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Replication Timing (RT) refers to the temporal order in which the genome is replicated during S phase. Early replicating regions correlate with the transcriptionally active, accessible euchromatin (A) compartment, while late replicating regions correlate with the heterochromatin (B) compartment and repressive histone marks. Previously, widespread A/B genome compartmentalization changes were reported following Brd2 depletion. Since RT and A/B compartmentalization are two of the most highly correlated chromosome properties, we evaluated the effects of Brd2 depletion on RT. We performed E/L Repli-Seq following Brd2 depletion in the previously described Brd2 conditional degron cell line and found no significant alterations in RT after Brd2 KD. This finding prompted us to re-analyze the Micro-C data from the previous publication. We report that we were unable to detect any compartmentalization changes in Brd2 depleted cells compared to DMSO control using the same data. Taken together, our findings demonstrate that Brd2 depletion alone does not affect A/B compartmentalization or RT in mouse embryonic stem cells.

T cells play key protective but also pathogenic roles in COVID-19. We studied expression of long non-coding RNAs (lncRNAs) in COVID-19 T cell transcriptomes by integrating previously published single-cell RNA sequencing datasets. The long intergenic non-coding RNA MALAT1 was the most highly transcribed lncRNA in T cells, with Th1 cells demonstrating the lowest and CD8+ resident memory cells the highest MALAT1 expression, amongst CD4+ and CD8+ T cells populations, respectively. We then identified gene signatures that covaried with MALAT1 in single T cells. A significantly higher number of transcripts correlated negatively with MALAT1 than those that correlated. Enriched functional annotations of the MALAT1-anti-correlating gene signature included processes associated with T cell activation such as cell division, oxidative phosphorylation and response to cytokine. The MALAT1 anti-correlating gene signature shared by both CD4+ and CD8+ T cells marked dividing T cells in both lung and blood of COVID-19 patients. Focussing on the tissue, we used an independent patient cohort of post-mortem COVID-19 lung samples and demonstrated that MALAT1 suppression was indeed a marker of MKI67+ proliferating CD8+ T cells. Our results reveal MALAT1 suppression and its associated gene signature are a hallmark of human proliferating T cells.

Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1.5 GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.

Proteins of the cytohesin family are known for their guanine-nucleotide exchange factor function for ARF-GTPases, mainly for ARF1 and ARF6. While Arf1 and Arf6 deficiency results in embryonic lethality, in vivo functions of cytohesins are rarely described and mostly inconspicuous. We analyzed the role of cytohesin-2 in vivo and in vitro and found that cytohesin-2 full knockout mice die within one day after birth. Mass spectrometry-based organellar proteomics in wildtype and CRISPR-Cas9-generated cytohesin-2-/- C2 myoblasts revealed a markedly altered Golgi compartment. Golgi volumes were reduced in different cytohesin-2-/- cell lines compared to wildtype cells as revealed by immunofluorescence. Reduced Golgi volumes were rescued by introducing cytohesin-2. Finally, we observed that typical functions of the Golgi apparatus were disrupted in cytohesin-2-deficient cells. Cytohesin2-/- C2 myoblasts exhibited significant changes in the galactose / N-acetyl-galactosamine glycosylation on the cell surface compared to wildtype cells when stained with peanut agglutinin. Further, protein secretion was overall reduced in neonatal cytohesin-2-/- mice compared to wildtype as determined by mass spectrometry-based proteomics. This study describes the essential role of cytohesin-2 in neonatal development and a novel function of the protein in Golgi regulation.

