4601

MXI1

MAD2|MXD2|MXI|bHLHc11;MAX interactor 1;MXI1;MAX interactor 1

MAX dimerization protein 2|Max-related transcription factor|class C basic helix-loop-helix protein 11|max-interacting protein 1

10q24-q25

protein-coding

BACKGROUND: Autoregulation of the myc gene family is a negative feedback mechanism known to occur at high levels of Myc expression. Loss of this mechanism and associated Myc overexpression has been observed in human tumors, thereby contributing to tumorigenesis. The childhood tumor neuroblastoma is characterized by N-myc amplification in aggressive and highly proliferative tumors that occur in a subset of patients. The precise molecular mechanism of autoregulation is unknown, and previous observations indicated that N-myc autoregulation was intact only in single-copy neuroblastoma cell lines. METHODS: Transient reporter assaysand trichostatin A (TSA) experiments were performed to evaluate several candidate genes, including Mxi1, c-myc promoter binding protein 1 (MBP-1), Miz, and histone deacetylase 2 (HDAC2), for their involvement in N-myc autoregulation. Mxi1 and HDAC2 were examined further for their expression levels and effects on endogenous N-myc levels. Finally, their recruitments to the N-myc promoter were investigated by chromatin immunoprecipitation (ChIP). RESULTS: The autoregulatory circuit wasoperative, even in amplified cell lines. Mxi1 consistently showed a modest effect in down-regulating N-myc in transient reporter assays. Overexpression of the c-myc, Mxi1, and mHDAC2 genes resulted in a threefold to fourfold decrease in endogenous N-myc levels. Mxi1 and HDAC2 were up-regulated by N-Myc in an myc-inducible cell line and in N-myc-expressing cell lines. In addition, down-regulation of the N-myc promoter was relieved in the presence of TSA. Increased association of HDAC2 with the autoregulatory region within the N-myc promoter by ChIP was observed upon down-regulation of endogenous N-myc. CONCLUSIONS: The autoregulatory circuit was intact in both amplified and single-copy neuroblastoma cell lines. Furthermore, myc gene autoregulation occurred through histone deacetylation.

Abnormalities of chromosome 10 are frequently observed in the development of medulloblastoma, the most common malignant brain tumor of childhood. To identifycritical genetic loci involved, we performed detailed physical mapping of regions of allelic loss on this chromosome. 18% of cases (5/32 primary tumors, 2/8 cell lines) harbored allelic losses on 10q. Refined mapping identified a 21.7Mb common interval, affecting the region 10q23.3-10q25.3. This region contains three genes, MXI1, SUFU and BTRC, which represent putative medulloblastoma tumor suppressor (TS) genes on the basis of either (i) negative regulation of critical medulloblastoma pathways, or (ii) mutation in other cancer types. We therefore sought evidence of their genetic inactivation in 46 cases, by mutational analysis of their entire coding regions. A MXI1 mutation was identified which abolishes its translation initiation site (A1G; MET1VAL), however no further tumor-specific sequence variations were detected. We next identified and characterised CpG islands associated with 5' regions of the MXI1, SUFU and BTRC genes; analysis ofthese regions for evidence of DNA hypermethylation, alongside expression analysis of their respective transcripts, revealed no evidence to support epigenetic inactivation of any gene. These findings implicate the inactivation of critical TS loci at 10q23.3-25.3 in medulloblastoma, however comprehensive analysis of SUFU, BTRC and MXI1 indicates they are unlikely to represent major targets of these allelic losses. MXI1 mutation appears to play a role in the pathogenesis of a small subset of cases, and suggests an alternative mechanism to MYC amplification for disruption of the MYC/MAD/MAX network in medulloblastoma.