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Abstract Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.

Abstract Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

Abstract DNA hydroxymethylation (5hmC), the most abundant oxidative derivative of DNA methylation, is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in age-related diseases, its functional role in aging remains unknown. Here, using mouse liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and restricts the magnitude of gene expression changes with age. Mechanistically, 5hmC decreases the binding of splicing associated factors and correlates with age-related alternative splicing events. We found that various age-related contexts, such as prolonged quiescence and senescence, drive the accumulation of 5hmC with age. We provide evidence that this age-related transcriptionally restrictive function is conserved in mouse and human tissues. Our findings reveal that 5hmC regulates tissue-specific function and may play a role in longevity.

Aims: Cytoplasmic dynein heavy chain (DYNC1H1) is a multi-subunit protein complex that provides motor force for movement of cargo on microtubules and traffics them back to the soma. In humans, mutations along the DYNC1H1 gene result in intellectual disabilities, cognitive delays, and neurologic and motor deficits. The aim of the study was to generate a mouse model to a newly identified de novo heterozygous DYNC1H1 mutation, within a functional ATPase domain (c9052C > T(P3018S)), identified in a child with motor deficits, and intellectual disabilities. Results: P3018S heterozygous (HET) knockin mice are viable; homozygotes are lethal. Metabolic and EchoMRI™ testing show that HET mice have a higher metabolic rate, are more active, and have less body fat compared to wildtype mice. Neurobehavioral studies show that HET mice perform worse when traversing elevated balance beams, and on the negative geotaxis test. Immunofluorescent staining shows neuronal migration abnormalities in the dorsal and lateral neocortex with heterotopia in layer I. Neuron-subtype specific transcription factors CUX1 and CTGF identified neurons from layers II/III and VI respectively in cortical layer I, and abnormal pyramidal neurons with MAP2+ dendrites projecting downward from the pial surface. Conclusion: The HET mice are a good model for the motor deficits seen in the child, and highlights the importance of cytoplasmic dynein in the maintenance of cortical function and dendritic orientation relative to the pial surface. Our results are discussed in the context of other dynein mutant mice and in relation to clinical presentation in humans with DYNC1H1 mutations.

Abstract Aging, chronic high-fat diet feeding, or housing at thermoneutrality induces brown adipose tissue (BAT) involution, a process characterized by reduction of BAT mass and function with increased lipid droplet size. Single nuclei RNA sequencing of aged mice identifies a specific brown adipocyte population of Ucp1-low cells that are pyroptotic and display a reduction in the longevity gene syntaxin 4 (Stx4a). Similar to aged brown adipocytes, Ucp1-STX4KO mice display loss of brown adipose tissue mass and thermogenic dysfunction concomitant with increased pyroptosis. Restoration of STX4 expression or suppression of pyroptosis activation protects against the decline in both mass and thermogenic activity in the aged and Ucp1-STX4KO mice. Mechanistically, STX4 deficiency reduces oxidative phosphorylation, glucose uptake, and glycolysis leading to reduced ATP levels, a known triggering signal for pyroptosis. Together, these data demonstrate an understanding of rapid brown adipocyte involution and that physiologic aging and thermogenic dysfunction result from pyroptotic signaling activation.

Neurexins are highly spliced transmembrane cell adhesion molecules that bind an array of partners via their extracellular domains. However, much less is known about the signaling pathways downstream of neurexin's largely invariant intracellular domain (ICD). Caenorhabditis elegans contains a single neurexin gene that we have previously shown is required for presynaptic assembly and stabilization. To gain insight into the signaling pathways mediating neurexin's presynaptic functions, we employed a proximity ligation method, endogenously tagging neurexin's intracellular domain with the promiscuous biotin ligase TurboID, allowing us to isolate adjacent biotinylated proteins by streptavidin pull-down and mass spectrometry. We compared our experimental strain to a control strain in which neurexin, endogenously tagged with TurboID, was dispersed from presynaptic active zones by the deletion of its C-terminal PDZ-binding motif. Selection of this control strain, which differs from the experimental strain only in its synaptic localization, was critical to identifying interactions specifically occurring at synapses. Using this approach, we identified both known and novel intracellular interactors of neurexin, including active zone scaffolds, actin-binding proteins (including almost every member of the Arp2/3 complex), signaling molecules, and mediators of RNA trafficking, protein synthesis and degradation, among others. Characterization of mutants for candidate neurexin interactors revealed that they recapitulate aspects of the nrx-1(-) mutant phenotype, suggesting they may be involved in neurexin signaling. Finally, to investigate a possible role for neurexin in local actin assembly, we endogenously tagged its intracellular domain with actin depolymerizing and sequestering peptides (DeActs) and found that this led to defects in active zone assembly. Together, these results suggest neurexin's intracellular domain may be involved in presynaptic actin-assembly, and furthermore highlight a novel approach to achieving high specificity for in vivo proteomics experiments.

