Epigenetic aging of human blood cells is influenced by the age of the host body.

Allogenic hematopoietic stem cell transplantation is a therapeutic procedure performed over a wide range of donor and recipient age combinations, representing natural experiments of how the age of the recipient affects aging in transplanted donor cells in vivo. We measured DNA methylation and epigenetic aging in donors and recipients and found that biological epigenetic clocks are accelerated in cells transplanted into an older body and decelerated in a younger body. This is the first evidence that the age of the circulating environment influences human epigenetic aging in vivo.

Mitotic chromosome condensation resets chromatin to safeguard transcriptional homeostasis during interphase.

Mitotic entry correlates with the condensation of the chromosomes, changes in histone modifications, exclusion of transcription factors from DNA, and the broad downregulation of transcription. However, whether mitotic condensation influences transcription in the subsequent interphase is unknown. Here, we show that preventing one chromosome to condense during mitosis causes it to fail resetting of transcription. Rather, in the following interphase, the affected chromosome contains unusually high levels of the transcription machinery, resulting in abnormally high expression levels of genes in cis, including various transcription factors. This subsequently causes the activation of inducible transcriptional programs in trans, such as the GAL genes, even in the absence of the relevant stimuli. Thus, mitotic chromosome condensation exerts stringent control on interphase gene expression to ensure the maintenance of basic cellular functions and cell identity across cell divisions. Together, our study identifies the maintenance of transcriptional homeostasis during interphase as an unexpected function of mitosis and mitotic chromosome condensation.

Post-acute symptoms 3-15 months after COVID-19 among unvaccinated and vaccinated individuals with a breakthrough infection.

OBJECTIVE: We aimed to describe post-acute sequelae of SARS-CoV-2 infection (PASC) related symptoms 3-15 months after a positive test in SARS-CoV-2 unvaccinated and vaccinated participants with a breakthrough infection. METHODS: Participants of the Norwegian COVID-19 cohort, without a positive SARS-CoV-2 test, completed a questionnaire about PASC-related symptoms between November 2020 and January 2021. About a year later, a second questionnaire (which also included the Everyday Memory Questionnaire [EMQ]-13) was completed by the same participants, most still without a positive SARS-CoV-2 test, but also by unvaccinated and vaccinated participants with a positive test 3-15 months before the questionnaire. Laboratory-confirmed SARS-CoV-2 status (positive or negative swab test determined by reverse transcriptase quantitative polymerase chain reaction) at the time of completing the questionnaire was ascertained from the Mandatory Norwegian Surveillance System for Communicable Diseases. RESULTS: No differences were found in the self-reported PASC symptoms, dyspnea, fatigue, smell/taste changes, concentration problems, or the EMQ-13 score between unvaccinated and vaccinated participants 3-15 months after the positive test. Fewer memory problems were reported among vaccinated than unvaccinated participants. CONCLUSION: SARS-CoV-2 vaccines offer minor protection against PASC symptoms, although fewer memory problems were reported among the vaccinated than the unvaccinated participants.

Cri du chat syndrome patients have DNA methylation changes in genes linked to symptoms of the disease.

BACKGROUND: Cri du chat (also called 5p deletion, or monosomy 5p) syndrome is a genetic disease caused by deletions of various lengths in the short (p) arm of chromosome 5. Genetic analysis and phenotyping have been used to suggest dose-sensitive genes in this region that may cause symptoms when a gene copy is lost, but the heterogeneity of symptoms for patients with similar deletions complicates the picture. The epigenetics of the syndrome has only recently been looked at with DNA methylation measurements of blood from a single patient, suggesting epigenetic changes in these patients. Here, we conduct the deepest epigenetic analysis of the syndrome to date with DNA methylation analysis of eight Cri du chat patients with sibling- and age-matched controls. RESULTS: The genome-wide patterns of DNA methylation in the blood of Cri du chat patients reveal distinct changes compared to controls. In the p-arm of chromosome 5 where patients are hemizygous, we find stronger changes in methylation of CpG sites than what is seen in the rest of the genome, but this effect is less pronounced in gene regulatory sequences. Gene set enrichment analysis using patient DNA methylation changes in gene promoters revealed enrichment of genes controlling embryonic development and genes linked to symptoms which are among the most common symptoms of Cri du chat syndrome: developmental delay and microcephaly. Importantly, this relative enrichment is not driven by changes in the methylation of genes on chromosome 5. CpG sites linked to these symptoms where Cri du chat patients have strong DNA methylation changes are enriched for binding of the polycomb EZH2 complex, H3K27me3, and H3K4me2, indicating changes to bivalent promoters, known to be central to embryonic developmental processes. CONCLUSIONS: Finding DNA methylation changes in the blood of Cri du chat patients linked to the most common symptoms of the syndrome is suggestive of epigenetic changes early in embryonic development that may be contributing to the development of symptoms. However, with the present data we cannot conclude about the sequence of events between DNA methylation changes and other cellular functions-the observed differences could be directly driving epigenetic changes, a result of other epigenetic changes, or they could be a reflection of other gene regulatory changes such as changed gene expression levels. We do not know which gene(s) on the p-arm of chromosome 5 that causes epigenetic changes when hemizygous, but an important contribution from this work is making the pool of possible causative genes smaller.

