PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

Loss of EIF4G2 mediates aggressiveness in distinct human endometrial cancer subpopulations with poor survival outcome in patients.

The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we stably knocked-down EIF4G2 in two human EC cell lines in parallel. EIF4G2 depletion resulted in increased resistance to conventional therapies and increased the prevalence of molecular markers for aggressive cell subsets, altering their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins, KIF5B and KLC1, 2, 3. Multiplexed imaging of the EC patient tumor cohort showed a correlation between decreased expression of the kinesin proteins, and poor survival in patients with tumors of certain grades and stages. These findings reveal potential novel biomarkers for Grade 2 EC with ramifications for patient stratification and therapeutic interventions.

Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae.

The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. Our findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating our findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.

Metabolic inputs in the probiotic bacterium Lacticaseibacillus rhamnosus contribute to cell-wall remodeling and increased fitness.

Lacticaseibacillus rhamnosus GG (LGG) is a Gram-positive beneficial bacterium that resides in the human intestinal tract and belongs to the family of lactic acid bacteria (LAB). This bacterium is a widely used probiotic and was suggested to provide numerous benefits for human health. However, as in most LAB strains, the molecular mechanisms that mediate the competitiveness of probiotics under different diets remain unknown. Fermentation is a fundamental process in LAB, allowing the oxidation of simple carbohydrates (e.g., glucose, mannose) for energy production under oxygen limitation, as in the human gut. Our results indicate that fermentation reshapes the metabolome, volatilome, and proteome architecture of LGG. Furthermore, fermentation alters cell envelope remodeling and peptidoglycan biosynthesis, which leads to altered cell wall thickness, aggregation properties, and cell wall composition. In addition, fermentable sugars induced the secretion of known and novel metabolites and proteins targeting the enteric pathogens Enterococcus faecalis and Salmonella enterica Serovar Typhimurium. Overall, our results link simple carbohydrates with cell wall remodeling, aggregation to host tissues, and biofilm formation in probiotic strains and connect them with the production of broad-spectrum antimicrobial effectors.

PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways.

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.

Global analysis of contact-dependent human-to-mouse intercellular mRNA and lncRNA transfer in cell culture.

Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically,<1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

The LINC Complex Inhibits Excessive Chromatin Repression.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. We show here that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, we suggest that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.

rG4detector, a novel RNA G-quadruplex predictor, uncovers their impact on stress granule formation.

RNA G-quadruplexes (rG4s) are RNA secondary structures, which are formed by guanine-rich sequences and have important cellular functions. Existing computational tools for rG4 prediction rely on specific sequence features and/or were trained on small datasets, without considering rG4 stability information, and are therefore sub-optimal. Here, we developed rG4detector, a convolutional neural network to identify potential rG4s in transcriptomics data. rG4detector outperforms existing methods in both predicting rG4 stability and in detecting rG4-forming sequences. To demonstrate the biological-relevance of rG4detector, we employed it to study RNAs that are bound by the RNA-binding protein G3BP1. G3BP1 is central to the induction of stress granules (SGs), which are cytoplasmic biomolecular condensates that form in response to a variety of cellular stresses. Unexpectedly, rG4detector revealed a dynamic enrichment of rG4s bound by G3BP1 in response to cellular stress. In addition, we experimentally characterized G3BP1 cross-talk with rG4s, demonstrating that G3BP1 is a bona fide rG4-binding protein and that endogenous rG4s are enriched within SGs. Furthermore, we found that reduced rG4 availability impairs SG formation. Hence, we conclude that rG4s play a direct role in SG biology via their interactions with RNA-binding proteins and that rG4detector is a novel useful tool for rG4 transcriptomics data analyses.

DAP5 drives translation of specific mRNA targets with upstream ORFs in human embryonic stem cells.

