Abstract The highly conserved Dbf4-Dependent Kinase (DDK) plays a pivotal role during S phase. It phosphorylates the replicative helicase (minichromosome maintenance, MCM complex), which leads to the initiation of replication. However, few other targets, besides the MCM complex, are known, leaving DDK an understudied kinase. Here, we determine the nuclear DDK-dependent phosphoproteome by a two-pronged mass spectrometry approach. Among ~ 400 DDK-dependent phosphorylation targets, we find the Arp8 subunit of the INO80 chromatin remodeling complex. Arp8 phosphorylation stabilises INO80’s complex integrity, finetunes its nucleosome spacing at replication origins, stimulates replication and improves the replication stress response. Taken together, we report the regulation of a chromatin remodeler with nucleosome-spacing activity by the cell-cycle machinery. DDK not only regulates the core replication machinery but also regulates a factor that generates replication-conducive chromatin architecture at replication origins.

Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.

Rett Syndrome (RTT), a severe neurological disorder caused by loss-of-function mutations in the X-linked MECP2 gene, results in profound life-long neurological dysfunction. RTT patients live an apparently normal initial life until 12-18 months of age following which, a progressive accumulation of a wide range of phenotypic manifestations sets in. While MeCP2 is known to regulate chromatin, its impact on global histone composition and dynamics remains poorly understood. Here, we combine mass spectrometry imaging (MSI) and laser capture microdissection (LCM) coupled to LC-MS/MS to systematically profile histone proteoforms in three key brain regions: the dentate gyrus (DG) and cornu ammonis (CA) of the hippocampus, and the cerebellum (Cb). Our analysis reveals striking neuron-specific differences in histone composition between Mecp2-deficient and wildtype (WT) mice. Interestingly, the expression of a pathogenic Mecp2 missense mutant (Y120D) results in subtler changes in histone composition that are distinct from the null mutations. This study provides the first spatially resolved epigenetic atlas of histone proteoforms in RTT and suggests that Mecp2 loss perturbs chromatin homeostasis in a neuron- and mutation-dependent manner. Our findings underscore the critical need for cell-type-resolved analyses to unravel the mechanistic underpinnings of RTT and emphasize the importance of personalised therapeutic strategies that consider both the affected cell-type and particular Mecp2 mutation.

BACKGROUND:As key constituents of cellular sphingolipid pools, sphingomyelin (SM) and ceramide (CER) are central to the regulation of cancer cell death and survival. The metabolic flux between these two lipids is a vital component of the cellular stress response, yet the underlying regulatory mechanisms in cancer remain elusive. Acid sphingomyelinase (SMPD1) facilitates the conversion of SM to CER, functioning as a key enzymatic driver of CER-mediated signalling. OBJECTIVES:Herein, we aim to evaluate the role of SMPD1-driven sphingolipid metabolism in pancreatic carcinogenesis. DESIGN:A targeted quantitative analysis of the plasma metabolome was conducted involving patients with pancreatic ductal adenocarcinoma (PDAC, n=202) and matched control subjects (n=204). Multiplex immunohistochemistry was performed on resected PDAC (n=122) to identify expression of SMPD1 with tumour and immune cell markers. CRISPR/Cas9 driven Smpd1-deleted murine cell lines were generated and subsequently assessed for their carcinogenic potential in vitro. The effects of Smpd1 deletion on tumour formation were evaluated using both syngeneic orthotopic and metastatic murine models. RESULTS:Here, we demonstrate that tumour cell-autonomous expression of SMPD1, in pancreatic ductal adenocarcinoma (PDAC), is associated with poorer patients' outcomes. Smpd1 ablation in murine PDAC cells resulted in reduced proliferation and migration in vitro and decreased metastases and tumour burden in vivo. Integrated transcriptomic, metabolomic and proteomic studies revealed that SMPD1 abrogation impairs KrasG12D oncogenic signalling and, thus, reduces tumour burden. Reduced plasma membrane interaction of KrasG12D was associated with SMPD1-dependent sphingolipid metabolism. Notably, the SMPD1 inhibitor (ARC39) potently synergised with the KrasG12D inhibitor (MRTX1133). CONCLUSION:In summary, SMPD1 regulated plasma membrane sequestration of KrasG12D represents a potential therapeutic target within the Kras signalling pathway for intractable PDAC.

