RORα contributes to the maintenance of genome ploidy in the liver of mice with diet-induced nonalcoholic steatohepatitis

Hepatic polyploidization is closely linked to the progression of nonalcoholic fatty liver disease (NAFLD); however, the underlying molecular mechanism is not clearly understood. In this study, we demonstrated the role of RORα in the maintenance of genomic integrity, particularly in the pathogenesis of NAFLD, using the high-fat diet (HFD)-fed liver-specific RORα knockout (RORα-LKO) mouse model. First, we observed that the loss of hepatic retinoic acid receptor-related orphan receptor α (RORα) accelerated hepatocyte nuclear polyploidization after HFD feeding. In 70% partial hepatectomy experiments, enrichment of hepatocyte polyploidy was more obvious in the RORα-LKO animals, which was accompanied by early progression to the S phase and blockade of the G2/M transition, suggesting a potential role of RORα in suppressing hepatocyte polyploidization in the regenerating liver. An analysis of a publicly available RNA-seq and chromatin immunoprecipitation-seq dataset, together with the Search Tool of the Retrieval of Interacting Genes/Proteins database resource, revealed that DNA endoreplication was the top enriched biological process gene ontology term. Furthermore, we found that E2f7 and E2f8, which encode key transcription factors for DNA endoreplication, were the downstream targets of RORα-induced transcriptional repression. Finally, we showed that the administration of JC1-40, an RORα activator (5 mg/kg body weight), significantly reduced hepatic nuclear polyploidization in the HFD-fed mice. Together, our observations suggest that the RORα-induced suppression of hepatic polyploidization may provide new insights into the pathological polyploidy of NAFLD and may contribute to the development of therapeutic strategies for the treatment of NAFLD.

Recruitment of BAF to the nuclear envelope couples the LINC complex to endoreplication

ABSTRACTDNA endoreplication has been implicated as a cell strategy for cell growth and in tissue injury. Here, we demonstrate that barrier-to-autointegration factor (BAF) represses endoreplication in Drosophila myofibers. We show that BAF localization at the nuclear envelope is eliminated in flies with mutations of the linker of nucleoskeleton and cytoskeleton (LINC) complex in which the LEM-domain protein Otefin is excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope requires the activity of the BAF kinase VRK1/Ball, and, consistently, non-phosphorylatable BAF-GFP is excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication, overlaps BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope results in increased E2F1 levels in the nucleoplasm and subsequent elevated DNA content. We suggest that LINC-dependent and phosphosensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation in the nucleoplasm.

Mechanosensitive recruitment of BAF to the nuclear membrane inhibits nuclear E2F1 and Yap levels

Mechanotransduction has been implicated as an important factor in regulating cell cycle progression; however, the underlying mechanism has not been fully elucidated. Here, we describe a novel mechano-sensitive component, namelybarrier to autointegration factor, (BAF), which regulates DNA endocycling inDrosophilamuscle fibers. We show that BAF negatively regulates DNA endoreplication by inhibiting of the nuclear entrance of E2F1 and Yap/Yorkie, two key components in cell cycle control. Furthermore, BAF localization at the nuclear membrane is mechanosensitive, as it was downregulated in LINC mutant larval muscles, or following nuclear deformation caused by disruption of nucleus-sarcomere connections. BAF forms a protein complex with E2F1, which is sensitive to BAF phosphorylation. Knockdown of BAF kinase VRK1/Ball disrupted localization of BAF at the nuclear membrane and resulted in increased E2F1 nuclear levels. Taken together, our results reveal a novel mechanosensitive pathway controlling BAF phosphorylation and localization at the nuclear membrane, which in turn, represses nuclear accumulation of positive cell cycle regulators.

Muscle tensions merge to cause a DNA replication crisis

In this issue, Wang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201708137) show that disruption to different mechanical domains of muscle cells converge at the linker of nucleoskeleton to cytoskeleton complex to affect DNA endoreplication potentially via barrier to autointegration factor–mediated epigenetic mechanisms.

Mechanotransduction via the LINC complex regulates DNA replication in myonuclei

Nuclear mechanotransduction has been implicated in the control of chromatin organization; however, its impact on functional contractile myofibers is unclear. We found that deleting components of the linker of nucleoskeleton and cytoskeleton (LINC) complex in Drosophila melanogaster larval muscles abolishes the controlled and synchronized DNA endoreplication, typical of nuclei across myofibers, resulting in increased and variable DNA content in myonuclei of individual myofibers. Moreover, perturbation of LINC-independent mechanical input after knockdown of β-Integrin in larval muscles similarly led to increased DNA content in myonuclei. Genome-wide RNA-polymerase II occupancy analysis in myofibers of the LINC mutant klar indicated an altered binding profile, including a significant decrease in the chromatin regulator barrier-to-autointegration factor (BAF) and the contractile regulator Troponin C. Importantly, muscle-specific knockdown of BAF led to increased DNA content in myonuclei, phenocopying the LINC mutant phenotype. We propose that mechanical stimuli transmitted via the LINC complex act via BAF to regulate synchronized cell-cycle progression of myonuclei across single myofibers.

DNA endoreplication level in endosperm during seed development in three monocotyledonous species

The DNA content after the Feulgen reaction in the endosperm of three monocotyledonous plant species (<i>Asparagus officinalis</i>, <i>Muscari comosom</i>, <i>Haemanthus kurharinae</i>) differing in their 2C DNA content, was cytophotometrically measured. During endosperm development 1-6 endoreplication cycles take place, depending on the species. Differences in nuclear DNA endoreplication dynamics in the tested species are similar to those occurring in root parenchyma, but the endoreplication level in the endosperm is higher.

DNA endoreplication level in endosperm and in cotyledons during seed development in three dicotyledonous species

The DNA content of nuclear and cellular endosperm and in cotyledons in the course of seed development in <i>Helianthus annuus</i>, <i>Pisum sativum</i> and <i>Vicia faba</i> was cytophotometrically measured after Faulgen's reaction. <i>Helianthus annuus</i> is characterized by the lowest endoreplication dynamics attaining a maximum DNA content of 48C in nuclear endosperm, which corresponds to four endoreplication rounds, and 16C DNA (three rounds) in cotyledons. In <i>Pisum sativum</i> cellular endosperm in the studied stages of seed development was absent and the highest DNA content in cotyledons was 512C DNA; this corresponds to eight endoreplication rounds. In <i>Vicia faba</i>, the maximum DNA endoreplication level was 1536C in nuclear endosperm, which corresponds to nine endoreplication rounds; in cotyledons the maximum DNA content was 64C (five rounds). In the course of seed development, differences in the dynamics of DNA endoreplication in throphic tissues of the examined species seem to be similar to differences in the endoreplication level in root and leaf parenchyma, but the DNA endoreplication level is much higher in throphic tissues. Therefore, the endoreplication pattern might be species-specific and genetically conditioned.

Nuclear DNA endoreplication and plastid index in mesophyll of some dicotyledonous species

Cytophotometric studies of nuclear DNA content after Feulgen procedure indicate that in mesophyll of all the seven studied species the highest nuclear DNA endoreplication level occurs in II or III leaf and it varies for particular species. No differences were found in nuclear DNA endoreplication dynamics between the basal and apical parts of the leaf blade. Chloroplast number per cell generally decreases in the successive leaves, and the plastid index is the smallest in the first (oldest) leaves, being similar in both zones. In four species chloroplast number and plastid index show relatively low negative correlation with nuclear DNA contents (expressed as endoreplication index), in two species this correlation is positive, and one species displays very low r value.