Genomic Structure of Hstx2 Modifier of Prdm9-Dependent Hybrid Male Sterility in Mice.

F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male sterility can be fully reconstituted from three components of this model, the Prdm9 gene, intersubspecific homeology of Mus musculus musculus and Mus musculus domesticus autosomes, and the X-linked Hstx2 locus. Hstx2 modulates the extent of Prdm9-dependent meiotic arrest and harbors two additional factors responsible for intersubspecific introgression-induced oligospermia (Hstx1) and meiotic recombination rate (Meir1). To facilitate positional cloning and to overcome the recombination suppression within the 4.3 Mb encompassing the Hstx2 locus, we designed Hstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-strand breaks specifically within the Hstx2 locus. The resulting recombinant reduced the Hstx2 locus to 2.70 Mb (chromosome X: 66.51-69.21 Mb). The newly defined Hstx2 locus still operates as the major X-linked factor of the F1 hybrid sterility, and controls meiotic chromosome synapsis and meiotic recombination rate. Despite extensive further crosses, the 2.70 Mb Hstx2 interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4me3 hotspots and absence of DMC1-defined DNA double-strand-break hotspots. To search for structural anomalies as a possible cause of recombination suppression, we used optical mapping and observed high incidence of subspecies-specific structural variants along the X chromosome, with a striking copy number polymorphism of the microRNA Mir465 cluster. This observation together with the absence of a strong sterility phenotype in Fmr1 neighbor (Fmr1nb) null mutants support the role of microRNA as a likely candidate for Hstx2.

Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica.

Inspection of the complete genome of the yeast Yarrowia lipolytica for the presence of genes encoding homologues of known telomere-binding proteins surprisingly revealed no counterparts of typical yeast Myb domain-containing telomeric factors including Rap1 or Taz1. Instead, we identified a gene, YALIOD10923g, encoding a protein containing two Myb domains, exhibiting a high degree of similarity to the Myb domain of human telomeric proteins TRF1 and TRF2 and homologous to an essential fission yeast protein Mug152 whose expression is elevated during meiosis. The protein, which we named Tay1p (telomere-associated in Yarrowia lipolytica 1), was purified for biochemical studies. Using a model Y. lipolytica telomere, we demonstrate that the protein preferentially binds to Y. lipolytica telomeric tracts. Tay1p binds along the telomeric tract as dimers and larger oligomers, and it is able to remodel the telomeric DNA into both looped structures and synaptic complexes of two model telomere DNAs. The ability of Tay1p to induce dimerization of telomeres in vitro goes in line with its oligomeric nature, where each oligomer can employ several Myb domains to form intermolecular telomere clusters. We also provide experimental evidence that Tay1p may be associated with Y. lipolytica telomeres in vivo. Together with its homologues from Schizosaccharomyces pombe and several basidiomycetous fungi (Sánchez-Alonso, P., and Guzman, P. (2008) Fungal Genet. Biol. 45, S54-S62), Tay1p constitutes a novel family of putative telomeric factors whose analysis may be instrumental in understanding the function and evolution of double-stranded DNA telomeric proteins.

Lack of the catalytic subunit of telomerase leads to growth defects accompanied by structural changes at the chromosomal ends in Yarrowia lipolytica.

Comparative analysis of the telomeres of distantly related species has proven to be helpful for identifying novel components involved in telomere maintenance. We therefore initiated such a study in the nonconventional yeast Yarrowia lipolytica. Its genome encodes only a small fraction of the proteins that are typically associated with telomeres in other yeast models, indicating that its telomeres may employ noncanonical means for their stabilization and maintenance. In this report, we have measured the size of the telomeric fragments in wild-type strains, and characterized the catalytic subunit of telomerase (YlEst2p). In silico analysis of the YlEst2 amino acid sequence revealed the presence of domains typical for telomerase reverse transcriptases. Disruption of YlEST2 is not lethal, but results in retarded growth accompanied by a rapid loss of the telomeric sequences. This phenotype is associated with structural changes at the chromosomal ends in the ΔYlest2 mutants, likely the circularization of all six chromosomes. An apparent absence of several typical telomere-associated factors, as well as the presence of an efficient means of telomerase-independent telomere maintenance, qualify Y. lipolytica as an attractive model for the study of telomere maintenance mechanisms and a promising source of novel players in telomere dynamics.