Abstract Pregnancy is a unique period regarding immune cell regulation. Within the placenta, maternal immune cells play a central role in immune surveillance and tissue remodeling. However, regulatory mechanisms of systemic immunity during pregnancy are less clear. Here, we show that neutrophil function is altered in pregnant mice (E13.5), indicated by increased slow rolling velocity and reduced adhesion. Mechanistically, PreImplantation factor (PIF), a 15 amino acid peptide which is produced by human and murine trophoblast cells of the placenta, is continuously secreted into the maternal circulation and plays a key role in modulating neutrophil function via blocking the voltage-gated potassium channel K V 1.3. This resulted in impaired intracellular Ca 2+ signaling and subsequently disturbance of neutrophil post-arrest modifications and a higher susceptibility to physiological shear forces in vivo and in vitro . Furthermore, PIF-mediated K V 1.3 blockade impaired E-selectin-mediated release of S100A8/A9 and phagocytosis. Taken together, we have identified PIF as an important modulator of neutrophil function during pregnancy suggesting a critical role in regulating innate immune responses throughout gestation.

The acetylation of histones is a central component of reversible chromatin modification that governs genome regulation. Understanding the complex histone acetylation network requires knowledge about the contributions of individual acetyltransferases. These are not easily determined through perturbation studies in cells, due to indirect effects and limited selectivity of the antibodies that detect site-specific histone acetylation. The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histones H4 and the variant H2A.V, but the precise positions of substrate lysines and their relative acetylation rates were unknown. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V, and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns, and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzyme's mode of action and to evaluate its contribution to histone acetylation patterns in cells.

Lysine demethylases of the KDM4 subfamily are epigenetic regulators frequently dysregulated in cancer, yet the identification of cell-active inhibitors remains challenging due to assay interference and limited chemotype diversity. Here, we report the discovery of 2-substituted-3-hydroxy-2,3-dihydroquinazolin-4(1H)-ones as a new class of KDM4A inhibitors. A focused library was identified through a screening cascade integrating biochemical, biophysical, and mass spectrometry-based assays to minimize false positives. Orthogonal validation using surface plasmon resonance, nanodifferential scanning fluorimetry, and MALDI-TOF-MS enabled reliable compound prioritization, leading to the identification of compound 6i as a validated KDM4A binder and inhibitor. Cellular target engagement was demonstrated by quantitative label-free proteomics, revealing a dose-dependent accumulation of H3K9me2 and H3K9me3 in HEK293T cells, consistent with on-target inhibition of KDM4-family demethylases. Compound 6i further exhibited favorable solubility, permeability, and metabolic stability, supporting its qualification as a promising cell-active KDM4 inhibitor scaffold.

Histone post-translation modifications change dramatically as cells enter mitosis, Indeed, histone phosphorylation and deacetylation have been proposed to be major factors driving the compaction of mitotic chromosomes. However, whether these modification changes directly regulate mitotic chromatin compaction remains a subject of debate. Here, we use a CDK1as chemical-genetic system to accumulate cells in G2 phase. Treatment of these G2 cells with the phosphatase inhibitor calyculin A efficiently causes them to undergo premature chromosome condensation, even when the CDK1as allele is inhibited with 1NM-PP1. Surprisingly, simultaneous treatment of the cells with R547, an inhibitor of CDK1, 2 and 4 kinases, causes them to remain phenotypically in G2 phase. Strikingly, although mitotic-like chromatin condensation fails to occur in these cells, LC-MS reveals that histone H3 phosphorylation occurs even more efficiently than in a normal mitosis. Furthermore, histone deacetylation also occurs in those interphase-looking calyculin A-treated cells. These results reveal that histone phosphorylation and deacetylation are not sufficient to drive mitotic-like chromosome condensation and compaction.