The role of a scientist is at first not so different from a philosopher. They both need to question common thinking and evaluate whether reality is not as we always thought. Based on this, we need to design hypotheses, experiments, and analyses to prove our alternative vision. Artificial Intelligence (AI) is rapidly moving from an “assistant” into a proper “colleague” for literature mining, data analysis and interpretation, and literally having (almost) real scientific conversations. However, being AI based on existing information, if we rely on it excessively will we still be able to question the status quo? In this article, we are particularly interested in discussing the future of proteomics and mass spectrometry with our new electronic collaborator. We leave to the reader the judgement whether the answers we received are satisfactory or superficial. What we were mostly interested in was laying down what we think are critical questions that the proteomics community should occasionally ask to itself. Proteomics has been around for more than 30 years, but it is still missing a few critical steps to fully address its promises as being the new genomics for clinical diagnostics and fundamental science, while becoming a user-friendly tool for every lab. Will we get there with the help of AI? And will these answers change in a short period, as AI continues to advance?

Summary: Antibody-dependent cellular cytotoxicity (ADCC) is associated with protection against neonatal herpes. We hypothesized that placental transfer of ADCC-mediating herpes simplex virus (HSV) immunoglobulin G (IgG) is influenced by antigenic target, function, glycans, gestational age, and maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Maternal and cord blood were collected from HSV-seropositive (HSV+) mothers pre-COVID and HSV+/SARS-CoV-2+ mothers during the pandemic. Transfer of HSV neutralizing IgG was significantly lower in preterm versus term dyads (transfer ratio [TR] 0.84 vs. 2.44) whereas the TR of ADCC-mediating IgG was

Abstract Background KDM5 family proteins are multi-domain regulators of transcription that when dysregulated contribute to cancer and intellectual disability. KDM5 proteins can regulate transcription through their histone demethylase activity in addition to demethylase-independent gene regulatory functions that remain less characterized. To expand our understanding of the mechanisms that contribute to KDM5-mediated transcription regulation, we used TurboID proximity labeling to identify KDM5-interacting proteins. Results Using Drosophila melanogaster, we enriched for biotinylated proteins from KDM5-TurboID-expressing adult heads using a newly generated control for DNA-adjacent background in the form of dCas9:TurboID. Mass spectrometry analyses of biotinylated proteins identified both known and novel candidate KDM5 interactors, including members of the SWI/SNF and NURF chromatin remodeling complexes, the NSL complex, Mediator, and several insulator proteins. Conclusions Combined, our data shed new light on potential demethylase-independent activities of KDM5. In the context of KDM5 dysregulation, these interactions may play key roles in the alteration of evolutionarily conserved transcriptional programs implicated in human disorders.

Abstract Background Three-dimensional (3D) cell culture has emerged as an alternative approach to 2D flat culture to model more accurately the phenotype of solid tissue in laboratories. Culturing cells in 3D more precisely recapitulates physiological conditions of tissues, as these cells reduce activities related to proliferation, focusing their energy consumption toward metabolism and homeostasis. Results Here, we demonstrate that 3D liver spheroids are a suitable system to model chromatin dynamics and response to epigenetics inhibitors. To delay necrotic tissue formation despite proliferation arrest, we utilize rotating bioreactors that apply active media diffusion and low shearing forces. We demonstrate that the proteome and the metabolome of our model resemble typical liver functions. We prove that spheroids respond to sodium butyrate (NaBut) treatment, an inhibitor of histone deacetylases (HDACi), by upregulating histone acetylation and transcriptional activation. As expected, NaBut treatment impaired specific cellular functions, including the energy metabolism. More importantly, we demonstrate that spheroids reestablish their original proteome and transcriptome, including pre-treatment levels of histone acetylation, metabolism, and protein expression once the standard culture condition is restored after treatment. Given the slow replication rate (> 40 days) of cells in 3D spheroids, our model enables to monitor the recovery of approximately the same cells that underwent treatment, demonstrating that NaBut does not have long-lasting effects on histone acetylation and gene expression. These results suggest that our model system can be used to quantify molecular memory on chromatin. Conclusion Together, we established an innovative cell culture system that can be used to model anomalously decondensing chromatin in physiological cell growth and rule out epigenetics inheritance if cells recover the original phenotype after treatment. The transient epigenetics effects demonstrated here highlight the relevance of using a 3D culture model system that could be very useful in studies requiring long-term drug treatment conditions that would not be possible using a 2D cell monolayer system.

