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Histone post-translational modifications (PTMs) regulate gene expression patterns through epigenetic mechanisms. The 5 histone proteins (H1, H2A, H2B, H3, and H4) are extensively modified, with over 75 distinct modification types spanning more than 200 sites. Despite strong advances in mass spectrometry-based approaches, identification and quantification of modified histone peptides remains challenging due to factors such as isobaric peptides, pseudo-isobaric PTMs, and low stoichiometry of certain marks. Here we describe the development of a new high-throughput method to identify and quantify over 150 modified histone peptides by liquid chromatography-mass spectrometry (LC-MS). Fast gradient microflow liquid chromatography and variable window SWATH data-independent acquisition on a new quadrupole time-of-flight platform is compared to a previous method using nanoflow LC-MS on an Orbitrap hybrid. Histones extracted from cells treated with either a histone deacetylase inhibitor (HDACi) or TGF-beta 1 were analyzed by data-independent acquisition (DIA) on two mass spectrometers: an Orbitrap Exploris 240 with a 55-minute nanoflow LC gradient, and the SCIEX ZenoTOF 7600 with a 10-minute microflow gradient. To demonstrate the reproducibility and speed advantage of the method, 100 consecutive injections of one sample were performed in less than 2 days on the QTOF platform. The result is the comprehensive characterization of histone PTMs achieved in less than 20 minutes of total run time using only 200 ng of sample. Results for drug-treated histone samples are comparable to those produced by the previous method and can be achieved using less than one-third of the instrument time., Competing Interests: Conflict of Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Reversible modification of the histone H3 N-terminal tail is critical in regulating the chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here, we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle-down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetic discovery and therapeutic development.

The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here, we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones, followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nano-LC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.

Understanding the ontogeny of teleost mucosa-associated lymphoid tissues (MALT) is critical for determining the earliest timepoint for effective mucosal vaccination of young fish. Here, we describe the developmental sequence that leads to the formation of an organized MALT structure in rainbow trout, the organized nasopharynx-associated lymphoid tissue (O-NALT). Control rainbow trout were sampled between 340 and 1860 degree days (DD) and routine histology and immunofluorescence staining were used to determine cellular changes in immune cells in the nasal cavity as well as O-NALT formation. We identified that O-NALT is first seeded by CD8α + T cells at 700 DD followed by IgM + B cells and cd4-2b +  cells at 1000 DD. Histomorphologically, trout O-NALT is fully formed at 1400 DD. Whole body gene expression analyses uncovered waves of igmh, cd4-2b, and cd8a expression that recapitulate the cellular seeding sequence of O-NALT by specific lymphocyte subsets. Our results indicate that 1) O-NALT formation results from a specific sequence of lymphocyte subset colonization pioneered by CD8α + T cells and 2) the presence of the full O-NALT structure at 1400 DD may mark this timepoint as the earliest developmental stage at which mucosal vaccines can induce long lasting, specific immune responses., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Our genome is not made of naked DNA but a fiber (chromatin) composed of DNA and proteins packaged into our chromosomes. The