MNM and SNM maintain but do not establish achiasmate homolog conjunction during Drosophila male meiosis.

The first meiotic division reduces genome ploidy. This requires pairing of homologous chromosomes into bivalents that can be bi-oriented within the spindle during prometaphase I. Thereafter, pairing is abolished during late metaphase I, and univalents are segregated apart onto opposite spindle poles during anaphase I. In contrast to canonical meiosis, homologous chromosome pairing does not include the formation of a synaptonemal complex and of cross-overs in spermatocytes of Drosophila melanogaster. The alternative pairing mode in these cells depends on mnm and snm. These genes are required exclusively in spermatocytes specifically for successful conjunction of chromosomes into bivalents. Available evidence suggests that MNM and SNM might be part of a physical linkage that directly conjoins chromosomes. Here this notion was analyzed further. Temporal variation in delivery of mnm and snm function was realized by combining various transgenes with null mutant backgrounds. The observed phenotypic consequences provide strong evidence that MNM and SNM contribute directly to chromosome linkage. Premature elimination of these proteins results in precocious bivalent splitting. Delayed provision results in partial conjunction defects that are more pronounced in autosomal bivalents compared to the sex chromosome bivalent. Overall, our findings suggest that MNM and SNM cannot re-establish pairing of chromosomes into bivalents if provided after a chromosome-specific time point of no return. When delivered before this time point, they fortify preformed linkages in order to preclude premature bivalent splitting by the disruptive forces that drive chromosome territory formation during spermatocyte maturation and chromosome condensation during entry into meiosis I.

Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes.

The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. Here we describe the identification and initial characterization of dany, a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC and tTAFs, the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, we propose that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization.

Separase Is Required for Homolog and Sister Disjunction during Drosophila melanogaster Male Meiosis, but Not for Biorientation of Sister Centromeres.

Spatially controlled release of sister chromatid cohesion during progression through the meiotic divisions is of paramount importance for error-free chromosome segregation during meiosis. Cohesion is mediated by the cohesin protein complex and cleavage of one of its subunits by the endoprotease separase removes cohesin first from chromosome arms during exit from meiosis I and later from the pericentromeric region during exit from meiosis II. At the onset of the meiotic divisions, cohesin has also been proposed to be present within the centromeric region for the unification of sister centromeres into a single functional entity, allowing bipolar orientation of paired homologs within the meiosis I spindle. Separase-mediated removal of centromeric cohesin during exit from meiosis I might explain sister centromere individualization which is essential for subsequent biorientation of sister centromeres during meiosis II. To characterize a potential involvement of separase in sister centromere individualization before meiosis II, we have studied meiosis in Drosophila melanogaster males where homologs are not paired in the canonical manner. Meiosis does not include meiotic recombination and synaptonemal complex formation in these males. Instead, an alternative homolog conjunction system keeps homologous chromosomes in pairs. Using independent strategies for spermatocyte-specific depletion of separase complex subunits in combination with time-lapse imaging, we demonstrate that separase is required for the inactivation of this alternative conjunction at anaphase I onset. Mutations that abolish alternative homolog conjunction therefore result in random segregation of univalents during meiosis I also after separase depletion. Interestingly, these univalents become bioriented during meiosis II, suggesting that sister centromere individualization before meiosis II does not require separase.

Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function.

Kinetochores allow attachment of chromosomes to spindle microtubules. Moreover, they host proteins that permit correction of erroneous attachments and prevent premature anaphase onset before bi-orientation of all chromosomes in metaphase has been achieved. Kinetochores are assembled from subcomplexes. Kinetochore proteins as well as the underlying centromere proteins and the centromeric DNA sequences evolve rapidly despite their fundamental importance for faithful chromosome segregation during mitotic and meiotic divisions. During evolution of Drosophila melanogaster, several centromere proteins were lost and a recent gene duplication has resulted in two Nnf1 paralogs, Nnf1a and Nnf1b, which code for alternative forms of a Mis12 kinetochore complex component. The rapid evolutionary divergence of centromere/kinetochore constituents in animals and plants has been proposed to be driven by an intragenome conflict resulting from centromere drive during female meiosis. Thus, a female meiosis-specific paralog might be expected to evolve rapidly under positive selection. While our characterization of the D. melanogaster Nnf1 paralogs hints at some partial functional specialization of Nnf1b for meiosis, we have failed to detect evidence for positive selection in our analysis of Nnf1 sequence evolution in the Drosophilid lineage. Neither paralog is essential, even though we find some clear differences in subcellular localization and expression during development. Loss of both paralogs results in developmental lethality. We therefore conclude that the two paralogs are still in early stages of differentiation.