Prevention of covid-19 and other acute respiratory infections with cod liver oil supplementation, a low dose vitamin D supplement: quadruple blinded, randomised placebo controlled trial.

OBJECTIVE: To determine if daily supplementation with cod liver oil, a low dose vitamin D supplement, in winter, prevents SARS-CoV-2 infection, serious covid-19, or other acute respiratory infections in adults in Norway. DESIGN: Quadruple blinded, randomised placebo controlled trial. SETTING: Norway, 10 November 2020 to 2 June 2021. PARTICIPANTS: 34 601 adults (aged 18-75 years), not taking daily vitamin D supplements. INTERVENTION: 5 mL/day of cod liver oil (10 µg of vitamin D, n=17 278) or placebo (n=17 323) for up to six months. MAIN OUTCOME MEASURES: Four co-primary endpoints were predefined: the first was a positive SARS-CoV-2 test result determined by reverse transcriptase-quantitative polymerase chain reaction and the second was serious covid-19, defined as self-reported dyspnoea, admission to hospital, or death. Other acute respiratory infections were indicated by the third and fourth co-primary endpoints: a negative SARS-CoV-2 test result and self-reported symptoms. Side effects related to the supplementation were self-reported. The fallback method was used to handle multiple comparisons. RESULTS: Supplementation with cod liver oil was not associated with a reduced risk of any of the co-primary endpoints. Participants took the supplement (cod liver oil or placebo) for a median of 164 days, and 227 (1.31%) participants in the cod liver oil group and 228 (1.32%) participants in the placebo group had a positive SARS-CoV-2 test result (relative risk 1.00, multiple comparison adjusted confidence interval 0.82 to 1.22). Serious covid-19 was identified in 121 (0.70%) participants in the cod liver oil group and in 101 (0.58%) participants in the placebo group (1.20, 0.87 to 1.65). 8546 (49.46%) and 8565 (49.44%) participants in the cod liver oil and placebo groups, respectively, had ≥1 negative SARS-CoV-2 test results (1.00, 0.97 to 1.04). 3964 (22.94%) and 3834 (22.13%) participants in the cod liver oil and placebo groups, respectively, reported ≥1 acute respiratory infections (1.04, 0.97 to 1.11). Only low grade side effects were reported in the cod liver oil and placebo groups. CONCLUSION: Supplementation with cod liver oil in the winter did not reduce the incidence of SARS-CoV-2 infection, serious covid-19, or other acute respiratory infections compared with placebo. TRIAL REGISTRATION: ClinicalTrials.gov NCT04609423.

Computed tomography with segmentation and quantification of individual organs in a D. melanogaster tumor model.

Drosophila melanogaster tumor models are growing in popularity, driven by the high degree of genetic as well as functional conservation to humans. The most common method to measure the effects of a tumor on distant organs of a human cancer patient is to use computed tomography (CT), often used in diagnosing cachexia, a debilitating cancer-induced syndrome most visibly characterized by loss of muscle mass. Successful application of high resolution micro-CT scanning of D. melanogaster was recently reported and we here present the segmentation of all visible larval organs at several stages of tumor development. We previously showed the strong expected reduction in muscle mass as the tumor develops, and we here report a surprisingly strong reduction also in gut and Malpighian tubules (kidney) volume. Time-point of tumor development was found to have a stronger correlation to cachectic organ volume loss than tumor volume, giving support to the previously proposed idea that tumor size does not directly determine degree of cachexia.

Host autophagy mediates organ wasting and nutrient mobilization for tumor growth.

During tumor growth-when nutrient and anabolic demands are high-autophagy supports tumor metabolism and growth through lysosomal organelle turnover and nutrient recycling. Ras-driven tumors additionally invoke non-autonomous autophagy in the microenvironment to support tumor growth, in part through transfer of amino acids. Here we uncover a third critical role of autophagy in mediating systemic organ wasting and nutrient mobilization for tumor growth using a well-characterized malignant tumor model in Drosophila melanogaster. Micro-computed X-ray tomography and metabolic profiling reveal that RasV12 ; scrib-/- tumors grow 10-fold in volume, while systemic organ wasting unfolds with progressive muscle atrophy, loss of body mass, -motility, -feeding, and eventually death. Tissue wasting is found to be mediated by autophagy and results in host mobilization of amino acids and sugars into circulation. Natural abundance Carbon 13 tracing demonstrates that tumor biomass is increasingly derived from host tissues as a nutrient source as wasting progresses. We conclude that host autophagy mediates organ wasting and nutrient mobilization that is utilized for tumor growth.