B cell receptor (BCR) signaling is required for the survival and maturation of B cells and is deregulated in B cell lymphomas. While proximal BCR signaling is well studied, little is known about the crosstalk of downstream effector pathways, and a comprehensive quantitative network analysis of BCR signaling is missing. Here, we semi-quantitatively modelled BCR signaling in Burkitt lymphoma (BL) cells using systematically perturbed phosphorylation data of BL-2 and BL-41 cells. The models unveiled feedback and crosstalk structures in the BCR signaling network, including a negative crosstalk from p38 to MEK/ERK. The relevance of the crosstalk was verified for BCR and CD40 signaling in different BL cells and confirmed by global phosphoproteomics on ERK itself and known ERK target sites. Compared to the starting network, the trained network for BL-2 cells was better transferable to BL-41 cells. Moreover, the BL-2 network was also suited to model BCR signaling in Diffuse large B cell lymphoma cells lines with aberrant BCR signaling (HBL-1, OCI-LY3), indicating that BCR aberration does not cause a major downstream rewiring.

Glucocorticoids (such as Dexamethasone) are commonly used immunomodulatory drugs with potent anti-inflammatory effects, whose mechanisms of action remain incompletely understood. They bind to the Glucocorticoid Receptor (GR), a nuclear hormone receptor that acts as a transcription factor to directly control the expression of inflammatory genes. To elucidate the complex molecular mechanisms employed by GR during the suppression of innate immune responses, we have performed proteomics, ChIP-seq, ATAC-seq, RNA-seq and bioinformatics together with genetic and pharmacological loss of function studies in primary mouse macrophages. We found that GR interacts with the ATP-dependent SWI/SNF chromatin remodeling complex to regulate a specific subset of target genes. Here we show that the central catalytic subunit BRG1 is required not only for the transcriptional activation of classical GR target genes such as Fkbp5 or Klf9, but also for the transcriptional repression of cytokines and chemokines such as Ccl2, Cxcl10 or Il1a. We demonstrate that loss of BRG1 activity leads to reduced histone deacetylase (HDAC) function, and consequently increased histone acetylation, at these repressive GR binding sites. Altogether, our findings suggest that GR interacts with BRG1 to assemble a functional co-repressor complex at a defined fraction of macrophage cis-regulatory elements. These results may indicate additional non-classical, remodeling-independent functions of the SWI/SNF complex and may have implications for the development of future immunomodulatory therapies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/472398v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1d9d238org.highwire.dtl.DTLVardef@1788bb1org.highwire.dtl.DTLVardef@11b3d4dorg.highwire.dtl.DTLVardef@155b9fc_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO In macrophages (m{Phi}) responding to bacterial LPS and Dexamethasone, the Glucocorticoid Receptor (GR) activates target genes like Klf9 or Fkbp5 via interaction with the BRG1-containing SWI/SNF complex, chromatin remodeling and Mediator recruitment. At the same time, GR represses the expression of inflammatory cytokines and chemokines such as Ccl2, Cxcl10, Il1a etc. by assembling a BRG1-containing co-repressor complex and de-acetylating surrounding histone tails. Loss of BRG1 activity affects both the transcriptional activation and repression of a subset of myeloid GR target genes via distinct mechanisms. (iTF: inflammatory transcription factor; Ac: histone acetylation) (Created with BioRender.com.) C_FIG

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 [A]2 and a smaller one of 415 [A]2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.