basic building block of chromatin is the nucleosome, which has two copies of each of the proteins called histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. Regions of our genetic material are found between the more open (euchromatin) and more compact (heterochromatin) regions of the genome that can be variably accessible to the underlying genes. Furthermore, post-translational modifications (PTMs) on histones, such as on H3, are critical for regulating chromatin accessibility and gene expression. While site-specific antibodies were the tool of choice for histone PTM analysis in the early days (pre-2000s), enter Don Hunt changing the histone PTM field forever. Don's clever thinking brought new innovative mass spectrometry-based approaches to the epigenetics field. His lab's effort led to the discovery of many new histone modifications and methods to facilitate the detection and quantification of histone PTMs, which are still considered state of the art in the proteomics field today. Due to Don's pioneering work in this area, many labs have been able to jump into the epigenetics field and "Hunt" down their own histone targets. A walkthrough of those early histone years in the Hunt Lab is described by three of us who were fortunate enough to be at the right place, at the right time., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer immunotherapies produce remarkable results in B cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor and normal tissues to identify biologically relevant cell surface immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer. Proteogenomic analyses reveal sixty high-confidence candidate immunotherapeutic targets, and we prioritize delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlates with a super-enhancer. Immunofluorescence, flow cytometry, and immunohistochemistry show robust cell surface expression of DLK1. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells results in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Since high DLK1 expression is found in several adult and pediatric cancers, our study demonstrates the utility of a proteogenomic approach and credentials DLK1 as an immunotherapeutic target., Competing Interests: Declaration of interests F. Zammarchi, K. Havenith, and P.H.v.B. are or were employed by ADC Therapeutics at the time the work was conducted and hold or previously held shares/stocks in ADC Therapeutics. The following patent is held: WO2018146199A1., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Social insects offer powerful models to investigate the mechanistic foundation of elaborate individual behaviors comprising a cooperative community. Workers of the leafcutter ant genus Atta provide an extreme example of behavioral segregation among many phenotypically distinct worker types. We utilize the complex worker system of Atta cephalotes to test the molecular underpinnings of behavioral programming and, in particular, the extent of plasticity to reprogramming. We identify specific neuropeptides as mediators of worker division of labor in A. cephalotes , finding two neuropeptides associated with characteristic behaviors of leafcutting and of brood care. Manipulation via genetic knockdown or by injection of these neuropeptides led to stark loss or gain of each behavior and to transcriptomic shifts in the predicted direction, that is, towards gene pathways expressed in the natural caste. We also compare specific A. cephalotes worker transcriptomes with those of orthologous workers of the eusocial mammal, the naked mole rat H. gaber , revealing global similarities between caste-biased expression and link to specific roles of our studied neuropeptides in ants. This work underscores the essential function of neuropeptides in establishing complex social behavior and a remarkable plasticity among individual behavioral types.

