The Airn lncRNA does not require any DNA elements within its locus to silence distant imprinted genes

Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use of model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing factors to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs for silencing distant genes in cis. In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model, we found interactions enriched on the maternal allele across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a2, Pde10a and Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Furthermore, we found that silent imprinted genes were covered with large blocks of H3K27me3 on the repressed paternal allele. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established

Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I.

Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females

Correction: The Airn lncRNA does not require any DNA elements within its locus to silence distant imprinted genes.

[This corrects the article DOI: 10.1371/journal.pgen.1008268.]

Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I

Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific α-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females

Red2Flpe-SCON: a versatile, multicolor strategy for generating mosaic conditional knockout mice

Abstract Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We develop the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We use the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which reveal that the stem cell gene, Sox2, is less essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation

Red2Flpe-SCON: a versatile, multicolor strategy for generating mosaic conditional knockout mice

Acknowledgements: We thank present and past members of the Koo, Elling and Urban labs at IMBA for valuable discussions and critical comments, Dr. Rike Zietlow and the Life Science Editors for reading and correcting the manuscript, VBC core facilities (especially the Histopathology facility, BioOptics and the animal caretakers). We thank Single Cell Discoveries for helping us with single cell RNA sequencing and offering technical advice on sample handling. This work was supported by core funding from the Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences; ERC starting grant, Troy Stem cells, 639050; Interpark Bio-Convergence Center Grant Program; and fellowship to S.W. (DOC Fellowship of the Austrian Academy of Sciences). G.C. was supported by a Lise Meitner Postdoctoral fellowship M2976, FWF, and by the FWF Standalone (P35694) and ERA PerMed (I 5900) grants. B.-K.K. and his team are supported by the Institute for Basic Science.Funder: This work was supported by core funding from the Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences; ERC starting grant, Troy Stem cells, 639050; Interpark Bio-Convergence Center Grant Program; and fellowship to first author Sam Wu (DOC Fellowship of the Austrian Academy of Sciences)Funder: FWF Lise Meitner Postdoctoral fellowship M2976 FWF Standalone P35694 FWF ERA PerMed I 5900AbstractImage-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We develop the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We use the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which reveal that the stem cell gene, Sox2, is less essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.</jats:p

Distinct functions of junD in cardiac hypertrophy and heart failure

Cardiac hypertrophic stimuli induce both adaptive and maladaptive growth response pathways in heart. Here we show that mice lacking junD develop less adaptive hypertrophy in heart after mechanical pressure overload, while cardiomyocyte-specific expression of junD in mice results in spontaneous ventricular dilation and decreased contractility. In contrast, fra-1 conditional knock-out mice have a normal hypertrophic response, whereas hearts from fra-1 transgenic mice decompensate prematurely. Moreover, fra-1 transgenic mice simultaneously lacking junD reveal a spontaneous dilated cardiomyopathy associated with increased cardiomyocyte apoptosis and a primary mitochondrial defect. These data suggest that junD promotes both adaptive-protective and maladaptive hypertrophy in heart, depending on its expression levels