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. Although condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, histone phosphorylation and deacetylation have been proposed to contribute to a further 2- to 4-fold reduction in mitotic chromatin volume. Here, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing highly synchronous mitotic entry. Our analysis reveals three temporally distinct programs of histone H3 phosphorylation on T3, S10, and S28 that could differentially regulate the association of readers with chromatin via methyl-phos switching. Mass spectrometry, quantitative chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR, and immunofluorescence analyses reveal that H3 T3 phosphorylation is a mitosis-specific marker of heterochromatin, whose deposition requires H3K9me3. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, deacetylation does not drive mitotic chromosome formation. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

Artery tertiary lymphoid organs (ATLOs) emerge in atherosclerosis, which is a chronic inflammatory artery disease with an autoimmune component. However, whether disease-relevant autoimmune B cells emerge in ATLOs remains unknown. In this study, we isolate germinal center (GC) B cells from ATLOs and lymph nodes from healthy and atherosclerosis-burdened mice, expression clone 60 autoantibodies and screen them for arterial wall reactivity. ATLO GC B cell-derived autoantibodies skew to atherosclerosis-relevant autoantigens versus their counterparts in lymph nodes of both genotypes. One ATLO GC B cell-derived autoantibody (termed A6) binds to histone 2B (H2B) with high affinity. Both vaccination with H2B and adoptive transfer of A6 accelerate atherosclerosis, revealing a pathogenic autoantibody-autoantigen pair. Mechanistically, ATLOs specifically show both distorted B cell activation and immune tolerance checkpoint-regulating gene expression profiles. In a human cohort, circulating anti-H2B antibody titers positively correlate with aortic calcification in humans. We suggest that ATLOs harbor a dysregulated immune tolerance environment permissive for autoreactive B cells that express pathogenic autoantibodies promoting atherosclerosis.

Advancing MS-based proteomics toward clinical applications evolves around developing standardized start-to-finish and fit-for-purpose workflows for clinical specimens. Steps along the method design involve the determination and optimization of several bioanalytical parameters such as selectivity, sensitivity, accuracy, and precision. In a joint effort, eight proteomics laboratories belonging to the MSCoreSys initiative including the CLINSPECT-M, MSTARS, DIASyM, and SMART-CARE consortia performed a longitudinal round-robin study to assess the analysis performance of plasma and serum as clinically relevant samples. A variety of LC-MS/MS setups including mass spectrometer models from ThermoFisher and Bruker as well as LC systems from ThermoFisher, Evosep, and Waters Corporation were used in this study. As key performance indicators, sensitivity, precision, and reproducibility were monitored over time. Protein identifications range between 300 and 400 IDs across different state-of-the-art MS instruments, with timsTOF Pro, Orbitrap Exploris 480, and Q Exactive HF-X being among the top performers. Overall, 71 proteins are reproducibly detectable in all setups in both serum and plasma samples, and 22 of these proteins are FDA-approved biomarkers, which are reproducibly quantified (CV < 20% with label-free quantification). In total, the round-robin study highlights a promising baseline for bringing MS-based measurements of serum and plasma samples closer to clinical utility.

Common genetic variants in a conserved cis-regulatory element (CRE) at histone deacetylase (HDAC)9 are a major risk factor for cardiovascular disease, including stroke and coronary artery disease. Given the consistency of this association and its proinflammatory properties, we examined the mechanisms whereby HDAC9 regulates vascular inflammation. HDAC9 bound and mediated deacetylation of NLRP3 in the NACHT and LRR domains leading to inflammasome activation and lytic cell death. Targeted deletion of the critical CRE in mice increased Hdac9 expression in myeloid cells to exacerbate inflammasome-dependent chronic inflammation. In human carotid endarterectomy samples, increased HDAC9 expression was associated with atheroprogression and clinical plaque instability. Incorporation of TMP195, a class IIa HDAC inhibitor, into lipoprotein-based nanoparticles to target HDAC9 at the site of myeloid-driven vascular inflammation stabilized atherosclerotic plaques, implying a lower risk of plaque rupture and cardiovascular events. Our findings link HDAC9 to atherogenic inflammation and provide a paradigm for anti-inflammatory therapeutics for atherosclerosis.