Detection of germline rearrangements in patients with α- and ß-thalassemia using high resolution array CGH.

Approximately 80% of α-thalassemia mutations are deletions in the α-globin cluster on chromosome 16 and about 10% of ß-thalassemia mutations are deletions in the ß-globin gene cluster on chromosome 11. Larger deletions involving the ß-globin gene cluster lead to (δß)-, (γδß)-, (εγδß)-thalassemia, or hereditary persistence of fetal hemoglobin (HPFH). Array comparative genomic hybridization (CGH) was applied to screen for deletions in the α- and ß-globin gene clusters not detected by routine gap-PCR. In total, in 13 patients with hypochromia and inclusion bodies (IBs) the α-globin gene cluster was analyzed and in 13 patients with increased fetal hemoglobin levels with or without hypochromia the ß-globin gene cluster was examined. All samples were subsequently investigated by multiplex ligation-dependent probe amplification (MLPA). In 9 out of 13 patients deletions of the α-globin gene cluster were identified; 5 of these deletions remove the entire α-globin cluster and extend to the telomere. Additional sequencing of the remaining 4 patients revealed polyadenylation mutation in 1 of them. 7 deletions were identified in the ß-globin gene cluster in 13 patients. Additional sequencing of the remaining 6 patients revealed mutations in one of the γ-globin gene promoters in 3 of them and a KLF1-mutation in 1 of them. Array CGH is a reliable method to screen for deletions in thalassemia and hemoglobinopathy. The method offers the advantage of a high resolution with the possibility to characterize breakpoints on sequence level.

Homozygous nonsense mutation in WNT10B and sporadic split-hand/foot malformation (SHFM) with autosomal recessive inheritance.

Split-hand/foot malformation (SHFM) is a limb malformation affecting the central rays of the hands and/or feet. Isolated SHFM occurs within families but more often sporadically. Since most families with more than one patient show dominant inheritance with reduced penetrance, sporadic SHFM is generally considered to be due to dominantly inherited new mutations. Recently, recessive inheritance of SHFM was proposed in a highly consanguineous family with a homozygous missense mutation in WNT10B. Nevertheless, the assumption of a second locus was necessary to explain the observed phenotypes in this family. To date, no other family and no case of sporadic SHFM with WNT10B mutations are known. By examining WNT10B in a patient with sporadic SHFM, we identified a homozygous 4-bp duplication resulting in a premature termination codon. Nine heterozygous relatives show no sign of SHFM. These findings have profound implications for genetic counseling. Obviously, sporadic SHFM may show recessive rather than dominant inheritance resulting in a 25% recurrence risk for sibs instead of a very low-recurrence risk as generally presumed. Likewise, there is a very low-recurrence risk for offspring of patients (unless there is consanguinity) instead of an estimated risk between 30% and 50%. It can be concluded that sporadic SHFM is not always a dominant trait. To determine the recurrence risk, patients affected with sporadic SHFM should be tested for mutations in WNT10B.

Regulated protein depletion by the auxin-inducible degradation system in Drosophila melanogaster.

The analysis of consequences resulting after experimental elimination of gene function has been and will continue to be an extremely successful strategy in biological research. Mutational elimination of gene function has been widely used in the fly Drosophila melanogaster. RNA interference is used extensively as well. In the fly, exceptionally precise temporal and spatial control over elimination of gene function can be achieved in combination with sophisticated transgenic approaches and clonal analyses. However, the methods that act at the gene and transcript level cannot eliminate protein products which are already present at the time when mutant cells are generated or RNA interference is started. Targeted inducible protein degradation is therefore of considerable interest for controlled rapid elimination of gene function. To this end, a degradation system was developed in yeast exploiting TIR1, a plant F box protein, which can recruit proteins with an auxin-inducible degron to an E3 ubiquitin ligase complex, but only in the presence of the phytohormone auxin. Here we demonstrate that the auxin-inducible degradation system functions efficiently also in Drosophila melanogaster. Neither auxin nor TIR1 expression have obvious toxic effects in this organism, and in combination they result in rapid degradation of a target protein fused to the auxin-inducible degron.