Chromosome-scale and haplotype-resolved genome assembly of a tetraploid potato cultivar.

Potato is the most widely produced tuber crop worldwide. However, reconstructing the four haplotypes of its autotetraploid genome remained an unsolved challenge. Here, we report the 3.1 Gb haplotype-resolved (at 99.6% precision), chromosome-scale assembly of the potato cultivar 'Otava' based on high-quality long reads, single-cell sequencing of 717 pollen genomes and Hi-C data. Unexpectedly, ~50% of the genome was identical-by-descent due to recent inbreeding, which was contrasted by highly abundant structural rearrangements involving ~20% of the genome. Among 38,214 genes, only 54% were present in all four haplotypes with an average of 3.2 copies per gene. Taking the leaf transcriptome as an example, 11% of the genes were differently expressed in at least one haplotype, where 25% of them were likely regulated through allele-specific DNA methylation. Our work sheds light on the recent breeding history of potato, the functional organization of its tetraploid genome and has the potential to strengthen the future of genomics-assisted breeding.

Telobox motifs recruit CLF/SWN-PRC2 for H3K27me3 deposition via TRB factors in Arabidopsis.

Polycomb repressive complexes (PRCs) control organismic development in higher eukaryotes through epigenetic gene repression1-4. PRC proteins do not contain DNA-binding domains, thus prompting questions regarding how PRCs find their target loci 5 . Here we present genome-wide evidence of PRC2 recruitment by telomere-repeat-binding factors (TRBs) through telobox-related motifs in Arabidopsis. A triple trb1-2, trb2-1, and trb3-2 (trb1/2/3) mutant with a developmental phenotype and a transcriptome strikingly similar to those of strong PRC2 mutants showed redistribution of trimethyl histone H3 Lys27 (H3K27me3) marks and lower H3K27me3 levels, which were correlated with derepression of TRB1-target genes. TRB1-3 physically interacted with the PRC2 proteins CLF and SWN. A SEP3 reporter gene with a telobox mutation showed ectopic expression, which was correlated with H3K27me3 depletion, whereas tethering TRB1 to the mutated cis element partially restored repression. We propose that telobox-related motifs recruit PRC2 through the interaction between TRBs and CLF/SWN, a mechanism essential for H3K27me3 deposition at a subset of target genes.

ADAM15 adds to apoptosis resistance of synovial fibroblasts by modulating focal adhesion kinase signaling.

OBJECTIVE: To study the contribution of ADAM15, a disintegrin metalloproteinase that is up-regulated in the rheumatoid arthritis (RA) synovial membrane, to the characteristic resistance of RA synovial fibroblasts (RASFs) to apoptosis induction by genotoxic stress or stimulation with proapoptotic FasL, which is present at high concentrations in RA synovial fluid. METHODS: Caspase 3/7 activity and the total apoptosis rate in RASFs upon exposure to the DNA-damaging agent camptothecin or FasL were determined using enzyme assays and annexin V staining. Phosphorylated signaling proteins were analyzed by immunoblotting. RNA interference was used to silence ADAM15 expression. NF-κB activity was determined by enzyme-linked immunosorbent assay. RESULTS: RASFs displayed significantly higher caspase 3/7 activity upon camptothecin and FasL exposure when ADAM15 had been down-regulated by specific small interfering RNAs. Upon FasL stimulation, RASFs phosphorylated focal adhesion kinase (FAK) and c-Src (Src), and activated phosphatidylinositol 3-kinase as well as the transcription factor NF-κB. This ADAM15-dependent, FasL-induced activation of antiapoptotic kinases and NF-κB was demonstrated by a marked reduction of apoptosis upon knockdown of ADAM15 protein expression. Inhibitors specifically interfering with FAK and Src signaling, such as FAK inhibitor 14 and dasatinib, potently induce apoptosis in RASFs, with significant enhancement by the silencing of ADAM15. CONCLUSION: ADAM15 contributes to apoptosis resistance in RASFs by activating the Src/FAK pathway upon FasL exposure, rendering the FAK/Src signaling pathway an interesting target for potential therapeutic intervention in RA.

