Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

A human progeria-associated BAF-1 mutation modulates gene expression and accelerates aging in C. elegans.

Alterations in the nuclear envelope are linked to a variety of rare diseases termed laminopathies. A single amino acid substitution at position 12 (A12T) of the human nuclear envelope protein BAF (Barrier to Autointegration Factor) causes Néstor-Guillermo Progeria Syndrome (NGPS). This premature ageing condition leads to growth retardation and severe skeletal defects, but the underlying mechanisms are unknown. Here, we have generated a novel in vivo model for NGPS by modifying the baf-1 locus in C. elegans to mimic the human NGPS mutation. These baf-1(G12T) mutant worms displayed multiple phenotypes related to fertility, lifespan, and stress resistance. Importantly, nuclear morphology deteriorated faster during aging in baf-1(G12T) compared to wild-type animals, recapitulating an important hallmark of cells from progeria patients. Although localization of BAF-1(G12T) was similar to wild-type BAF-1, lamin accumulation at the nuclear envelope was reduced in mutant worms. Tissue-specific chromatin binding and transcriptome analyses showed reduced BAF-1 association in most genes deregulated by the baf-1(G12T) mutation, suggesting that altered BAF chromatin association induces NGPS phenotypes via altered gene expression.

Loss of an H3K9me anchor rescues laminopathy-linked changes in nuclear organization and muscle function in an Emery-Dreifuss muscular dystrophy model.

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in Caenorhabditis elegans.

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the flippase (FLP) and cyclization recombination (Cre) enzymes has proved particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes, and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

Analysis of Nuclear Pore Complexes in Caenorhabditis elegans by Live Imaging and Functional Genomics.

Nuclear pore complexes (NPCs) are essential to communication of macromolecules between the cell nucleus and the surrounding cytoplasm. RNA synthesized in the nucleus is exported through NPCs to function in the cytoplasm, whereas transcription factors and other proteins are selectively and actively imported. In addition, many NPC constituents, known as nuclear pore proteins (nucleoporins or nups), also play critical roles in other processes, such as genome organization, gene expression, and kinetochore function. Thanks to its genetic amenability and transparent body, the nematode Caenorhabditis elegans is an attractive model to study NPC dynamics. We provide here an overview of available genome engineered strains and FLP/Frt-based tools to study tissue-specific functions of individual nucleoporins. We also present protocols for live imaging of fluorescently tagged nucleoporins in intact tissues of embryos, larvae, and adult and for analysis of interactions between nucleoporins and chromatin by DamID.

Nuclear Organization in Stress and Aging.

The eukaryotic nucleus controls most cellular processes. It is isolated from the cytoplasm by the nuclear envelope, which plays a prominent role in the structural organization of the cell, including nucleocytoplasmic communication, chromatin positioning, and gene expression. Alterations in nuclear composition and function are eminently pronounced upon stress and during premature and physiological aging. These alterations are often accompanied by epigenetic changes in histone modifications. We review, here, the role of nuclear envelope proteins and histone modifiers in the 3-dimensional organization of the genome and the implications for gene expression. In particular, we focus on the nuclear lamins and the chromatin-associated protein BAF, which are linked to Hutchinson-Gilford and Nestor-Guillermo progeria syndromes, respectively. We also discuss alterations in nuclear organization and the epigenetic landscapes during normal aging and various stress conditions, ranging from yeast to humans.