Natural abundance isotope ratios to differentiate sources of carbon used during tumor growth in vivo.

BACKGROUND: Radioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal. However, a labeled metabolite can be enzymatically changed in cellular metabolism, complicating the use of this experimental strategy to understand how a labeled metabolite moves between organs. These methods are also technically demanding, expensive and potentially toxic. To allow quantification of the bulk movement of metabolites between organs, we have developed a novel application of stable isotope ratio mass spectrometry (IRMS). RESULTS: We exploit natural differences in 13C/12C ratios of plant nutrients for a low-cost and non-toxic carbon labeling, allowing a measurement of bulk carbon transfer between organs in vivo. IRMS measurements were found to be sufficiently sensitive to measure organs from individual Drosophila melanogaster larvae, giving robust measurements down to 2.5 µg per sample. We apply the method to determine if carbon incorporated into a growing solid tumor is ultimately derived from food or host tissues. CONCLUSION: Measuring tumor growth in a D. melanogaster larvae tumor model reveals that these tumors derive a majority of carbon from host sources. We believe the low cost and non-toxic nature of this methodology gives it broad applicability to study carbon flows between organs also in other animals and for a range of other biological questions.

ChIP-exo analysis highlights Fkh1 and Fkh2 transcription factors as hubs that integrate multi-scale networks in budding yeast.

The understanding of the multi-scale nature of molecular networks represents a major challenge. For example, regulation of a timely cell cycle must be coordinated with growth, during which changes in metabolism occur, and integrate information from the extracellular environment, e.g. signal transduction. Forkhead transcription factors are evolutionarily conserved among eukaryotes, and coordinate a timely cell cycle progression in budding yeast. Specifically, Fkh1 and Fkh2 are expressed during a lengthy window of the cell cycle, thus are potentially able to function as hubs in the multi-scale cellular environment that interlocks various biochemical networks. Here we report on a novel ChIP-exo dataset for Fkh1 and Fkh2 in both logarithmic and stationary phases, which is analyzed by novel and existing software tools. Our analysis confirms known Forkhead targets from available ChIP-chip studies and highlights novel ones involved in the cell cycle, metabolism and signal transduction. Target genes are analyzed with respect to their function, temporal expression during the cell cycle, correlation with Fkh1 and Fkh2 as well as signaling and metabolic pathways they occur in. Furthermore, differences in targets between Fkh1 and Fkh2 are presented. Our work highlights Forkhead transcription factors as hubs that integrate multi-scale networks to achieve proper timing of cell division in budding yeast.

Predictive models of eukaryotic transcriptional regulation reveals changes in transcription factor roles and promoter usage between metabolic conditions.

Transcription factors (TF) are central to transcriptional regulation, but they are often studied in relative isolation and without close control of the metabolic state of the cell. Here, we describe genome-wide binding (by ChIP-exo) of 15 yeast TFs in four chemostat conditions that cover a range of metabolic states. We integrate this data with transcriptomics and six additional recently mapped TFs to identify predictive models describing how TFs control gene expression in different metabolic conditions. Contributions by TFs to gene regulation are predicted to be mostly activating, additive and well approximated by assuming linear effects from TF binding signal. Notably, using TF binding peaks from peak finding algorithms gave distinctly worse predictions than simply summing the low-noise and high-resolution TF ChIP-exo reads on promoters. Finally, we discover indications of a novel functional role for three TFs; Gcn4, Ert1 and Sut1 during nitrogen limited aerobic fermentation. In only this condition, the three TFs have correlated binding to a large number of genes (enriched for glycolytic and translation processes) and a negative correlation to target gene transcript levels.

Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions.

One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of TSSs at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup, we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the spatial distribution of transcription initiation events, described by the shape index, has a surprisingly strong negative correlation with measured gene expression levels, meaning that genes with higher expression levels tend to have a broader distribution of TSSs. Our analysis supplies a set of high-quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.

A bioinformatic pipeline to analyze ChIP-exo datasets.