Summary: Tet2 is a member of the Ten-eleven translocation (Tet1/2/3) family of enzymes and is expressed in embryonic stem cells (ESCs). It demethylates DNA (catalytic functions) and partners with chromatin modifiers (noncatalytic functions) to regulate genes. However, the significance of these functions in ESCs is less defined. Using Tet2 catalytic mutant (Tet2m/m) and knockout (Tet2−/−) ESCs, we identified Tet2 target genes regulated by its catalytic dependent versus independent roles. Tet2 was enriched at their active enhancers and promoters to demethylate them. We also identified the histone deacetylase component Sin3a as a Tet2 partner, co-localizing at promoters and active enhancers. Tet2 deficiency diminished Sin3a at these regions. Tet2 and Sin3a co-occupancy overlapped with Tet1. Combined loss of Tet1/2, but not of their catalytic activities, reduced Sin3a at active enhancers. These findings establish Tet2 catalytic and noncatalytic functions as regulators of DNA demethylation and Sin3a recruitment at active enhancers in ESCs.

Herpes simplex virus type 2 (HSV-2) coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2–infected and bystander 2D10 cells. Bulk and single-cell RNA-Seq studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2–infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms, including upregulation of MALAT1 to release epigenetic silencing.

Senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage and secrete factors that promote atherosclerotic plaque vulnerability and disease. Here, we report increased levels and activity of dipeptidyl peptidase 4 (DPP4), a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent VSMCs revealed a unique senescence-associated secretory phenotype (SASP) signature comprising many complement and coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Serum samples from persons with high risk for cardiovascular disease contained high levels of DPP4-regulated complement and coagulation factors. Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque stability, while single-cell resolution of senescent VSMCs reflected the senomorphic and senolytic effects of DPP4 inhibition in murine atherosclerosis. We propose that DPP4-regulated factors could be exploited therapeutically to reduce senescent cell function, reverse senohemostasis, and improve vascular disease.

Abstract The immune system has been described to play a role in the development of Alzheimer’s disease (AD), but the distribution of immunoglobulins and their subclasses in brain tissue has not been explored. In this study, examination of pathologically diagnosed frontal cortex gray matter revealed significantly higher levels of IgM and IgG in late-stage AD (Braak and Braak stages V and VI) compared to age-matched controls. While levels of IgG2 and IgG4 constant region fragments were higher in late-stage AD, concentration of native–state IgG4 with free Fc regions was increased in AD III and VI. RNA analysis did not support parenchymal B-cell production of IgG4 in AD III and V, indicating possible peripheral or meningeal B-cell involvement. Changes in the profile of IgM, IgG and IgG subclasses in AD frontal cortex may provide insight into understanding disease pathogenesis and progression.

During inflammatory, demyelinating diseases such as multiple sclerosis (MS), inflammation and axonal damage are prevalent early in the course. Axonal damage includes swelling, defects in transport, and failure to clear damaged intracellular proteins, all of which affect recovery and compromise neuronal integrity. The clearance of damaged cell components is important to maintain normal turnover and restore homeostasis. In this study, we used mass spectrometry to identify insoluble proteins within high-speed/mercaptoethanol/sarcosyl-insoluble pellets from purified white matter plaques isolated from the brains of individuals with relapsing–remitting MS (RRMS). We determined that the transmembrane protein 106B (TMEM106B), normally lysosome-associated, is insoluble in RRMS plaques relative to normal-appearing white matter from individuals with Alzheimer’s disease and non-neurologic controls. Relative to wild-type mice, hypomorphic mice with a reduction in TMEM106B have increased axonal damage and lipid droplet accumulation in the spinal cord following myelin-oligodendrocyte-glycoprotein-induced experimental autoimmune encephalomyelitis. Additionally, the corpora callosa from cuprizone-challenged hypomorphic mice fail to clear lipid droplets efficiently during remyelination, suggesting that when TMEM106B is compromised, protein and lipid clearance by the lysosome is delayed. As TMEM106B contains putative lipid- and LC3-binding sites, further exploration of these sites is warranted.