KDEL receptors (KDELRs) prevent the secretion of soluble chaperones and enzymes meant to reside in the endoplasmic reticulum. While a single KDELR exists in yeast (ERD2), three variants are present in mammals, displaying high sequence similarity (73-83%). However, the phylogenetic conservation of the differences and the diverse tissue distribution of the three KDELRs suggest functional specialization. Here we show that, while all three receptors can prevent the secretion of KDEL-bearing clients, KDELR1 and KDELR3 regulate the production of AGR2, a key assistant of mucin folding, in opposite ways. AGR2 transcripts increase dramatically upon silencing KDELR3 but decrease when KDELR1 is downregulated. Silencing ERp44, but not other ER residents, phenocopies KDELR3 knockdown, suggesting that AGR2 regulation depends on ERp44-KDELR3 interactions. Our findings identify a novel regulatory circuit that controls the molecular composition of the early secretory pathway based on specific interactions between KDELRs and ER residents.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in numerous cell types in vitro and in models of neurodegeneration and diabetes in vivo. So far, the exact mode of its action has remained elusive and plasma membrane or ER-located receptors of MANF have not been identified. We have found that MANF can directly interact with transmembrane unfolded protein response (UPR) receptor IRE1 and compete with the major ER chaperone BiP (GRP78) for the interaction with IRE1. With lower affinities MANF can also interact with other UPR receptors, PERK and ATF6. Using molecular modeling and mutagenesis analysis, we have identified the exact structural MANF regions involved in its binding to the luminal domain of IRE1. MANF attenuates UPR signaling by decreasing IRE1 oligomerization and IRE1 phosphorylation. MANF mutant deficient in IRE1 binding cannot regulate IRE1 oligomerization and fails to protect neurons from ER stress induced death. Importantly, we found that MANF-IRE1 interaction is also crucial for the survival promoting action of MANF for dopamine neurons in an animal model of Parkinsons disease. Our data reveal a novel mechanism of IRE1 regulation during ER stress and demonstrate the intracellular mode of action of MANF as a modulator of UPR and neuronal cell survival through the direct interaction with IRE1 and regulation of its activity. Furthermore, our data explain why MANF in contrast to other growth factors has no effects on naive cells and rescues only ER stressed or injured cells.

Naive B cells exit quiescence and enter a proliferative state upon activation, ultimately differentiating into antibody-secreting or memory B cells. Toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS), can serve as physiological stimuli to initiate this transition. Using quantitative proteomics, we show that TLR4 engagement induces metabolic reprogramming in murine B cells, increasing the expression of amino acid transporters and cholesterol biosynthetic enzymes. The amino acid transporter SLC7A5 is markedly upregulated following LPS stimulation, and conditional deletion of Slc7a5 impairs B cell proliferation, underscoring its essential role in B cell activation. LPS also elevates intracellular cholesterol levels, and inhibition of the rate-limiting enzyme HMG-CoA reductase blocks proliferation. This effect was mediated by a dual requirement for cholesterol metabolism and protein prenylation downstream of HMG-CoA reductase. Notably, this was not unique to TLR4 signalling but is also observed in B cells activated via TLR7, TLR9, CD40, or the B cell receptor. Together, these findings reveal that metabolic rewiring, including amino acid uptake and cholesterol metabolism, is an essential feature of B cell activation and proliferation.

Overexpression of RNase H1, a ribonuclease that degrades RNA:DNA hybrids and R-loops, can suppress genome instability phenotypes in a range of maladaptive conditions. This has been interpreted to suggest that genotoxic co-transcriptional R-loops arise under these conditions and are resolved by RNase H1. Here, we manipulated RNase H1 levels using conditional knockout and overexpression models in primary murine B cells and mapped the resulting genomic R-loop landscapes. Rnaseh1 deletion resulted in a dramatic loss of mitochondrial replication and compromised B cell responses, consistent with a critical mitochondrial function for RNase H1. Genome-wide R-loops were, however, not significantly affected. More surprisingly, overexpressing active nuclear RNase H1 did not lead to significant reduction of R-loop levels or change their distribution. These results were confirmed using a human cell line in which active, nuclear RNase H1 can be induced. Our findings indicate that co-transcriptional R-loops are not efficiently resolved by RNase H1 and suggest that the identity of the RNA/DNA hybrids at the root of the genome instability phenotypes suppressed by RNase H1 may need to be re-interpreted.