Death associated protein 5 (DAP5/eIF4G2/NAT1) is a member of the eIF4G translation initiation factors that has been shown to mediate noncanonical and/or cap-independent translation. It is essential for embryonic development and for differentiation of embryonic stem cells (ESCs), specifically its ability to drive translation of specific target mRNAs. In order to expand the repertoire of DAP5 target mRNAs, we compared ribosome profiles in control and DAP5 knockdown (KD) human ESCs (hESCs) to identify mRNAs with decreased ribosomal occupancy upon DAP5 silencing. A cohort of 68 genes showed decreased translation efficiency in DAP5 KD cells. Mass spectrometry confirmed decreased protein abundance of a significant portion of these targets. Among these was KMT2D, a histone methylase previously shown to be essential for ESC differentiation and embryonic development. We found that nearly half of the cohort of DAP5 target mRNAs displaying reduced translation efficiency of their main coding sequences upon DAP5 KD contained upstream open reading frames (uORFs) that are actively translated independently of DAP5. This is consistent with previously suggested mechanisms by which DAP5 mediates leaky scanning through uORFs and/or reinitiation at the main coding sequence. Crosslinking protein-RNA immunoprecipitation experiments indicated that a significant subset of DAP5 mRNA targets bound DAP5, indicating that direct binding between DAP5 protein and its target mRNAs is a frequent but not absolute requirement for DAP5-dependent translation of the main coding sequence. Thus, we have extended DAP5's function in translation of specific mRNAs in hESCs by a mechanism allowing translation of the main coding sequence following upstream translation of short ORFs.

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) safeguards the developing mouse cortex.

HNRNPU encodes the heterogeneous nuclear ribonucleoprotein U, which participates in RNA splicing and chromatin organization. Microdeletions in the 1q44 locus encompassing HNRNPU and other genes and point mutations in HNRNPU cause brain disorders, including early-onset seizures and severe intellectual disability. We aimed to understand HNRNPU's roles in the developing brain. Our work revealed that HNRNPU loss of function leads to rapid cell death of both postmitotic neurons and neural progenitors, with an apparent higher sensitivity of the latter. Further, expression and alternative splicing of multiple genes involved in cell survival, cell motility, and synapse formation are affected following Hnrnpu's conditional truncation. Finally, we identified pharmaceutical and genetic agents that can partially reverse the loss of cortical structures in Hnrnpu mutated embryonic brains, ameliorate radial neuronal migration defects and rescue cultured neural progenitors' cell death.

The roles of intracellular and extracellular calcium in Bacillus subtilis biofilms.

In nature, bacteria reside in biofilms- multicellular differentiated communities held together by an extracellular matrix. This work identified a novel subpopulation-mineral-forming cells-that is essential for biofilm formation in Bacillus subtilis biofilms. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. Consistently, biofilm development is prevented by the inhibition of calcium uptake. Our results provide a clear demonstration of the orchestrated production of calcite exoskeleton, critical to morphogenesis in simple prokaryotes.

Whole-genome sequencing reveals that variants in the Interleukin 18 Receptor Accessory Protein 3'UTR protect against ALS.

The noncoding genome is substantially larger than the protein-coding genome but has been largely unexplored by genetic association studies. Here, we performed region-based rare variant association analysis of >25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole genomes and the whole genomes of 70,403 non-ALS controls. We identified interleukin-18 receptor accessory protein (IL18RAP) 3' untranslated region (3'UTR) variants as significantly enriched in non-ALS genomes and associated with a fivefold reduced risk of developing ALS, and this was replicated in an independent cohort. These variants in the IL18RAP 3'UTR reduce mRNA stability and the binding of double-stranded RNA (dsRNA)-binding proteins. Finally, the variants of the IL18RAP 3'UTR confer a survival advantage for motor neurons because they dampen neurotoxicity of human induced pluripotent stem cell (iPSC)-derived microglia bearing an ALS-associated expansion in C9orf72, and this depends on NF-κB signaling. This study reveals genetic variants that protect against ALS by reducing neuroinflammation and emphasizes the importance of noncoding genetic association studies.

A new function for the serine protease HtrA2 in controlling radiation-induced senescence in cancer cells.

Radiation therapy can induce cellular senescence in cancer cells, leading to short-term tumor growth arrest but increased long-term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation-induced senescence in cultured cancer cells. The irradiated cells exhibited a typical senescent phenotype, including upregulation of p53 and its main target, p21, followed by a sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence-associated beta-galactosidase activity. Mass spectrometry-based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy. A functional siRNA screen using a cell death-related library identified mitochondrial serine protease HtrA2 as being necessary for sustained growth arrest of the senescent cells. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus, HtrA2 is involved in the cytoskeletal reorganization that accompanies radiation-induced senescence and the continuous maintenance of proliferation arrest.

Constitutive activation of canonical Wnt signaling disrupts choroid plexus epithelial fate.

The choroid plexus secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the choroid plexus epithelium arises from the Wnt- expressing cortical hem. Canonical Wnt signaling pathway molecules such as nuclear ß-CATENIN are expressed in the mouse and human embryonic choroid plexus epithelium indicating that this pathway is active. Point mutations in human ß-CATENIN are known to result in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of choroid plexus epithelial identity and an apparent transformation of this tissue to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell derived organoids. The choroid plexus is also disrupted when ß-Catenin is conditionally inactivated. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal choroid plexus development in the mammalian brain.

Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors.

The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

The extracellular matrix protein TasA is a developmental cue that maintains a motile subpopulation within Bacillus subtilis biofilms.

In nature, bacteria form biofilms-differentiated multicellular communities attached to surfaces. Within these generally sessile biofilms, a subset of cells continues to express motility genes. We found that this subpopulation enabled Bacillus subtilis biofilms to expand on high-friction surfaces. The extracellular matrix (ECM) protein TasA was required for the expression of flagellar genes. In addition to its structural role as an adhesive fiber for cell attachment, TasA acted as a developmental signal stimulating a subset of biofilm cells to revert to a motile phenotype. Transcriptomic analysis revealed that TasA stimulated the expression of a specific subset of genes whose products promote motility and repress ECM production. Spontaneous suppressor mutations that restored motility in the absence of TasA revealed that activation of the biofilm-motility switch by the two-component system CssR/CssS antagonized the TasA-mediated reversion to motility in biofilm cells. Our results suggest that although mostly sessile, biofilms retain a degree of motility by actively maintaining a motile subpopulation.

A unified nomenclature for vertebrate olfactory receptors.

BACKGROUND: Olfactory receptors (ORs) are G protein-coupled receptors with a crucial role in odor detection. A typical mammalian genome harbors ~ 1000 OR genes and pseudogenes; however, different gene duplication/deletion events have occurred in each species, resulting in complex orthology relationships. While the human OR nomenclature is widely accepted and based on phylogenetic classification into 18 families and further into subfamilies, for other mammals different and multiple nomenclature systems are currently in use, thus concealing important evolutionary and functional insights. RESULTS: Here, we describe the Mutual Maximum Similarity (MMS) algorithm, a systematic classifier for assigning a human-centric nomenclature to any OR gene based on inter-species hierarchical pairwise similarities. MMS was applied to the OR repertoires of seven mammals and zebrafish. Altogether, we assigned symbols to 10,249 ORs. This nomenclature is supported by both phylogenetic and synteny analyses. The availability of a unified nomenclature provides a framework for diverse studies, where textual symbol comparison allows immediate identification of potential ortholog groups as well as species-specific expansions/deletions; for example, Or52e5 and Or52e5b represent a rat-specific duplication of OR52E5. Another example is the complete absence of OR subfamily OR6Z among primate OR symbols. In other mammals, OR6Z members are located in one genomic cluster, suggesting a large deletion in the great ape lineage. An additional 14 mammalian OR subfamilies are missing from the primate genomes. While in chimpanzee 87% of the symbols were identical to human symbols, this number decreased to ~ 50% in dog and cow and to ~ 30% in rodents, reflecting the adaptive changes of the OR gene superfamily across diverse ecological niches. Application of the proposed nomenclature to zebrafish revealed similarity to mammalian ORs that could not be detected from the current zebrafish olfactory receptor gene nomenclature. CONCLUSIONS: We have consolidated a unified standard nomenclature system for the vertebrate OR superfamily. The new nomenclature system will be applied to cow, horse, dog and chimpanzee by the Vertebrate Gene Nomenclature Committee and its implementation is currently under consideration by other relevant species-specific nomenclature committees.

Human genetics and neuropathology suggest a link between miR-218 and amyotrophic lateral sclerosis pathophysiology.

Motor neuron-specific microRNA-218 (miR-218) has recently received attention because of its roles in mouse development. However, miR-218 relevance to human motor neuron disease was not yet explored. Here, we demonstrate by neuropathology that miR-218 is abundant in healthy human motor neurons. However, in amyotrophic lateral sclerosis (ALS) motor neurons, miR-218 is down-regulated and its mRNA targets are reciprocally up-regulated (derepressed). We further identify the potassium channel Kv10.1 as a new miR-218 direct target that controls neuronal activity. In addition, we screened thousands of ALS genomes and identified six rare variants in the human miR-218-2 sequence. miR-218 gene variants fail to regulate neuron activity, suggesting the importance of this small endogenous RNA for neuronal robustness. The underlying mechanisms involve inhibition of miR-218 biogenesis and reduced processing by DICER. Therefore, miR-218 activity in motor neurons may be susceptible to failure in human ALS, suggesting that miR-218 may be a potential therapeutic target in motor neuron disease.