The decrease of sequencing cost in the recent years has made genome-wide studies of transcription factor (TF) binding through chromatin immunoprecipitation methods like ChIP-seq and chromatin immunoprecipitation with lambda exonuclease (ChIP-exo) more accessible to a broader group of users. Especially with ChIP-exo, it is now possible to map TF binding sites in more detail and with less noise than previously possible. These improvements came at the cost of making the analysis of the data more challenging, which is further complicated by the fact that to this date no complete pipeline is publicly available. Here we present a workflow developed specifically for ChIP-exo data and demonstrate its capabilities for data analysis. The pipeline, which is completely publicly available on GitHub, includes all necessary analytical steps to obtain a high confidence list of TF targets starting from raw sequencing reads. During the pipeline development, we emphasized the inclusion of different quality control measurements and we show how to use these so users can have confidence in their obtained results.

Reconstruction of a Global Transcriptional Regulatory Network for Control of Lipid Metabolism in Yeast by Using Chromatin Immunoprecipitation with Lambda Exonuclease Digestion.

To build transcription regulatory networks, transcription factor binding must be analyzed in cells grown under different conditions because their responses and targets differ depending on environmental conditions. We performed whole-genome analysis of the DNA binding of five Saccharomyces cerevisiae transcription factors involved in lipid metabolism, Ino2, Ino4, Hap1, Oaf1, and Pip2, in response to four different environmental conditions in chemostat cultures, which allowed us to keep the specific growth rate constant. Chromatin immunoprecipitation with lambda exonuclease digestion (ChIP-exo) enabled the detection of binding events at a high resolution. We discovered a large number of unidentified targets and thus expanded functions for each transcription factor (e.g., glutamate biosynthesis as a target of Oaf1 and Pip2). Moreover, condition-dependent binding of transcription factors in response to cell metabolic state (e.g., differential binding of Ino2 between fermentative and respiratory metabolic conditions) was clearly suggested. Combining the new binding data with previously published data from transcription factor deletion studies revealed the high complexity of the transcriptional regulatory network for lipid metabolism in yeast, which involves the combinatorial and complementary regulation by multiple transcription factors. We anticipate that our work will provide insights into transcription factor binding dynamics that will prove useful for the understanding of transcription regulatory networks. IMPORTANCE Transcription factors play a crucial role in the regulation of gene expression and adaptation to different environments. To better understand the underlying roles of these adaptations, we performed experiments that give us high-resolution binding of transcription factors to their targets. We investigated five transcription factors involved in lipid metabolism in yeast, and we discovered multiple novel targets and condition-specific responses that allow us to draw a better regulatory map of the lipid metabolism.

Integrated analysis of the yeast NADPH-regulator Stb5 reveals distinct differences in NADPH requirements and regulation in different states of yeast metabolism.

The Saccharomyces cerevisiae transcription factor (TF) Stb5 is known to be involved in regulating NADPH generation. We explored its role by combining DNA binding studies with transcriptome analysis at four environmental conditions that were selected to cover a range of different metabolic states. Using ChIP-exo, DNA binding targets of Stb5 were found to confirm many previously proposed binding targets, in particular genes encoding enzymes involved in NADPH generation and the pentose-phosphate (PP) pathway. Transcriptome analysis of an STB5 deletion strain revealed transcriptional changes in direct regulation targets of Stb5, including several PP pathway genes as well as additional novel regulatory targets, but interestingly not including the proposed PP pathway flux controlling enzyme Zwf1. Consistently, NADPH levels were found to decrease significantly with STB5 deletion in cultures with aerobic, glucose metabolism. We also found reduced growth for the STB5 deletion strain in similar conditions as those with reduced NADPH levels, supporting a role for Stb5 in NADPH generation through the PP pathway. We finally explored the flux distribution by genome scale modelling simulations and found a decreased flux in both NADPH generating as well as consuming reactions in the STB5 deletion strain.

Rab7b modulates autophagic flux by interacting with Atg4B.

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co-localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B.

HS1BP3 inhibits autophagy by regulation of PLD1.

Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3'-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization.

HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

Complex Relations Between Phospholipids, Autophagy, and Neutral Lipids.

Research in the past decade has established the importance of autophagy to a large number of physiological processes and pathophysiological conditions. Originally characterized as a pathway responsible for protein turnover and recycling of amino acids in times of starvation, it has been recently recognized as a major regulator of lipid metabolism. Different lipid species play various roles in the regulation of autophagosomal biogenesis, both as membrane constituents and as signaling platforms. Distinct types of autophagy, in turn, facilitate specific steps in metabolic pathways of different lipid classes, best exemplified in recent studies on neutral lipid dynamics. We review the emerging notion of intricate links between phospholipids, autophagy, and neutral lipids.

RAB24 facilitates clearance of autophagic compartments during basal conditions.

RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.

Actin shapes the autophagosome.

Compared with most intracellular vesicles, the autophagosome is formed by an unusual event of vesicle budding involving an elusive sequence of membrane expansions that ends with a double membrane vesicle. It is now shown that actin polymerization inside the forming autophagosome is a driving force for the expansion and assembly of a functional autophagosome.