The RNA-binding protein Hangover (Hang) is essential for several stress responses in Drosophila melanogaster. Here, we discover a novel function of Hang in the regulation of gene expression. Hang binds to >2000 genes in the Drosophila genome and modulates transcription. We identify a diverse set of chromatin regulators as Hang interactors, including NSL, dMec, Sin3A, dREAM, and Ino80. Among these, the non-specific lethal complex (NSL) is the most prominent one. We show that Hang attenuates NSL-mediated H4K16 acetylation at transcriptional start sites to downregulate gene expression. Our work uncovers novel roles for Hang in epigenetic gene regulation and suggests that it coordinates the function of multiple chromatin regulators.

Alzheimer´s disease is a neurodegenerative disease with high global prevalence and no cure available. It is known that the microbiota-gut-brain-axis plays a role in the pathogenesis, but the pathways are not fully understood yet. To elucidate the role of dietary fibre supplementation on this axis in a 5xFAD mouse model of Alzheimer´s disease, a feeding trial with an inulin supplement was conducted. At the start (Basis, n = 11) and after 7 weeks with (AD + F; n = 15) and without (AD; n = 15) supplementation, the mice were sacrificed and the following samples were taken: ingesta for 16 S rRNA sequencing and short-chain fatty acid (SCFA) analysis, and brain tissue for amyloid-beta staining and proteome analysis. The microbiota patterns in stomach, small intestine, caecum and colon differed between AD and AD + F. SCFA concentrations were significantly higher in group AD + F as compared to AD and Basis. In the AD mice, plaque load was significantly increased as compared to Basis, while a reduction in AD + F as compared to AD was observed. The brain proteome also differed between AD + F and AD, indicating a beneficial effect of the inulin supplementation, possibly mediated in part by microbial acetate. Since prebiotic substances like inulin are also part of human diets, this should be investigated further in the translational context.

In vertebrate embryos, gene expression is first initiated at zygotic genome activation (ZGA). Maternally expressed transcription factors are essential for this process. However, it is unknown whether active chromatin modifications established in gametes are present in early embryos and contribute to ZGA and embryonic development. Here, we show that in Xenopus laevis, H3K4me3 occurs at common genomic loci in gametes, in transcriptionally quiescent pre-ZGA embryos, and in transcriptionally active ZGA embryos. These loci exhibit high H3K4me3 intensities and breadth, DNA hypomethylation, and elevated CpG content. We show that H3K4 methylation pre-marking is required for successful ZGA and development, including expression of the key ZGA transcription factor Pou5f3.2. We demonstrate that the H3K4 methyltransferase Cxxc1 ensures establishment of H3K4me3 and proper ZGA. These findings reveal a role for H3K4 methylation in defining active chromatin states in Xenopus laevis embryos and highlight its importance for accurate ZGA and embryonic development.

Human plasma is routinely collected during clinical care and constitutes a rich source of biomarkers for diagnostics and patient stratification. Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a key method for plasma biomarker discovery, but the high dynamic range of plasma proteins poses significant challenges for MS analysis and data processing. To benchmark the quantitative performance of neat plasma analysis, we introduce a multispecies sample set based on a human tryptic plasma digest containing varying low level spike-ins of yeast and E. coli tryptic proteome digests, termed PYE. By analysing the sample set on state-of-the-art LC-MS platforms across twelve different sites in data-dependent (DDA) and data-independent acquisition (DIA) modes, we provide a data resource comprising a total of 1116 individual LC-MS runs. Centralized data analysis shows that DIA methods outperform DDA-based approaches regarding identifications, data completeness, accuracy, and precision. DIA achieves excellent technical reproducibility, as demonstrated by coefficients of variation (CVs) between 3.3% and 9.8% at protein level. Comparative analysis of different setups clearly shows a high overlap in identified proteins and proves that accurate and precise quantitative measurements are feasible across multiple sites, even in a complex matrix such as plasma, using state-of-the-art instrumentation. The collected dataset, including the PYE sample set and strategy presented, serves as a valuable resource for optimizing the accuracy and reproducibility of LC-MS and bioinformatic workflows for clinical plasma proteome analysis.

The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histone N-terminal ‘tail’ domains. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A, or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzyme’s mode of action. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, CRC1064-A1 EMBO long-term fellowship, ALTF 168–2018