👤 S Debernardi

The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus. AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy. The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10. This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library. The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma. GAS41 shows significant homology to the Saccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL. The interaction was confirmed in vivo. Furthermore, the study showed by coimmunoprecipitation that GAS41 interacts with INI1 (Integrase Interactor 1) and that INI1 was present in the AF10 immunoprecipitate. INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel chromatin and to modulate transcription. The retention of the leucine zipper in the MLL and CALM fusions suggests that a key feature of these chimeric proteins may be their ability to interfere in normal gene regulation through interaction with the adenosine triphosphate-dependent chromatinremodeling complexes. Show less

As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias. Show less

The interstitial insertion of genetic material from one chromosome into another can achieve the type of gene-gene fusions more usually associated with chromosome translocations. An example of such an interstitial insertion, which has created an MLL-AF10 fusion in an acute myeloid leukaemia, has been analysed at the genomic level. The genomic fusion, which resulted in the juxtaposition of 3' AF10 sequence to 5' MLL sequence, was identified within MLL and AF10 intronic sequences. It was further established that the remaining 3' MLL sequence, from exon 6 onwards, was fused to novel sequence of unknown origin (named FM3 for fused to MLL 3'). The points of fusion of these 5' and 3' portions of MLL matched to adjacent nucleotides and lay between exons 5 and 6. The FM3 sequence was shown to be from chromosome arm 10p and located close to AF10 in a proximal position. It was subsequently demonstrated that in the leukaemia a third fusion existed between 5' AF10 and the FM3 sequence at a point immediately downstream from its fusion to MLL. It was therefore concluded that the MLL-AF10 gene fusion is the result of a simultaneous transposition of genetic material into the MLL gene and the joining of the remaining free ends on chromosome 10. This kind of event, characterised completely here for the first time, is a means to achieve a fusion when the genes involved lie in opposite orientations and results in three genomic junctions. Show less

Leukaemogenesis correlates with alterations in chromatin structure brought about by the gain or loss of interactive domains from regulatory factors that are disrupted by chromosomal translocations. The gene MLL, a target of such translocation events, forms chimaeric fusion products with a variety of partner genes. While MLL appears to be involved in chromatin-mediated gene regulation, the functions of its partner genes are largely speculative. We report the biochemical analysis of the MLL partner gene AF10 and its possible role in leukaemogenesis. AF10 has been reported to be re-arranged with genes other than MLL leading to the same phenotype, a myeloid leukaemia. We have identified a novel protein-protein interaction motif in the AF10 protein comprising the extended LAP/PHD-finger. This domain mediates homo-oligomerisation of recombinant AF10 and is conserved in several proteins, including MLL itself. AF10 binds cruciform DNA via a specific interaction with an AT-hook motif and is localised to the nucleus by a defined bipartite nuclear localisation signal in the N-terminal region. Show less