Exit from the endoplasmic reticulum is mediated by the Sar1/COPII machinery and a number of accessory factors. How the initial steps of cargo recruitment upstream of Sar1/COPII are mediated remains unclear, but the dihydropyridine FLI-06 inhibits cargo recruitment into ER exit sites. Here, we used chemical genetics screening approaches in conjunction with FLI-06 treatment and identified the ER membrane proteins YIPF5 and GOT1A/B as putative components of early export processes. Surprisingly, the two homologous proteins GOT1A and GOT1B, coded by GOLT1A and GOLT1B, respectively, exhibited opposite functions after treatment with FLI-06: increasing the expression of GOT1A or reducing the expression of GOT1B or YIPF5 prevented inhibition of ER-export by FLI-06. Inhibiting ER export with FLI-06 elicited a specific ER stress-related gene expression signature distinct from the ER-stress signature induced by Thapsigargin. The interactomes of GOT1A and GOT1B suggested a connection to ER-stress mediators. Moreover, RNA-Seq data showed that FLI-06-induced genes are strongly enriched for ATF6 target genes which are suppressed by GOLT1A overexpression or GOLT1B knock-down. This suggests that ATF6 signaling is involved in FLI-06-mediated toxicity, and we could demonstrate that siRNA-mediated knock-down or specific inhibitor of ATF6 rescued cells from FLI-06-mediated cell death. Knock-down or inhibition of ATF6 is sufficient to resume transport from the ER under FLI-06-treatment, suggesting that ATF6 is directly involved in the FLI-06-mediated ER-export block. Surprisingly, our data show that this ATF6 function is independent of de novo transcription, implying a novel, transcription-independent function of ATF6.

Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, has provided unique insights which have contributed to the emergence of the Ubiquitin code. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains less well understood. Similarly, the contribution of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains assembled by APC/c during mitosis, also remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell proliferation and we established that this is mediated through loss of its ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase). We confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. To explore the mechanisms involved, we used proteomics to explore the endogenous HECTD1 interactome in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity in mitosis.

Signal transducer and activator of transcription 5 (STAT5) is a key transcriptional regulator acting downstream of hematopoietic cytokines and hormones, such as erythropoietin (Epo), thrombopoietin or prolactin. STAT5-mediated gene regulation involves tyrosine phosphorylation at cytokine receptors and subsequent nuclear import. We studied STAT5 nucleocytoplasmic shuttling via live-cell imaging of fluorescent mutants in STAT5-/- HeLa EpoR cells. Unexpectedly, STAT5 mutants lacking the transactivation domain (TAD) were retained in the cytoplasm following Epo stimulation. Building upon this, we identified a 12-amino-acid stretch in the TAD sufficient to restore nuclear translocation. Further analysis revealed two residues within this 12-amino-acid stretch, D754 and D758, to be essential for nuclear import of phosphorylated full-length STAT5. Importantly, a single intact TAD in the STAT5 dimer is sufficient for nuclear import. Our findings reveal a unique role of the TAD in STAT5 nuclear trafficking distinct from other STATs, providing new mechanistic insight and potential targets for therapeutic intervention in STAT5-driven disease such as myeloproliferative neoplasms and leukemia.

BackgroundAutoinflammatory diseases (AID) result from dysregulation of the first lines of innate immune responses. Recently, development of high throughput genome sequencing technology led to the rapid emergence of important knowledge in the genetic field. About 20 genes have been identified so far in monogenic forms of distinct AID. However, 70-90 % of patients with AID remain without genetic diagnosis.\n\nObjectiveWe report the identification and characterization of a mutation in the C-terminal region of the Rho GTPase Cdc42 in a patient presenting a severe autoinflammatory phenotype.\n\nMethodsWe have analyzed the consequences of the mutation on the subcellular localization of the Cdc42 protein using imaging techniques. Molecular studies were performed using proteomic and biochemical experiments to provide mechanistic bases of the observed defects. Functional assays were also conducted using flow cytometry and cytokine production measurements.\n\nResultsWe show that mutant Cdc42 is trapped in the Golgi apparatus due to the aberrant addition of a palmitate that both enhances the interaction of mutant Cdc42 with Golgi membranes and inhibit its extraction by GDP dissociation inhibitor (GDI), thus impairing its cytosol/membrane shuttling. At the functional level, mutant Cdc42 fails to sustain actin filaments polymerization and induces an exacerbated profile of pro-inflammatory cytokine production due to increased NF-{kappa}B activation.\n\nConclusionsOur study now provides a molecular explanation for mutations that have been identified recently in our AID patient and others in the C-terminal part of Cdc42. Mutations located in this region of Cdc42 impair the intracellular localization of Cdc42, preventing its interaction with the plasma membrane. Thus, our results definitively link mutations in the CDC42 gene to a complex immune-hemato-autoinflammatory phenotype in humans.