ADAM15 protein amplifies focal adhesion kinase phosphorylation under genotoxic stress conditions.

ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.

A simple method of investigating mutations in CHEK2 by DHPLC: a study of the German populations of Saxony, Saxony-Anhalt, and Thuringia.

Allele variants of the CHEK2 gene have been found to be associated with several types of cancer, including cancer of the breast, prostate, lung, and ovary. In the Polish population, three founder mutations of CHEK2 have been identified: I157T, 444+1G>A (formerly IVS2+1G>A), and 1100delC. The aim of our study was to establish a simple method to identify founder CHEK2 mutations and determine the prevalence of these changes in the population of Eastern Germany (Saxony, Saxony-Anhalt, and Thuringia). We drew up denaturing high-performance liquid chromatography (DHPLC) conditions for analysis of intron 2 and exon 3 for two mutations (444+1G>A, I157T) and exon 10 for mutation 1100delC. We tested 251 patients and controls. Mutations show a similar frequency in the general population of Eastern Germany as in neighboring Poland (4.95% vs. 4.8% for the missense mutation I157T and 0.99% vs. 0.5% for the truncating mutations 444+1G>A and 1100delC). Investigation of these mutations by DHPLC is highly sensitive and less time-consuming compared to restriction fragment length polymorphism or allele-specific oligonucleotide polymerase chain reaction. It can be easily integrated into diagnostic testing.

ADAM15 exerts an antiapoptotic effect on osteoarthritic chondrocytes via up-regulation of the X-linked inhibitor of apoptosis.

OBJECTIVE: To investigate the capacity of ADAM15, a disintegrin metalloproteinase that is up-regulated in osteoarthritic (OA) cartilage, to protect chondrocytes against apoptosis induced by growth factor deprivation and genotoxic stress. METHODS: Caspase 3/7 activity was determined in primary OA and ADAM15-transfected T/C28a4 chondrocytes upon exposure to the DNA-damaging agent camptothecin or serum withdrawal. Camptothecin-induced cytotoxicity was determined by measuring cellular ATP content. (Anti-)apoptotic proteins were analyzed by immunoblotting, and levels of messenger RNA (mRNA) for X-linked inhibitor of apoptosis (XIAP) were determined using real-time polymerase chain reaction. RNA interference was applied for down-regulation of ADAM15 and XIAP expression. Immunohistochemistry analysis of normal and OA cartilage samples was performed using XIAP- and ADAM15-specific antibodies. RESULTS: ADAM15-transfected chondrocytes cultured on a collagen matrix displayed significantly reduced caspase 3/7 activity upon serum or intermittent matrix withdrawal, compared with vector-transfected control cells. Apoptosis induction by camptothecin exposure also led to significantly elevated caspase 3/7 activity and reduced cell viability of the vector-transfected compared with ADAM15-transfected chondrocytes. Increased levels of activated caspase 3 and cleaved poly(ADP-ribose) polymerase were detected in the vector controls. XIAP, an inhibitor of activated caspase 3, was significantly up-regulated ( approximately 3-fold) at the protein and mRNA levels in ADAM15-transfected chondrocytes upon camptothecin treatment. Specific down-regulation of either ADAM15 or XIAP in OA chondrocytes led to significant sensitization to camptothecin-induced caspase 3/7 activity. Immunohistochemical analysis revealed low to moderate XIAP expression in normal specimens and markedly increased XIAP staining, colocalizing with ADAM15, in OA cartilage. CONCLUSION: ADAM15 conveys antiapoptotic properties to OA chondrocytes that might sustain their potential to better resist the influence of death-inducing stimuli under pathophysiologic conditions.

A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina.

Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short ( approximately 250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5' and 3' flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.