari, Italy) for providing BAC/PAC FISH clones, and Laurence Lagneaux (Belgium) for NALM-6 cell line. Thanks to Jason Martin for reading the manuscript.
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