Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF

Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and an important oncogene in melanoma. MITF heterodimeric assembly with related basic helix-loop-helix leucine zipper transcription factors is highly restricted, and its binding profile to cognate DNA sequences is distinct. Here, we determined the crystal structure of MITF in its apo conformation and in the presence of two related DNA response elements, the E-box and M-box. In addition, we investigated mouse and human Mitf mutations to dissect the functional significance of structural features. Owing to an unusual three-residue shift in the leucine zipper register, the MITF homodimer shows a marked kink in one of the two zipper helices to allow an out-of-register assembly. Removal of this insertion relieves restricted heterodimerization by MITF and permits assembly with the transcription factor MAX. Binding of MITF to the M-box motif is mediated by an unusual nonpolar interaction by Ile212, a residue that is mutated in mice and humans with Waardenburg syndrome. As several related transcription factors have low affinity for the M-box sequence, our analysis unravels how these proteins discriminate between similar target sequences. Our data provide a rational basis for targeting MITF in the treatment of important hereditary diseases and cancer

Novel MITF targets identified using a two-step DNA microarray strategy

Malignant melanoma is a chemotherapy-resistant cancer with high mortality. Recent advances in our understanding of the disease at the molecular level have indicated that it shares many characteristics with developmental precursors to melanocytes, the mature pigment-producing cells of the skin and hair follicles. The development of melanocytes absolutely depends on the action of the microphthalmia-associated transcription factor (MITF). MITF has been shown to regulate a broad variety of genes, whose functions range from pigment production to cell-cycle regulation, migration and survival. However, the existing list of targets is not sufficient to explain the role of MITF in melanocyte development and melanoma progression. DNA microarray analysis of gene expression offers a straightforward approach to identify new target genes, but standard analytical procedures are susceptible to the generation of false positives and require additional experimental steps for validation. Here, we introduce a new strategy where two DNA microarray-based approaches for identifying transcription factor targets are combined in a cross-validation protocol designed to help control false-positive generation. We use this two-step approach to successfully re-identify thirteen previously recorded targets of MITF-mediated upregulation, as well as 71 novel targets. Many of these new targets have known relevance to pigmentation and melanoma biology, and further emphasize the critical role of MITF in these processes

Nonsense mutation in the LGR4 gene is associated with several human diseases and other traits

Low bone mineral density (BMD) is used as a parameter of osteoporosis. Genome-wide association studies of BMD have hitherto focused on BMD as a quantitative trait, yielding common variants of small effects that contribute to the population diversity in BMD. Here we use BMD as a dichotomous trait, searching for variants that may have a direct effect on the risk of pathologically low BMD rather than on the regulation of BMD in the healthy population. Through whole-genome sequencing of Icelandic individuals, we found a rare nonsense mutation within the leucine-rich-repeat-containing G-protein-coupled receptor 4 (LGR4) gene (c.376C>T) that is strongly associated with low BMD, and with osteoporotic fractures. This mutation leads to termination of LGR4 at position 126 and fully disrupts its function. The c.376C>T mutation is also associated with electrolyte imbalance, late onset of menarche and reduced testosterone levels, as well as an increased risk of squamous cell carcinoma of the skin and biliary tract cancer. Interestingly, the phenotype of carriers of the c.376C>T mutation overlaps that of Lgr4 mutant mice. © 2013 Macmillan Publishers Limited. All rights reserved