Shinji Hirotsune

Refinement of a 400-kb Critical Region Allows Genotypic Differentiation between Isolated Lissencephaly, Miller-Dieker Syndrome, and Other Phenotypes Secondary to Deletions of 17p13.3

Deletions of 17p13.3, including the LIS1 gene, result in the brain malformation lissencephaly, which is characterized by reduced gyration and cortical thickening; however, the phenotype can vary from isolated lissencephaly sequence (ILS) to Miller-Dieker syndrome (MDS). At the clinical level, these two phenotypes can be differentiated by the presence of significant dysmorphic facial features and a more severe grade of lissencephaly in MDS. Previous work has suggested that children with MDS have a larger deletion than those with ILS, but the precise boundaries of the MDS critical region and causative genes other than LIS1 have never been fully determined. We have completed a physical and transcriptional map of the 17p13.3 region from LIS1 to the telomere. Using fluorescence in situ hybridization, we have mapped the deletion size in 19 children with ILS, 11 children with MDS, and 4 children with 17p13.3 deletions not involving LIS1. We show that the critical region that differentiates ILS from MDS at the molecular level can be reduced to 400 kb. Using somatic cell hybrids from selected patients, we have identified eight genes that are consistently deleted in patients classified as having MDS. In addition, deletion of the genes CRK and 14-3-3 epsilon delineates patients with the most severe lissencephaly grade. On the basis of recent functional data and the creation of a mouse model suggesting a role for 14-3-3 epsilon in cortical development, we suggest that deletion of one or both of these genes in combination with deletion of LIS1 may contribute to the more severe form of lissencephaly seen only in patients with MDS.

Deletion of Mnt leads to disrupted cell cycle control and tumorigenesis

Mnt is a Max‐interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function we deleted the Mnt gene in mice. Since mice lacking Mnt were born severely runted and typically died within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely entered the S phase of the cell cycle and proliferated more rapidly than Mnt+/+ MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt–Myc antagonism. Like MEFs that overexpress Myc, Mnt−/− MEFs were prone to apoptosis, efficiently escaped senescence and could be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium led to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis.

Evidence of Mnt-Myc antagonism revealed by Mnt gene deletion

Myc proteins play a central role in promoting cell proliferation and contribute to a diverse array of cancers. Myc function appears completely dependent on heterodimerization with Max through related bHLHZip regions. Max interaction with Myc is required for DNA binding at so-called E-box sequences and Myc-dependent transcriptional activation. The identification of Mnt as a ubiquitously expressed Max-interacting transcriptional repressor with similar DNA binding specificity raised the possibility that Mnt may serve a general role as a Myc antagonist.