Mitochondrial nucleoids from the yeast Candida parapsilosis: expansion of the repertoire of proteins associated with mitochondrial DNA.

Molecules of mitochondrial DNA (mtDNA) are packed into nucleic acid-protein complexes termed mitochondrial nucleoids (mt-nucleoids). In this study, we analysed mt-nucleoids of the yeast Candida parapsilosis, which harbours a linear form of the mitochondrial genome. To identify conserved as well as specific features of mt-nucleoids in this species, we employed two strategies for analysis of their components. First, we investigated the protein composition of mt-nucleoids isolated from C. parapsilosis mitochondria, determined N-terminal amino acid sequences of 14 proteins associated with the mt-nucleoids and identified corresponding genes. Next, we complemented the list of mt-nucleoid components with additional candidates identified in the complete genome sequence of C. parapsilosis as homologues of Saccharomyces cerevisiae mt-nucleoid proteins. Our approach revealed several known mt-nucleoid proteins as well as additional components that expand the repertoire of proteins associated with these cytological structures. In particular, we identified and purified the protein Gcf1, which is abundant in the mt-nucleoids and exhibits structural features in common with the mtDNA packaging protein Abf2 from S. cerevisiae. We demonstrate that Gcf1p co-localizes with mtDNA, has DNA-binding activity in vitro, and is able to stabilize mtDNA in the S. cerevisiae Deltaabf2 mutant, all of which points to a role in the maintenance of the C. parapsilosis mitochondrial genome. Importantly, in contrast to Abf2p, in silico analysis of Gcf1p predicted the presence of a coiled-coil domain and a single high-mobility group (HMG) box, suggesting that it represents a novel type of mitochondrial HMG protein.

Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans.

Mitochondria of eukaryotic organisms contain populations of DNA molecules that are packed into higher-order structures called mitochondrial nucleoids (mt-nucleoids). In Saccharomyces cerevisiae, the compaction of mitochondrial DNA (mtDNA) into mt-nucleoids is mediated primarily by the high-mobility group (HMG) box-containing protein Abf2, which is an important player in stabilization and metabolism of mtDNA. Although it is evident that analogous proteins must exist in other yeast species, an apparently fast divergence rate has precluded their identification, characterization and comparative analysis. Using in silico analysis of the complete genome sequence of the pathogenic yeast Candida albicans we predicted that the ORF 19.400/19.8030 assigned as GCF1 encodes a putative mitochondrial HMG box-containing protein. In contrast to Abf2p, which contains two HMG boxes, Gcf1p contains only one C-terminal HMG box. In addition, it contains one putative coiled-coil domain with a potential role in protein dimerization. Fluorescence microscopy analysis of a C-terminally tagged Gcf1p with green fluorescent protein (GFP) revealed its mitochondrial localization in both heterologous (S. cerevisiae) and native (C. albicans) hosts. Biochemical analyses of DNA-binding properties indicate that Gcf1p is, similarly to Abf2p, a non-specific DNA-binding protein. To analyse the role of Gcf1p in mtDNA metabolism, we constructed strains lacking one functional allele of the GCF1 gene and carrying one GCF1 allele under the control of the MET3 promoter. Under repressible conditions this strain exhibited a more than 3000-fold decrease in levels of GCF1 mRNA, which was correlated with a substantial decrease in the number of mtDNA copies as well as recombination intermediates. The dramatic effect of reduced levels of Gcf1p on mtDNA metabolism indicates that the protein is involved in essential molecular transactions that relate to the mitochondrial genome.