Protein-protein interactions (PPIs) provide essential insights into the complex molecular mechanisms and signaling pathways within cells that regulate development and disease-related phenotypes. However, for membrane proteins, the impact of various forms of glycosylation has often been overlooked in PPI studies. In this study, we introduce a novel approach, glycan-dependent affinity purification followed by mass spectrometry (GAP-MS), to assess variations in PPIs for any glycoprotein of interest under different glycosylation conditions. As a proof of principle, we selected four glycoproteins-BSG, CD44, EGFR, and SLC3A2-as baits to compare their co-purified partners across five metabolically controlled glycan conditions. The findings demonstrate the capability of GAP-MS to identify PPIs influenced by altered glycosylation states, establishing a foundation for systematically exploring the Glycan-Dependent Protein Interactome (GDPI) for other glycoproteins of interest., Competing Interests: DECLARATION OF INTERESTS The authors declare that they have no conflicts of interest with the contents of this article.

Proneural gliomas are brain tumors characterized by enrichment of oligodendrocyte progenitor cell (OPC) transcripts and genetic alterations. In this study we sought to identify transcriptional and epigenetic differences between OPCs with Trp53 deletion and PDGF-BB overexpression (BB-p53n) and those carrying only p53 deletion (p53n). In culture, the BB-p53n OPCs display growth characteristics more similar to glioma cells than p53n OPCs. When injected in mouse brains, BB-p53n OPC form tumors, while the p53n OPCs do not. Unbiased histone proteomics and transcriptomic analysis on these OPC populations identified higher levels of the histone H3K27me3 mark and lower levels of the histone H4K20me3. The transcriptome of the BB-p53n OPCs was characterized by higher levels of transcripts related to proliferation and cell adhesion compared to p53n OPCs. Pharmacological inhibition of the enzyme responsible for histone H3K27 trimethylation (EZH2i) in BB-p53n OPCs, reduced cell cycle transcripts and increased the expression of differentiation markers, but was not sufficient to restore their growth characteristics. This suggests that PDGF-BB overexpression in p53n OPCs favors the early stages of transformation, by promoting proliferation and halting differentiation in a H3K27me3-dependent pathway, and favoring growth characteristics in a H3K27me3 independent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The "Histone Code" is comprised of specific types and positions of post-translational modifications (PTMs) which produce biological signals for gene regulation and have potential as biomarkers for medical diagnostics. Previous work has shown that electron-based fragmentation improves the sequence coverage and confidence of labile PTM position assignment. Here, we evaluated two derivatization methods (e.g., irreversible - propionylation and reversible-citraconylation) for bottom-up analysis of histone H4 4-17 proteoforms using online liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), and electron-based dissociation (ExD) in tandem with mass spectrometry. Two platforms were utilized: a custom-built LC-TIMS-q-ExD-ToF MS/MS based on a Bruker Impact and a commercial μLC-EAD-ToF MS/MS SCIEX instrument. Complementary LC-TIMS preseparation of H4 4-17 0-4ac positional isomer standards showed that they can be resolved in their endogenous form, while positional isomers cannot be fully resolved in their propionylated form; online LC-ExD-MS/MS provided high sequence coverage (>90%) for all H4 4-17 (0-4ac) proteoforms in both instrumental platforms. When applied to model cancer cells treated with a histone deacetylase inhibitor (HeLa + HDACi), both derivatization methods and platforms detected and confirmed H4 4-17 (0-4ac) proteolytic peptides based on their fragmentation pattern. Moreover, a larger number of HeLa + HDACi H4 4-17 proteoforms were observed combining LC-TIMS and LC-q-ExD-ToF MS/MS due to the positional isomer preseparation in the LC-TIMS domain of citraconylated H4 4-17 (0-4ac) peptides.

Background: Methylation of histone 3 lysine 36 (H3K36me) has emerged as an essential epigenetic component for the faithful regulation of gene expression. Despite its importance in development and disease, how the molecular agents collectively shape the H3K36me landscape is unclear., Results: We use mouse mesenchymal stem cells to perturb the H3K36me methyltransferases (K36MTs) and infer the activities of the five most prominent enzymes: SETD2, NSD1, NSD2, NSD3, and ASH1L. We find that H3K36me2 is the most abundant of the three methylation states and is predominantly deposited at intergenic regions by NSD1, and partly by NSD2. In contrast, H3K36me1/3 are most abundant within exons and are positively correlated with gene expression. We demonstrate that while SETD2 deposits most H3K36me3, it may also deposit H3K36me2 within transcribed genes. Additionally, loss of SETD2 results in an increase of exonic H3K36me1, suggesting other (K36MTs) prime gene bodies with lower methylation states ahead of transcription. While NSD1/2 establish broad intergenic H3K36me2 domains, NSD3 deposits H3K36me2 peaks on active promoters and enhancers. Meanwhile, the activity of ASH1L is restricted to the regulatory elements of developmentally relevant genes, and our analyses implicate PBX2 as a potential recruitment factor., Conclusions: Within genes, SETD2 primarily deposits H3K36me3, while the other K36MTs deposit H3K36me1/2 independently of SETD2 activity. For the deposition of H3K36me1/2, we find a hierarchy of K36MT activities where NSD1 > NSD2 > NSD3 > ASH1L. While NSD1 and NSD2 are responsible for most genome-wide propagation of H3K36me2, the activities of NSD3 and ASH1L are confined to active regulatory elements., (© 2024. The Author(s).)

