Point Mutations in GLI3 Lead to Misregulation of its Subcellular Localization

Background Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. Principal Findings We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-α4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. Conclusions The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations

Präimplantationsdiagnostik für monogene Erkrankungen am PID-Zentrum Regensburg

Here we report on the results of 316 diagnostic cycles of preimplantation genetic diagnosis (PGD) at our PGD center for 149 families at high risk of transmitting a monogenic disease to their offspring. Since 2001, we have performed a total of 251 polar body diagnosis (PBD) cycles. After obtaining the license as PGD center in June 2015, we subsequently performed 65 trophectoderm diagnostic (TED) cycles after trophectoderm (TE) biopsy (1.4/family). Our preliminary data confirm a high diagnostic efficiency of both procedures, with duplication of the pregnancy rate per transfer after TED to 48.2% compared with 22.9% after PBD. At an average rate of 4.3 available blastocysts per TED cycle, the rate of cycles without transferable embryos (27.7% for 6/45 families & x202f;= 13.3%) was, as expected, higher compared with PBD (14.7%) for an average of 8 oocytes per cycle with successful biopsy of both polar bodies. As early as the first transfer after TED, however, a clinical pregnancy could be achieved for 55.3% of the couples. Overall, TED enabled a clinical pregnancy for 68.5% of the families with transfer, after an average of 1.4 transfers and, overall a milder treatment with fewer TED diagnostic cycles (PBD: cumulatively 40.3% after 2.3 transfers), less hormone stimulation and correspondingly lower costs. In particular, the subgroup of female carriers of pathogenic repeat expansions, with lower PGD pregnancy rates overall, appears to benefit from TED, according to our preliminary data

The E3 Ubiquitin Ligase MID1 Catalyzes Ubiquitination and Cleavage of Fu

SHH (Sonic Hedgehog)-GLI signaling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signaling. A protein complex containing the ubiquitin ligase MID1 and protein phosphatase 2A regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signaling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyzes the ubiquitination and proteasomal cleavage of the GLI3 regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1-PP2A protein complex with GLI3 activity control