Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Impaired age-associated mitochondrial translation is mitigated by exercise and PGC-1α.

Sarcopenia, the age-related loss of skeletal muscle mass and function, can dramatically impinge on quality of life and mortality. While mitochondrial dysfunction and imbalanced proteostasis are recognized as hallmarks of sarcopenia, the regulatory and functional link between these processes is underappreciated and unresolved. We therefore investigated how mitochondrial proteostasis, a crucial process that coordinates the expression of nuclear- and mitochondrial-encoded mitochondrial proteins with supercomplex formation and respiratory activity, is affected in skeletal muscle aging. Intriguingly, a robust mitochondrial translation impairment was observed in sarcopenic muscle, which is regulated by the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) with the estrogen-related receptor α (ERRα). Exercise, a potent inducer of PGC-1α activity, rectifies age-related reduction in mitochondrial translation, in conjunction with quality control pathways. These results highlight the importance of mitochondrial proteostasis in muscle aging, and elucidate regulatory interactions that underlie the powerful benefits of physical activity in this context.

Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation.

Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis.

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3' UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.

The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins.

BACKGROUND: The behavior of cells in vivo is complex and highly dynamic, as it results from an interplay between intercellular matrix proteins with surface receptors and other microenvironmental cues. Although the effects of the cellular niche have been investigated for a number of cell types using different molecular approaches, comprehensive assessments of how the global transcriptome responds to 3D scaffolds composed of various extracellular matrix (ECM) constituents at different concentrations are still lacking. RESULTS: In this study, we explored the effects of two diverse extracellular matrix (ECM) components, Collagen I and Matrigel, on the transcriptional profile of cells in a cell culture system. Culturing Huh-7 cells on traditional cell culture plates (Control) or on the ECM components at different concentrations to modulate microenvironment properties, we have generated transcriptomics data that may be further explored to understand the differentiation and growth potential of this cell type for the development of 3D cultures. Our analysis infers transcription factors that are most responsible for the transcriptome response to the extracellular cues. CONCLUSION: Our data indicates that the Collagen I substrate induces a robust transcriptional response in the Huh-7 cells, distinct from that induced by Matrigel. Enhanced hepatocyte markers (ALB and miR-122) reveal a potentially robust remodelling towards primary hepatocytes. Our results aid in defining the appropriate culture and transcription pathways while using hepatoma cell lines. As systems mimicking the in vivo structure and function of liver cells are still being developed, our study could potentially circumvent bottlenecks of limited availability of primary hepatocytes for preclinical studies of drug targets.

Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2.

The overexpression of sialic acids on glycans, called hypersialylation, is a common alteration found in cancer cells. Sialylated glycans can enhance immune evasion by interacting with sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on tumor-infiltrating immune cells. Here, we investigated the effect of sialylated glycans and their interaction with Siglec receptors on myeloid-derived suppressor cells (MDSCs). We found that MDSCs derived from the blood of lung cancer patients and tumor-bearing mice strongly express inhibitory Siglec receptors and are highly sialylated. In murine cancer models of emergency myelopoiesis, Siglec-E knockout in myeloid cells resulted in prolonged survival and increased tumor infiltration of activated T cells. Targeting suppressive myeloid cells by blocking Siglec receptors or desialylation strongly reduced their suppressive potential. We further identified CCL2 as a mediator involved in T-cell suppression upon interaction between sialoglycans and Siglec receptors on MDSCs. Our results demonstrated that sialylated glycans inhibit anticancer immunity by modulating CCL2 expression.

Combined multidimensional single-cell protein and RNA profiling dissects the cellular and functional heterogeneity of thymic epithelial cells.

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T cell development and selection. Recent single-cell RNA sequencing studies have uncovered previously unappreciated transcriptional heterogeneity among thymic epithelial cells (TEC). However, there are only very few cell markers that allow a comparable phenotypic identification of TEC. Here, using massively parallel flow cytometry and machine learning, we deconvoluted known TEC phenotypes into novel subpopulations. Using CITEseq, these phenotypes were related to corresponding TEC subtypes defined by the cells' RNA profiles. This approach allowed the phenotypic identification of perinatal cTEC and their physical localisation within the cortical stromal scaffold. In addition, we demonstrate the dynamic change in the frequency of perinatal cTEC in response to developing thymocytes and reveal their exceptional efficiency in positive selection. Collectively, our study identifies markers that allow for an unprecedented dissection of the thymus stromal complexity, as well as physical isolation of TEC populations and assignment of specific functions to individual TEC subtypes.

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.

Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making "CR mimetics" of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.

Transcription factor motif activity as a biomarker of muscle aging.

In prior work, we analyzed gene expression profiles of mouse, rat and human gastrocnemius muscles to identify conserved regulators of muscle aging processes. By further comparing data obtained from different muscles we found stronger conservation of aging-related factors at the level of molecular pathways than at the level of individual genes. Here we compared the predictive power of models based on gene expression levels and those based on transcription factor motif activities for an individual's age. Although somewhat less accurate than models based on gene expression, models based on motif activities achieve good prediction of muscle age, further providing insights into aging-related molecular pathways.

Molecular and phenotypic analysis of rodent models reveals conserved and species-specific modulators of human sarcopenia.

Sarcopenia, the age-related loss of skeletal muscle mass and function, affects 5-13% of individuals aged over 60 years. While rodents are widely-used model organisms, which aspects of sarcopenia are recapitulated in different animal models is unknown. Here we generated a time series of phenotypic measurements and RNA sequencing data in mouse gastrocnemius muscle and analyzed them alongside analogous data from rats and humans. We found that rodents recapitulate mitochondrial changes observed in human sarcopenia, while inflammatory responses are conserved at pathway but not gene level. Perturbations in the extracellular matrix are shared by rats, while mice recapitulate changes in RNA processing and autophagy. We inferred transcription regulators of early and late transcriptome changes, which could be targeted therapeutically. Our study demonstrates that phenotypic measurements, such as muscle mass, are better indicators of muscle health than chronological age and should be considered when analyzing aging-related molecular data.

The neuromuscular junction is a focal point of mTORC1 signaling in sarcopenia.

With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (https://sarcoatlas.scicore.unibas.ch/), as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging.

How time delay and network design shape response patterns in biochemical negative feedback systems.

BACKGROUND: Negative feedback in combination with time delay can bring about both sustained oscillations and adaptive behaviour in cellular networks. Here, we study which design features of systems with delayed negative feedback shape characteristic response patterns with special emphasis on the role of time delay. To this end, we analyse generic two-dimensional delay differential equations describing the dynamics of biochemical signal-response networks. RESULTS: We investigate the influence of several design features on the stability of the model equilibrium, i.e., presence of auto-inhibition and/or mass conservation and the kind and/or strength of the delayed negative feedback. We show that auto-inhibition and mass conservation have a stabilizing effect, whereas increasing abruptness and decreasing feedback threshold have a de-stabilizing effect on the model equilibrium. Moreover, applying our theoretical analysis to the mammalian p53 system we show that an auto-inhibitory feedback can decouple period and amplitude of an oscillatory response, whereas the delayed feedback can not. CONCLUSIONS: Our theoretical framework provides insight into how time delay and design features of biochemical networks act together to elicit specific characteristic response patterns. Such insight is useful for constructing synthetic networks and controlling their behaviour in response to external stimulation.