A hallmark of addiction is the ability of drugs of abuse to trigger relapse after periods of prolonged abstinence. Here, we describe an epigenetic mechanism whereby chronic cocaine exposure causes lasting chromatin and downstream transcriptional modifications in the nucleus accumbens (NAc), a critical brain region controlling motivation. We link prolonged withdrawal from cocaine to the depletion of the histone variant H2A.Z, coupled with increased genome accessibility and latent priming of gene transcription, in D1 dopamine receptor-expressing medium spiny neurons (D1 MSNs) that relate to aberrant gene expression upon drug relapse. The histone chaperone ANP32E removes H2A.Z from chromatin, and we demonstrate that D1 MSN-selective Anp32e knockdown prevents cocaine-induced H2A.Z depletion and blocks cocaine's rewarding actions. By contrast, very different effects of cocaine exposure, withdrawal, and relapse were found for D2 MSNs. These findings establish histone variant exchange as an important mechanism and clinical target engaged by drugs of abuse to corrupt brain function and behavior.

Epigenetic inhibitors exhibit powerful antiproliferative and anticancer activities. However, cellular responses to small-molecule epigenetic inhibition are heterogenous and dependent on factors such as the genetic background, metabolic state, and on-/off-target engagement of individual small-molecule compounds. The molecular study of the extent of this heterogeneity often measures changes in a single cell line or using a small number of compounds. To more comprehensively profile the effects of small-molecule perturbations and their influence on these heterogeneous cellular responses, we present a molecular resource based on the quantification of chromatin, proteome, and transcriptome remodeling due to histone deacetylase inhibitors (HDACi) in non-isogenic cell lines. Through quantitative molecular profiling of 10,621 proteins, these data reveal coordinated molecular remodeling of HDACi treated cancer cells. HDACi-regulated proteins differ greatly across cell lines with consistent (JUN, MAP2K3, CDKN1A) and divergent (CCND3, ASF1B, BRD7) cell-state effectors. Together these data provide valuable insight into cell-type driven and heterogeneous responses that must be taken into consideration when monitoring molecular perturbations in culture models., Competing Interests: Conflict of Interest: We acknowledge our ongoing collaborations and sponsored agreements with Thermo Fisher Scientific, Genentech, and AI Proteins.

Nucleic acids are fundamental biological molecules that encode and convey genetic information within living organisms. Over 150 modifications have been found in nucleic acids, which are involved in critical biological functions, including regulating gene expression, stabilizing nucleic acid structure, modulating protein translation, and so on. The dysregulation of nucleic acid modifications is correlated with many diseases such as cancers and neurological disorders. However, it is still challenging to simultaneously characterize and quantify diverse modifications using traditional genomic methods. Mass spectrometry (MS) has served as a crucial tool to solve this issue, and can directly identify the modified species through their distinct mass differences compared to the canonical ones and provide accurate quantitative information. This review surveys the history of nucleic acid modification discovery, advancements in MS-based methods, nucleic acid sample preparation, and applications in biological and medical research. We expect the high-throughput and valuable quantitative information from MS analysis will be more broadly applied to studying nucleic acid modification status in different pathological conditions, which is key to filling gaps in traditional genomics and transcriptomics research and enabling researchers to gain insights into epigenetics and epitranscriptomics., (© 2024 John Wiley & Sons Ltd.)

GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp 3 U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology., Competing Interests: Declaration of interests R.A.F. is a stockholder of ORNA Therapeutics and is a board of directors’ member and stockholder of Chronus Health and Blue Planet Systems. C.R.B. is a cofounder of Firefly Biologics and a cofounder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), ReNAgade Therapeutics, and InterVenn Biosciences., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and- paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development., Competing Interests: Competing Interests Y.Z. is a founder, board member, advisor to, and inventor on patents licensed to PTM Bio Inc. (Hangzhou, China and Chicago, IL) and Maponos Therapeutics Inc. (Chicago, IL). P.A.C. is a founder of Acylin Therapeutics and has been a consultant for Abbvie and Constellation and Epizyme. He is an inventor of an issued U.S. patent for Corin.

The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K 0 ) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me 1-3 ) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutate histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleash cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupts the deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal levels of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs., (© 2024. The Author(s).)