ABSTRACT The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In an attempt to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a transmembrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrated that GRA64 appears to interact with components of the host endosomal sorting complexes required for transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence or encystation in mice, as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of Δgra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection. IMPORTANCE Toxoplasma gondii is a widespread foodborne parasite that causes congenital disease and life-threatening complications in immunocompromised individuals. Part of this parasite’s success lies in its ability to infect diverse organisms and host cells and to persist as a latent infection within parasite-constructed structures called tissue cysts. In this study, we characterized a protein that is secreted by T. gondii into its parasitophorous vacuole during intracellular infection, which we dub GRA64. On the vacuolar membrane, this protein is exposed to the host cell cytosol and interacts with specific host ESCRT proteins. Parasites lacking the GRA64 protein exhibit ultrastructural changes in tissue cysts during chronic infection. This study lays the foundation for future studies on the mechanics and consequences of host ESCRT-parasite protein interactions.

ATRX is an RNA binding protein that mediates targeting of polycomb repressive complex 2 (PRC2) to genomic sites. Here the authors identify the RNA binding region and show that the RNA binding is required for ATRX localization and for its recruitment of PRC2 to a subset of polycomb targets.

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Taken together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis.

Summary: Protein arginine methyltransferases (PRMTs) catalyze the post-translational monomethylation (Rme1), asymmetric (Rme2a), or symmetric (Rme2s) dimethylation of arginine. To determine the cellular consequences of type I (Rme2a) and II (Rme2s) PRMTs, we developed and integrated multiple approaches. First, we determined total cellular dimethylarginine levels, revealing that Rme2s was ∼3% of total Rme2 and that this percentage was dependent upon cell type and PRMT inhibition status. Second, we quantitatively characterized in vitro substrates of the major enzymes and expanded upon PRMT substrate recognition motifs. We also compiled our data with publicly available methylarginine-modified residues into a comprehensive database. Third, we inhibited type I and II PRMTs and performed proteomic and transcriptomic analyses to reveal their phenotypic consequences. These experiments revealed both overlapping and independent PRMT substrates and cellular functions. Overall, this study expands upon PRMT substrate diversity, the arginine methylome, and the complex interplay of type I and II PRMTs.

OBJECTIVES/GOALS: Murine and clinical data suggest that antibody-dependent cellular cytotoxicity (ADCC) is associated with greater protection against disseminated neonatal HSV disease. To quantify the relative transfer of Abs with different functions and targets, we conducted a prospective study of mother-infant term and preterm dyads pre and during COVID-19 METHODS/STUDY POPULATION: Total and HSV lysate, glycoprotein D (gD) and glycoprotein B (gB)-specific IgG, IgG1 and IgG3 as well as HSV neutralizing Abs (nAbs) and ADCC were quantified in paired 3rd-trimester pregnant women and their newborns (cord) blood. Transfer ratios (TR) were defined as cord:maternal Ab levels. IgG1 and IgG3 subclass and gD or gB-specific Abs were isolated by column purification and glycan profiles were assessed by mass spectrometry. The study population included 21 term and 15 preterm dyads who were HSV-1 (+/- HSV-2) seropositive enrolled between 2018-2019 (pre-COVID) and 25 additional HSV-1 (+/- HSV-2) seropositive term dyads whose mothers were SARS-CoV-2 PCR and COVID Ab+ at delivery; 14 were asymptomatic and 11 had mild-moderate COVID disease. None of the mothers had active genital HSV lesions during delivery RESULTS/ANTICIPATED RESULTS: Anti-HSV IgG, IgG1 and IgG3 TR were higher in term vs. preterm dyads (p

