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The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity. Author summary: Chromatin structure alterations are central for cells to efficiently respond to DNA damage since DNA assaults do not simply occur on 'naked' DNA but in the context of chromatin. Cells have developed an integral process to mitigate DNA damage, known as the DNA damage response (DDR), which involves instrumental chromatin dynamic changes often regulated by histone-modifying enzymes. Our work demonstrates that N-terminal acetyltransferase Nat4 regulates the response to DNA damage. Importantly, cells lacking Nat4 are more susceptible to genotoxic-induced DNA damage, as well as accumulate higher number of DNA breaks. Accordingly, Nat4 expression is induced by DNA damage and specifically localizes at DNA double-strand breaks. In agreement with this, the absence of Nat4 impairs the DDR signaling, since the distribution of DNA damage-induced phosphorylation on serine 129 of histone H2A and the subsequent binding of Rad9 surrounding the break are robustly reduced. Both events are regulated by Mec1 kinase, whose recruitment at the DNA double-strand break is significantly decreased when Nat4 is absent. Consistently, downstream activation of the DNA damage checkpoint, indicative by Rad53 phosphorylation, is significantly reduced. Finally, this work supports that Nat4 functions in DNA damage response through its N-terminal acetyltransferase activity, specifically towards histone H4. Collectively, our data reveal a novel molecular and biological role for Nat4 in the response to DNA damage, and thus implicating a new player in genome integrity. [ABSTRACT FROM AUTHOR]

Histone modifications commonly integrate environmental cues with cellular metabolic outputs by affecting gene expression. However, chromatin modifications such as acetylation do not always correlate with transcription, pointing towards an alternative role of histone modifications in cellular metabolism. Using an approach that integrates mass spectrometry-based histone modification mapping and metabolomics with stable isotope tracers, we demonstrate that elevated lipids in acetyltransferase-depleted hepatocytes result from carbon atoms derived from deacetylation of hyperacetylated histone H4 flowing towards fatty acids. Consistently, enhanced lipid synthesis in acetyltransferase-depleted hepatocytes is dependent on histone deacetylases and acetyl-CoA synthetase ACSS2, but not on the substrate specificity of the acetyltransferases. Furthermore, we show that during diet-induced lipid synthesis the levels of hyperacetylated histone H4 decrease in hepatocytes and in mouse liver. In addition, overexpression of acetyltransferases can reverse diet-induced lipogenesis by blocking lipid droplet accumulation and maintaining the levels of hyperacetylated histone H4. Overall, these findings highlight hyperacetylated histones as a metabolite reservoir that can directly contribute carbon to lipid synthesis, constituting a novel function of chromatin in cellular metabolism. Synopsis: It remains unclear if chromatin modifications directly influence cellular metabolic pathways. This study reveals that hyperacetylated histone H4 acts as an acetate reservoir that contributes carbon atoms to lipid synthesis in hepatocytes. Depletion of histone and non-histone acetyltransferases in hepatocytes leads to elevated lipids, with hyperacetylated histone H4 contributing carbon atoms to fatty acids. Enhanced lipid synthesis in acetyltransferase-depleted hepatocytes depends on histone deacetylation and acetyl-CoA synthetase ACSS2. Overexpression of acetyltransferases inhibits diet-induced lipogenesis and maintains hyperacetylated histone H4 levels in vitro and mouse liver. Acetylated histones represent a reservoir for acetyl-CoA, via which acetyltransferases and deacetylases can directly modulate lipid accumulation. [ABSTRACT FROM AUTHOR]

Background: Epigenetic regulation relies on the activity of enzymes that use sentinel metabolites as cofactors to modify DNA or histone proteins. Thus, fluctuations in cellular metabolite levels have been reported to affect chromatin modifications. However, whether epigenetic modifiers also affect the levels of these metabolites and thereby impinge on downstream metabolic pathways remains largely unknown. Here, we tested this notion by investigating the function of N-alpha-acetyltransferase 40 (NAA40), the enzyme responsible for N-terminal acetylation of histones H2A and H4, which has been previously implicated with metabolic-associated conditions such as age-dependent hepatic steatosis and calorie-restriction-mediated longevity. Results: Using metabolomic and lipidomic approaches, we found that depletion of NAA40 in murine hepatocytes leads to significant increase in intracellular acetyl-CoA levels, which associates with enhanced lipid synthesis demonstrated by upregulation in de novo lipogenesis genes as well as increased levels of diglycerides and triglycerides. Consistently, the increase in these lipid species coincide with the accumulation of cytoplasmic lipid droplets and impaired insulin signalling indicated by decreased glucose uptake. However, the effect of NAA40 on lipid droplet formation is independent of insulin. In addition, the induction in lipid synthesis is replicated in vivo in the Drosophila melanogaster larval fat body. Finally, supporting our results, we find a strong association of NAA40 expression with insulin sensitivity in obese patients. Conclusions: Overall, our findings demonstrate that NAA40 affects the levels of cellular acetyl-CoA, thereby impacting lipid synthesis and insulin signalling. This study reveals a novel path through which histone-modifying enzymes influence cellular metabolism with potential implications in metabolic disorders. [ABSTRACT FROM AUTHOR]