The unfolded protein response (UPR) is a key adaptive pathway that controls endoplasmic reticulum (ER) homeostasis. The UPR is transduced by three ER-resident sensors of ER homeostasis disruption in the lumen of this compartment. They trigger select downstream signaling pathways in the cytosol and nucleus. Among them, IRE1 (referred to as IRE1 hereafter), a type I transmembrane protein, senses accumulation of improperly folded proteins in the ER lumen and transduces signals through both kinase and endoribonuclease (RNase) activities in the cytosol. IRE1 catalyzes XBP1 mRNA unconventional splicing and RNA degradation (Regulated IRE1 Dependent Decay, termed RIDD). Recent studies have reported that IRE1-dependent protein-protein interactions (PPi) drive additional non-canonical IRE1 functions. Herein, we define the IRE1 signalosome as a list of IRE1 binding partners (direct or not) which alter IRE1 signaling towards XBP1 mRNA splicing and RIDD. Here we determined the IRE1 in situ interactome using BioID, putatively connecting IRE1 to previously unrecognized cellular functions. In addition, we link the binding of several IRE1 partners to the regulation of its RNase. Furthermore, we identify HNRNPL as an IRE1-interacting partner, previously unrecognized, which stabilizes IRE1 under basal conditions by counteracting ERAD-mediated degradation. Overall, the characterization of the IRE1 signalosome not only reveals the multi-faceted control of IRE1 RNase activity and stability by its interacting partners and allow us to discuss putative additional IRE1 regulators and cellular functions based on the nature of its interactome and its localization.

NEK1 is a pleiotropic protein kinase implicated in mitosis, ciliogenesis and DNA repair but little is known about its regulation or targets. Its relevance for human health is underscored by the association of NEK1 mutations with human diseases including axial spondylometaphyseal dyplasia (SMD) and amyotrophic lateral sclerosis (ALS). Mutations in the C21ORF2 gene are associated with a similar pattern of human diseases, suggesting close functional links with NEK1. Here we report that in unperturbed, untransformed cells, endogenous NEK1 and C21ORF2 form a tight complex that does not appear to contain other proteins. A small acidic domain "CID: C21ORF2 interaction domain" at the C-terminus of NEK1 is necessary and sufficient to interact with C21ORF2, and pathogenic mutations in this region disrupt the complex. AlphaFold modelling predicts with high confidence an extended binding interface between a leucine-rich repeat (LRR) domain in the N-terminal half of C21ORF2 and a stretch of the NEK1-CID; mutating residues mediating electrostatic interactions within this interface disrupts the NEK1-C21ORF2 interaction. This model also explains why pathogenic mutations disrupt the complex. We go on to show that the kinase activity of NEK1 and its interaction with C21ORF2 is critical for NEK1 function in cells. These data reveal C21ORF2 as a regulatory subunit of NEK1, illuminating our understanding of how this kinase is regulated and NEK1-C21ORF2-associated diseases.

