The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis. Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

Premature Termination Codon in 5′ Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation

Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.

Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival

ABSTRACT Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

Identification of dwarfism loci Dw1 and Dw2 in clonal apple rootstocks using molecular markers

The paper presents the results of identification of quantitative trait loci (QTL) of Dw1 and Dw2 involved in the control of dwarf growth in clonal apple rootstocks using the molecular markers. In total, 14 forms of rootstocks were analyzed. The microsatellite markers Hi01c04, Hi04a08, CH03a09 (for the Dw1 locus), MDP0000365711, and MDP0000243703 (for the Dw2 locus) were used in the study. Analysis of the results did not reveal a relationship between the presence of a marker and the manifestation of a sign. Of the three markers of the Dw1 locus, only Hi01c04 amplifies a fragment of the expected size of 120 bp. It was found in 9 forms of rootstocks (PB-4, 57-491, 83-1-15, M9, G16, 2-12-10, 2-9-102, 4-6-5 and 70-20-20). The Hi01c04 marker was identified in both dwarf and medium-sized forms. Of the two markers of the Dw2 locus, the desired fragment is amplified in MDP0000365711. It is typical for almost all genotypes. The exception is 83-1-15, 2-12-10 and 70-20-20, which have a null allele. To assess the genetic diversity of the Dw1 and Dw2 loci, 6 microsatellite sequences Hi01c04, Hi04a08, CH03a09, CH02d08, MDP0000365711, and MDP0000243703 were used. In 14 studied samples, 29 allelic variants ranging in size from 102 bp. up to 170 bp were identified. The number of alleles per locus varied from 1 (for the MDP0000365711 locus) to 7 (for the Hi04a08 locus). No rare alleles were identified. All alleles were observed more than three times. Based on the analysis of SSR spectra, a dendrogram reflecting the similarity of the genotypes under study was built.

Progress towards completing the mutant mouse null resource

The generation of a comprehensive catalog of null alleles covering all protein-coding genes is the goal of the International Mouse Phenotyping Consortium. Over the past 20 years, significant progress has been made towards achieving this goal through the combined efforts of many large-scale programs that built an embryonic stem cell resource to generate knockout mice and more recently employed CRISPR/Cas9-based mutagenesis to delete critical regions predicted to result in frameshift mutations, thus, ablating gene function. The IMPC initiative builds on prior and ongoing work by individual research groups creating gene knockouts in the mouse. Here, we analyze the collective efforts focusing on the combined null allele resource resulting from strains developed by the research community and large-scale production programs. Based upon this pooled analysis, we examine the remaining fraction of protein-coding genes focusing on clearly defined mouse–human orthologs as the highest priority for completing the mutant mouse null resource. In summary, we find that there are less than 3400 mouse–human orthologs remaining in the genome without a targeted null allele that can be further prioritized to achieve our overall goal of the complete functional annotation of the protein-coding portion of a mammalian genome.

A Null Allele of the Pol IV Second Subunit is Viable in Oryza sativa

All eukaryotes possess three DNA-dependent RNA polymerases, Pols I-III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24-nt siRNAs that interact with Pol V transcripts to target de novo DNA methylation and silence transcription of transposons. Members of the grass family encode additional duplicated subunits of Pol IV and V, raising questions regarding the function of each paralog. In this study, we identify a null allele of the putative Pol IV second subunit, NRPD2, and demonstrate that NRPD2 is the sole subunit functioning with NRPD1 in small RNA production and CHH methylation in leaves. Homozygous nrpd2 mutants have neither gametophytic defects, nor embryo lethality, although adult plants are dwarf and sterile.