How organisms control when to transition between different stages of development is a key question in biology. In plants, epigenetic silencing by Polycomb repressive complex 1 (PRC1) and PRC2 plays a crucial role in promoting developmental transitions, including from juvenile-to-adult phases of vegetative growth. PRC1/2 are known to repress the master regulator of vegetative phase change, miR156, leading to the transition to adult growth, but how this process is regulated temporally is unknown. Here we investigate whether transcription factors in the VIVIPAROUS/ABI3-LIKE (VAL) gene family provide the temporal signal for the epigenetic repression of miR156. Exploiting a novel val1 allele, we found that VAL1 and VAL2 redundantly regulate vegetative phase change by controlling the overall level, rather than temporal dynamics, of miR156 expression. Furthermore, we discovered that VAL1 and VAL2 also act independently of miR156 to control this important developmental transition. In combination, our results highlight the complexity of temporal regulation in plants.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification, yet this epigenetic mark remains poorly understood. Here, the authors identify the native MORF complex as a histone H3K23-specific acetyltransferase and show that interaction of the MORF subunit with acylated H3K14 promotes acetylation of H3K23 by this complex to activate transcription.

Abstract Background Epithelial to mesenchymal transition (EMT) plays a crucial role in cancer propagation. It can be orchestrated by the activation of multiple signaling pathways, which have been found to be highly coordinated with many epigenetic regulators. Although the mechanism of EMT has been studied over decades, cross talk between signaling and epigenetic regulation is not fully understood. Results Here, we present a time-resolved multi-omics strategy, which featured the identification of the correlation between protein changes (proteome), signaling pathways (phosphoproteome) and chromatin modulation (histone modifications) dynamics during TGF-β-induced EMT. Our data revealed that Erk signaling was activated in 5-min stimulation and structural proteins involved in cytoskeleton rearrangement were regulated after 1-day treatment, constituting a detailed map of systematic changes. The comprehensive profiling of histone post-translational modifications identified H3K27me3 as the most significantly up-regulated mark. We thus speculated and confirmed that a combined inhibition of Erk signaling and Ezh2 (H3K27me3 methyltransferase) was more effective in blocking EMT progress than individual inhibitions. Conclusions In summary, our data provided a more detailed map of cross talk between signaling pathway and chromatin regulation comparing to previous EMT studies. Our findings point to a promising therapeutic strategy for EMT-related diseases by combining Erk inhibitor (singling pathway) and Ezh2 inhibitor (epigenetic regulation).

Objective: Increasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. Methods: In this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. Results: Our proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. Conclusion: Our study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage.

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.

DNA modifications are small covalent chemical groups that modify nucleotides to regulate DNA readout. Anomalous abundance and genome-wide localization of these modifications can negatively tune gene expression and propagate into unbalanced epigenetics regulation, which is known to be associated with multiple conditions such as cancer, diabetes and aging. We present a direct injection mass spectrometry (DI-MS) platform that offers fast, accurate and precise quantitation of global levels of DNA cytidine methylation (mC) and hydroxymethylation (hmC) in less than one minute per sample. On the contrary to most methods adopting mass spectrometry for the analysis of nucleotide modifications, in this DI-MS approach we eliminate the use of liquid chromatography, increasing throughput, eliminating issues of carryover and batch effects caused by column contamination across samples. In addition, potential biases in detection efficiency of modified nucleotides with different binding efficiency to stationary phases is eliminated, as no chromatographic separation is adopted. This method can analyze >1000 samples per day, overcoming the throughput of next-generation sequencing. • Direct injection mass spectrometry improves throughput and precision compared to liquid chromatography. • Direct injection can be used to quantify in less than one minute global levels of DNA methylation and hydroxymethylation. • The unbiased acquisition can be potentially utilized to analyze other nucleotide modifications.

The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.

HIV-neurocognitive impairment (HIV-NCI) can be a debilitating condition for people with HIV (PWH), despite the success of antiretroviral therapy (ART). Substance use disorder is often a comorbidity with HIV infection. The use of methamphetamine (meth) increases systemic inflammation and CNS damage in PWH. Meth may also increase neuropathogenesis through the functional dysregulation of cells that harbor HIV. Perivascular macrophages are long-lived reservoirs for HIV in the CNS. The impaired clearance of extracellular debris and increased release of reactive oxygen species (ROS) by HIV-infected macrophages cause neurotoxicity. Macroautophagy is a vital intracellular pathway that can regulate, in part, these deleterious processes. We found in HIV-infected primary human macrophages that meth inhibits phagocytosis of aggregated amyloid-β, increases total ROS, and dysregulates autophagic processes. Treatment with widely prescribed ART drugs had minimal effects, although there may be an improvement in phagocytosis when co-administered with meth. Pharmacologically inhibited lysosomal degradation, but not induction of autophagy, further increased ROS in response to meth. Using mass spectrometry, we identified the differentially expressed proteins in meth-treated, HIV-infected macrophages that participate in phagocytosis, mitochondrial function, redox metabolism, and autophagy. Significantly altered proteins may be novel targets for interventional strategies that restore functional homeostasis in HIV-infected macrophages to improve neurocognition in people with HIV-NCI using meth.

Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.

Nuclear RNAi provides a highly tractable system to study RNA-mediated chromatin changes and epigenetic inheritance. Recent studies have indicated that the regulation and function of nuclear RNAi-mediated heterochromatin are highly complex. Our knowledge of histone modifications and the corresponding histonemodifying enzymes involved in the system remains limited. In this study, we show that the heterochromatin mark, H3K23me3, is induced by nuclear RNAi at both exogenous and endogenous targets in C. elegans. In addition, dsRNA-induced H3K23me3 can persist for multiple generations after the dsRNA exposure has stopped. We demonstrate that the histone methyltransferase SET-32, methylates H3K23 in vitro. Both set-32 and the germline nuclear RNAi Argonaute, hrde-1, are required for nuclear RNAi-induced H3K23me3 in vivo. Our data poise H3K23me3 as an additional chromatin modification in the nuclear RNAi pathway and provides the field with a new target for uncovering the role of heterochromatin in transgenerational epigenetic silencing.

A number of histone lysine modifications related to acetylation have been identified, but their functional significance is unclear. Here, the authors use in vitro and in vivo assays to characterize eight acyl histone post-translational modifications and link their abundance with metabolism.

HDAC3 is a critical mediator of hepatic lipid metabolism and its loss leads to fatty liver. Here, the authors characterize the liver HDAC3 interactome in vivo, provide evidence that HDAC3 interacts with PROX1, and show that HDAC3 and PROX1 control expression of genes regulating lipid homeostasis.

Abstract Background Middle-down mass spectrometry (MS), i.e., analysis of long (~50–60 aa) polypeptides, has become the method with the highest throughput and accuracy for the characterization of combinatorial histone posttranslational modifications (PTMs). The discovery of histone readers with multiple domains, and overall the cross talk of PTMs that decorate histone proteins, has revealed that histone marks have synergistic roles in modulating enzyme recruitment and subsequent chromatin activities. Here, we demonstrate that the middle-down MS strategy can be combined with metabolic labeling for enhanced quantification of histone proteins and their combinatorial PTMs in a dynamic manner. Methods We used a nanoHPLC-MS/MS system consisting of hybrid weak cation exchange–hydrophilic interaction chromatography combined with high resolution MS and MS/MS with ETD fragmentation. After spectra identification, we filtered confident hits and quantified polypeptides using our in-house software isoScale. Results We first verified that middle-down MS can discriminate and differentially quantify unlabeled from heavy labeled histone N-terminal tails (heavy lysine and arginine residues). Results revealed no bias toward identifying and quantifying unlabeled versus heavy labeled tails, even if the heavy labeled peptides presented the typical skewed isotopic pattern typical of long protein sequences that hardly get 100% labeling. Next, we plated epithelial cells into a media with heavy methionine-(methyl-13CD3), the precursor of the methyl donor S-adenosylmethionine and stimulated epithelial to mesenchymal transition (EMT). We assessed that results were reproducible across biological replicates and with data obtained using the more widely adopted bottom-up MS strategy, i.e., analysis of short tryptic peptides. We found remarkable differences in the incorporation rate of methylations in non-confluent cells versus confluent cells. Moreover, we showed that H3K27me3 was a critical player during the EMT process, as a consistent portion of histones modified as H3K27me2K36me2 in epithelial cells were converted into H3K27me3K36me2 in mesenchymal cells. Conclusions We demonstrate that middle-down MS, despite being a more scarcely exploited MS technique than bottom-up, is a robust quantitative method for histone PTM characterization. In particular, middle-down MS combined with metabolic labeling is currently the only methodology available for investigating turnover of combinatorial histone PTMs in dynamic systems.