In the arms race between a pathogen and the host, many bacteria have acquired a sizable armory to counteract or change the defense mechanisms of the host cell, including the eukaryotic ubiquitin system. Simkania negevensis, an obligate intracellular Chlamydia-like bacterium, is one such example. S. negevensis has a biphasic developmental cycle, similar to other members of the Chlamydiales order. The bacterium develops inside a tubular membranous compartment called Simkania-containing vacuole, which is in close contact with the host endoplasmic reticulum (ER) and mitochondria. It can infect a wide range of hosts, with a long infection cycle lasting up to 15 days, and is associated with respiratory tract diseases. Recently, S. negevensis has been discovered to possess an unusually large number of deubiquitinating enzymes, but not much is known about the variety of other ubiquitin-modifying enzymes in this bacterium. Our study provides an initial description of the activity of a so far uncharacterized S. negevensis ubiquitin E3 RING-ligase (SNE_A12920 or SneRING). We report that SneRING primarily generates K63- and K-11 linked ubiquitin chains and preferentially interacts with UbcH5b and UBE2T E2 ubiquitin ligases among the ones we tested. Bacteria express SneRING upon infection of various human cell lines, as well as amoeba. In addition, we demonstrate that a portion of the expressed SneRING co-localizes with mitochondria and ER. Mass spectrometry studies of the SneRING interactome show enrichment in mitochondrial and ER proteins containing a prohibitin domain and involved in organelle morphology, respiration, and stress response. Our work offers the first insights into the function of an S. negevensis RING ligase, an enzyme potentially involved in organelle remodeling to accommodate the unique lifestyle of this intracellular bacterium.

Skeletal muscle atrophy and weakness are major contributors to morbidity, prolonged recovery, and long-term disability across a wide range of diseases. Atrophy is caused by breakdown of sarcomeric proteins resulting in loss of muscle mass and strength. Molecular mechanism underlying the onset of muscle atrophy and its progression have been analysed in patients, mice, and cell culture but the complementarity of these model systems remains to be explored. Here, we applied deep-coverage transcriptomic and proteomic profiling to characterize dynamic changes during dexamethasone-induced atrophy in the widely used murine skeletal muscle cell line C2C12. Comparison with published datasets confirmed that muscle differentiation is well recapitulated in C2C12 myotubes. Under dexamethasone treatment, this model was particularly suited to capture early atrophy events. We identified alterations in mitochondrial gene expression and differential alternative splicing events during early-stage myotube atrophy. This dataset complements existing in vivo data and provides novel insights into the regulatory processes during skeletal muscle wasting.

BackgroundThe increase in human lifespan without a proportional increase in healthspan imposes a substantial burden on individuals and society. Exceptionally long-lived individuals and members of long-lived families exhibit compression of multi-morbidity. Genetics, and in particular rare protein-altering variants, appear to play an important role in their longevity. MethodsIn this study, we employed a targeted pathway approach to provide functional evidence of the significance of rare variants in the insulin/insulin-like growth factor 1 signalling - mechanistic target of rapamycin (IIS/mTOR) signalling pathway identified in long-lived individuals. To this end, we used CRISPR/Cas9 to introduce these rare genetic variants into mouse embryonic stem cells (mESCs). We subsequently assessed several functional readouts that have previously been associated with lifespan regulation in model organisms and/or IIS/mTOR and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling pathway activity. ResultsFunctional characterisation revealed that the variants exhibit both shared and distinct effects on the signalling pathways. Principal component analysis of omics-based datasets showed that the variants clustered into two groups, a distribution that corresponds with the grouping observed for a subset of functional readouts. All variant mESC lines exhibited a downregulation in IIS/mTOR and MAPK/ERK signalling pathway activity as well as an increase in Foxo3 expression and FOXO3 binding activity. We identified alterations in lipid and mitochondrial metabolism, including a reduction in mitochondrial DNA levels, which were mostly shared among all variants. All variant mESC lines exhibited a signature implying increased pluripotency. The effects on stress resistance and growth rate diverged between the two variant groups, with partially opposing effects. Group 1 demonstrated a reduced growth rate and increased resistance to a subset of stressors, while Group 2 demonstrated an increased growth rate and reduced resistance to a subset of stressors. ConclusionsHere, we provide evidence that rare genetic variants in the IIS/mTOR and MAPK/ERK signalling pathways identified in long-lived human individuals result in shared functional effects associated with longevity in model organisms. These insights can serve as a foundation to better understand the role of rare variants in the insulin signalling network in the regulation of human longevity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/728260v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1bf5ebdorg.highwire.dtl.DTLVardef@e4e5dcorg.highwire.dtl.DTLVardef@1aee276org.highwire.dtl.DTLVardef@95f170_HPS_FORMAT_FIGEXP M_FIG C_FIG