The growing prevalence of metabolic diseases including fatty liver disease and Type 2 diabetes has increased the emphasis on understanding metabolism at the mechanistic level and how it is perturbed in disease. Metabolomics is a continually expanding field that seeks to measure metabolites in biological systems during a physiological stimulus or a genetic alteration. Typically, metabolomics studies provide total pool sizes of metabolites rather than dynamic flux measurements. More recently there has been a resurgence in approaches that use stable isotopes (e.g. 2 H and 13 C) for the unambiguous tracking of individual atoms through compartmentalised metabolic networks in humans to determine underlying mechanisms. This is known as metabolic flux analysis and enables the capture of a dynamic picture of the metabolome and its interactions with the genome and proteome. In this review, we describe current approaches using stable isotope labelling in the field of metabolomics and provide examples of studies that led to an improved understanding of glucose, fatty acid and amino acid metabolism in humans, particularly in relation to metabolic disease. Examples include the use of stable isotopes of glucose to study tumour bioenergetics as well as brain metabolism during traumatic brain injury. Lipid tracers have also been used to measure non-esterified fatty acid production whilst amino acid tracers have been used to study the rate of protein digestion on whole body postprandial protein metabolism. In addition, we illustrate the use of stable isotopes for measuring flux in human physiology by providing examples of breath tests to measure insulin resistance and gastric emptying rates. [ABSTRACT FROM AUTHOR]

Context Over the past few decades, traditional foods have been displaced by ultra-processed foods (UPFs), with the latter being associated with health problems. Objective This scoping systematic review aimed to identify the relationship between UPF intake and overweight/obesity as well as other cardiometabolic risk factors during childhood and adolescence. Data Sources The guidance for this protocol is the Preferred Reporting Items for Systematic review and Meta-Analysis Protocols (PRISMA-P). A systematic search was undertaken on PubMed, Scopus, and Cochrane Library electronic databases based on prespecified inclusion and exclusion criteria up to 6 February 2022. Data Extraction A total of 17 observational studies—9 cross-sectional, 7 cohort-longitudinal, and 1 study reporting both cross-sectional and longitudinal outcomes—among children and adolescents aged ≤18 years were eligible for inclusion in this review. Fourteen studies evaluated the consumption of UPFs in association with overweight/obesity and 9 studies examined the association of UPF consumption and cardiometabolic-related risk factors. Data Analysis Most studies (14/17) showed that an increase in UPFs was associated with a higher prevalence of overweight/obesity and cardiometabolic comorbidities among children and adolescents, whereas 4 of 17 studies (3 cross-sectional and 1 cohort) found no association. Most cohort and cross-sectional studies showed good quality according to the National Institutes of Health and Newcastle-Ottawa quality assessment, respectively. Conclusion The positive association found between UPFs and overweight/obesity and cardiometabolic comorbidities among children and adolescents raises concerns for future health. Further investigation is recommended to explore the role of specific types of UPFs on cardiometabolic conditions and to identify the amount of daily intake that increase risk in order to shape appropriate public health policies. Systematic Review Registration PROSPERO registration no. CRD42022316432. [ABSTRACT FROM AUTHOR]

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment. [ABSTRACT FROM AUTHOR]

The structurally and functionally complex endoplasmic reticulum (ER) hosts critical processes including lipid synthesis. Here, we focus on the functional characterization of transmembrane protein TMEM147, and report that it localizes at the ER and nuclear envelope in HeLa cells. Silencing of TMEM147 drastically reduces the level of lamin B receptor (LBR) at the inner nuclear membrane and results in mistargeting of LBR to the ER. LBR possesses a modular structure and corresponding bifunctionality, acting in heterochromatin organization via its N-terminus and in cholesterol biosynthesis via its sterol-reductase C-terminal domain. We show that TMEM147 physically interacts with LBR, and that the C-terminus of LBR is essential for their functional interaction. We find that TMEM147 also physically interacts with the key sterol reductase DHCR7, which is involved in cholesterol biosynthesis. Similar to what was seen for LBR, TMEM147 downregulation results in a sharp decline of DHCR protein levels and co-ordinate transcriptional decreases of LBR and DHCR7 expression. Consistent with this, lipidomic analysis upon TMEM147 silencing identified changes in cellular cholesterol levels, cholesteryl ester levels and profile, and in cellular cholesterol uptake, raising the possibility that TMEM147 is an important new regulator of cholesterol homeostasis in cells. [ABSTRACT FROM AUTHOR]