Abstract Gene expression in Plasmodia integrates post-transcriptional regulation with epigenetic marking of active genomic regions through histone post-translational modifications (PTMs). To generate insights into the importance of histone PTMs to the entire asexual and sexual developmental cycles of the parasite, we used complementary and comparative quantitative chromatin proteomics to identify and functionally characterise histone PTMs in 8 distinct life cycle stages of P. falciparum parasites. ~500 individual histone PTMs were identified of which 106 could be stringently validated. 46 individual histone PTMs and 30 co-existing PTMs were fully quantified with high confidence. Importantly, 15 of these histone PTMs are novel for Plasmodia (e.g. H3K122ac, H3K27me3, H3K56me3). The comparative nature of the data revealed a highly dynamic histone PTM landscape during life cycle development, with a set of histone PTMs (H3K4ac, H3K9me1 and H3K36me2) displaying a unique and conserved abundance profile exclusively during gametocytogenesis (P

Summary: Global changes in chromatin organization and the cessation of transcription during mitosis are thought to challenge the resumption of appropriate transcription patterns after mitosis. The acetyl-lysine binding protein BRD4 has been previously suggested to function as a transcriptional “bookmark” on mitotic chromatin. Here, genome-wide location analysis of BRD4 in erythroid cells, combined with data normalization and peak characterization approaches, reveals that BRD4 widely occupies mitotic chromatin. However, removal of BRD4 from mitotic chromatin does not impair post-mitotic activation of transcription. Additionally, histone mass spectrometry reveals global preservation of most posttranslational modifications (PTMs) during mitosis. In particular, H3K14ac, H3K27ac, H3K122ac, and H4K16ac widely mark mitotic chromatin, especially at lineage-specific genes, and predict BRD4 mitotic binding genome wide. Therefore, BRD4 is likely not a mitotic bookmark but only a “passenger.” Instead, mitotic histone acetylation patterns may constitute the actual bookmarks that restore lineage-specific transcription patterns after mitosis. : Chromatin reader protein BRD4 is thought to bookmark mitotic chromatin to propagate transcriptional states across mitosis. Behera et al. profiled and perturbed mitotic BRD4 chromatin occupancy to show that BRD4 is dispensable for this process. Instead, BRD4 mitotic chromatin association is likely a mere reflection of mitotically stable histone marks. Keywords: erythroid cells, mitotic bookmarking, mitosis, cell identity, BRD4, histone marks

Summary: The polycomb repressive complex 2 (PRC2) methylates lysine 27 of histone H3 (H3K27) through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3) have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs) with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state. : Juan et al. use genome editing to subtly modify H3K27 methylation states (H3K27me2/H3K27me3) and report that such perturbation influences the ability of ESCs to differentiate into preferential cell lineages and acquire a “ground state.” Keywords: polycomb proteins, H3K27 methylation, embryonic stem cells

Chromatin accessibility is a major regulator of gene expression. Histone writers/erasers have a critical role in chromatin compaction, as they “flag” chromatin regions by catalyzing/removing covalent post-translational modifications on histone proteins. Anomalous chromatin decondensation is a common phenomenon in cells experiencing aging and viral infection. Moreover, about 50% of cancers have mutations in enzymes regulating chromatin state. Numerous genomics methods have evolved to characterize chromatin state, but the analysis of (in)accessible chromatin from the protein perspective is not yet in the spotlight. We present an overview of the most used approaches to generate data on chromatin accessibility and then focus on emerging methods that utilize mass spectrometry to quantify the accessibility of histones and the rest of the chromatin bound proteome. Mass spectrometry is currently the method of choice to quantify entire proteomes in an unbiased large-scale manner; accessibility on chromatin of proteins and protein modifications adds an extra quantitative layer to proteomics dataset that assist more informed data-driven hypotheses in chromatin biology. We speculate that this emerging new set of methods will enhance predictive strength on which proteins and histone modifications are critical in gene regulation, and which proteins occupy different chromatin states in health and disease.

