👤 C V Vignon

The early events in the nuclear reprogramming process during somatic cell nuclear transfer (SCNT) consist of morphological remodeling of the donor nucleus including premature chromosome condensation (PCC). In the present study, the objective was to increase oocyte M-Phase Promoting Factor (MPF) kinase activity and to examine the fate of the donor nucleus and the development of SCNT embryos thereafter. Indeed, in controls, recipient oocytes activated upon nuclear transfer, undergo a decrease in MPF activity, responsible for the inability to promote PCC in 77.8% of reconstituted embryos. Here we showed that exposure of the recipient oocyte to the proteasome inhibitor MG132 prior to fusion inhibited the degradation of cyclin B, which normally occurred immediately after activation by electro stimulation, and therefore sustained a high level of MPF. Treatment with MG132 also significantly increased the percentage of SCNT embryos with PCC when compared to the nontreated SCNT control embryos (94.1 vs. 22.2%, respectively, p < 0.01). The frequency of development to the blastocyst stage did not differ between MG132-treated or untreated recipient oocytes. However, we observed a significant increase of the total cells number in embryos produced after MG132 treatment. Investigation of the global nuclear organization by immunodetection of heterochromatin protein 1 (CBX1) showed that SCNT embryos derived from MG132-treated recipient oocytes displayed organization patterns similar to the ones observed in IVF embryos in contrast to the nontreated SCNT controls. Taken together, these results suggest that the PCC induced by MG132 treatment allows reorganization of the chromatin at an appropriate time potentially, leading to better reprogramming. Show less

Efficient reprograming of the donor cell genome in nuclear transfer (NT) embryos is linked to the ability of the embryos to sustain full-term development. As the nuclear architecture has recently emerged as a key factor in the regulation of gene expression, we questioned whether early bovine embryos obtained from transfer of cultured fibroblasts into enucleated oocytes would adopt an embryo-like nuclear organization. We studied the dynamics of constitutive heterochromatin in the stages prior to embryonic genome activation by distribution analysis of heterochromatin protein CBX1 (HP1), centromeric proteins CENPA and CENPB, and histone H3 three-methylated at lysine 9. Then we applied descriptive, quantitative, and co-localization analyses. A dramatic reorganization of heterochromatic blocks of somatic donor cells was first observed in the late one-cell stage NT embryos. Then at two- and four-cell stages, we found two types of NT embryos: one displaying noncondensed heterochromatin patches similar to IVF embryos, whereas the second type displayed condensed heterochromatin blocks, normally observed in IVF embryos only after the eight-cell stage. These analyses discriminate for the first time two contrasted types of nuclear organization in NT embryos, which may correspond to different functional states of the nuclei. The relationship with the somatic nucleus reprograming efficiency is discussed. Show less

Invins(10;11)(p12;q23q12) is one of the rare but recurring chromosome rearrangements seen in acute monoblastic leukemia. We cloned the proximal 10p breakpoint from one patient and showed that the MLL gene at 11q23 was fused to the 3' portion of AF10 at 10p12. In addition, we cloned the telomeric 10p junction and we found that the 5' portion of AF10 was juxtaposed to a previously unidentified gene at 11q12, which we call HEAB (a human homolog to a hypothetical Caenorhabditis elegans ATP/GTP-binding protein). These results indicate that the AF10 gene is split into a 5' AF10 and a 3' AF10 portion by the 11q23q12 chromosome segment and that both breakpoint junctions result in fusion transcripts of 5' AF10/HEAB and MLL/3' AF10. Only the MLL/3' AF10 fusion mRNA results in an in-frame fusion. Northern blot analysis of HEAB expression shows that a 2.0-kb major transcript is expressed ubiquitously in human tissues and is especially abundant in testis and skeletal muscle, whereas a 3.2-kb minor transcript is noted with the highest level of expression in thymus and peripheral blood leukocytes. The HEAB gene encodes a 425-amino acid protein that is rich in valine and leucine. HEAB protein shows high homology in its entire amino acid sequence to a putative C elegans protein and contains an adenosine triphosphate (ATP)/guanosine triphosphate (GTP)-binding motif that has homology to the ATP-binding transporter superfamily or to GTP-binding proteins. Our results could explain the high frequency of complex insertion and other rearrangement events that involve 10p12 and 11q12 and 11q23. The finding that different portions of a single gene are involved in fusions with two independent genes in the same leukemic cell is unique in the analysis of chromosome translocations. Show less