Intestinal ischemia and reperfusion injury is a model system of possible consequences of severe trauma and surgery, which might result into tissue dysfunction and organ failure. Neutrophils contribute to the injuries preceded by ischemia and reperfusion. However, the mechanisms by which intestinal ischemia and reperfusion stimulate and activate circulating neutrophils is still not clear. In this work, we used proteomics approach to explore the underlying regulated mechanisms in Wistar rat neutrophils after ischemia and reperfusion. We isolated neutrophils from three different biological groups; control, sham laparotomy, and intestinal ischemia/reperfusion. In the workflow, we included iTRAQ-labeling quantification and peptide fractionation using HILIC prior to LC-MS/MS analysis. From proteomic analysis, we identified 2,045 proteins in total that were grouped into five different clusters based on their regulation trend between the experimental groups. A total of 417 proteins were found as significantly regulated in at least one of the analyzed conditions. Interestingly, the enzyme prediction analysis revealed that ischemia/reperfusion significantly reduced the relative abundance of most of the antioxidant and pro-survival molecules to cause more tissue damage and ROS production whereas some of the significantly up regulated enzymes were involved in cytoskeletal rearrangement, adhesion and migration. Clusters based KEGG pathways analysis revealed high motility, phagocytosis, directional migration, and activation of the cytoskeletal machinery in neutrophils after ischemia and reperfusion. Increased ROS production and decreased phagocytosis were experimentally validated by microscopy assays. Taken together, our findings provide a characterization of the rat neutrophil response to intestinal ischemia and reperfusion and the possible mechanisms involved in the tissue injury by neutrophils after intestinal ischemia and reperfusion.

Calmodulin (CaM) is a Ca2+-binding signaling protein that binds to and activates many target proteins, known as calmodulin-binding proteins (CaM-BPs). They are involved in multiple cellular processes. Despite the diversity and importance of CaM-BPs, many remain to be identified and characterized. We performed extensive optimization of a CaM-affinity capture method, using commercial CaM-chromatographic material. We identify both the Ca2+-dependent and -independent CaM binding proteomes in both mouse brain and in rat brain neuronal organelles, synaptosomes, and compared cytosolic with membrane associated targets. Fractionation of peptides, derived from on-resin tryptic digestion, using hydrophilic interaction liquid chromatography (HILIC) was combined with reversed-phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to improve identification of low abundance CaM-BPs in a reproducible and sensitive manner. Various detergents were tested for the most efficient membrane protein solubilization for pull-down of membrane-associated CaM-BPs. We identified 3529 putative mouse brain CaM-BPs, of which 1629 were integral membrane or membrane-associated. Among them, 170 proteins were known CaM-BPs or previously reported as potential CaM-BPs while 696 contained predicted CaM binding motifs. In synaptosomes we identified 2698 CaM-BPs and 2783 unique phosphopeptides derived from 984 of the potential synaptosomal CaM-BPs. Overall, our improved workflow provides unmatched sensitivity for the identification of the CaM binding proteome and its associated phosphoproteome and this now enables sensitive analysis of organelle-specific CaM-BPs.

It is becoming increasingly clear that transcription factors operate in complex networks through thousands of genomic binding sites, many of which bind several transcription factors. However, the extent and mechanisms of crosstalk between transcription factors at these hotspots remain unclear. Using a combination of advanced proteomics and genomics approaches, we identify ∼12,000 transcription factor hotspots (∼400 bp) in the early phase of adipogenesis, and we find evidence of both simultaneous and sequential binding of transcription factors at these regions. We demonstrate that hotspots are highly enriched in large super-enhancer regions (several kilobases), which drive the early adipogenic reprogramming of gene expression. Our results indicate that cooperativity between transcription factors at the level of hotspots as well as super-enhancers is very important for enhancer activity and transcriptional reprogramming. Thus, hotspots and super-enhancers constitute important regulatory hubs that serve to integrate external stimuli on chromatin.

Epigenetic modifications influence gene expression and provide a unique mechanism for fine-tuning cellular differentiation and development in multicellular organisms. Here we report on the biological functions of UTX-1, the Caenorhabditis elegans homologue of mammalian UTX, a histone demethylase specific for H3K27me2/3. We demonstrate that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Consistent with its homology to UTX, UTX-1 regulates global levels of H3K27me2/3 in C. elegans. Surprisingly, we found that the catalytic activity is not required for the developmental function of this protein. Biochemical analysis identified UTX-1 as a component of a complex that includes SET-16(MLL), and genetic analysis indicates that the defects associated with loss of UTX-1 are likely mediated by compromised SET-16/UTX-1 complex activity. Taken together, these results demonstrate that UTX-1 is required for many aspects of nematode development; but, unexpectedly, this function is independent of its enzymatic activity.