There is an increasing appreciation for the role of cell surface glycans in modulating interactions with extracellular ligands and participating in intercellular communication. We recently reported the existence of sialoglycoRNAs, where mammalian small RNAs are covalently linked to N-glycans through the modified base acp 3 U and trafficked to the cell surface. However, little is currently known about the role for O-glycosylation, another major class of carbohydrate polymer modifications. Here, we use parallel genetic, enzymatic, and mass spectrometry approaches to demonstrate that O-linked glycan biosynthesis is responsible for the majority of sialoglycoRNA levels. By examining the O-glycans associated with RNA from cell lines and colon organoids we find known and previously unreported O-linked glycan structures. Further, we find that O-linked glycans released from small RNA from organoids derived from ulcerative colitis patients exhibit higher levels of sialylation than glycans from healthy organoids. Together, our work provides flexible tools to interrogate O-linked glycoRNAs (O-glycoRNA) and suggests that they may be modulated in human disease., Competing Interests: DECLARATION OF INTERESTS R.A.F. is a stockholder of ORNA Therapeutics. R.A.F. is a board of directors member and stockholder of Chronus Health and Blue Planet Systems. The other authors declare no competing interests.

Metabolic control of chromatin and gene expression is emerging as a key, but largely unexplored aspect of gene regulation. In the brain, metabolic-epigenetic interactions can influence critical neuronal functions. Here, we use a combination of behavioral, proteomic and genomic approaches to demonstrate that the intermediary metabolite acetate enhances memory in a brain region- and sex-specific manner. We show that acetate facilitates the formation of dorsal hippocampus-dependent spatial memories in female but not in male mice, while having no effect on cortex-dependent non-spatial memories in either sex. Acetate-enhanced spatial memory is driven by increased acetylation of histone variant H2A.Z, and upregulation of genes implicated in spatial learning in the dorsal hippocampus of female mice. In line with the sex-specific behavioral outcomes, the effect of acetate on dorsal hippocampal histone modifications and gene expression shows marked differences between the sexes during critical windows of memory formation (consolidation and recall). Overall, our findings elucidate a novel role for acetate, a ubiquitous and abundant metabolite, in regulating dorsal hippocampal chromatin, gene expression and learning, and outline acetate exposure as a promising new approach to enhance memory formation.

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers., (© 2024. The Author(s).)

Automated particle analysis (APA) provides a vast amount of compositional data via energy-dispersive X-ray spectroscopy along with size and shape data via scanning electron microscopy for individual particles in a sample. In many instances, APA data are leveraged to support identification of the source of a sample based on the detection of particles of a specific composition. Often, the particles that provide context make up a minuscule portion of the sample. Additionally, the interpretation of complex samples can be difficult due to the diversity of compositions both in the mixture and within a particle. In this work, we demonstrate a method to compute and cluster similarity graphs that describe inter-particle relationships within a sample using a multi-modal few-shot learning neural network. As a proof-of-concept, we show that samples known to have been exposed to gunshot residue can be distinguished from samples occasionally mistaken for gunshot residue. Our workflow builds upon standard APA techniques and data processing methods to unveil additional information in a readily interpretable and quantitatively comparable format., Competing Interests: Conflict of Interest: The authors declare that they have no competing interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome., Competing Interests: Competing interests statement:The authors declare no competing interest.

HDAC8 plays crucial roles in biological processes, from gene regulation to cell motility, making it a highly desirable target for therapeutic intervention. HDAC8 also has deacetylase-independent activity which cannot be blocked by a conventional inhibitor. In this study, we report the discovery of YX862, a highly potent and selective hydrazide-based HDAC8-proteolysis targeting chimera (PROTAC) degrader. The selectivity is achieved through rational design of the warhead to spare HDAC3 activity from the previous HDAC3/8 dual degrader YX968. We demonstrate that the degradation of HDAC8 by YX862 increases acetylation levels of its nonhistone substrates such as SMC3 without significantly triggering histone PTM, supporting HDAC8's major role in nonhistone PTM regulation. YX862 exhibits promising on-target antiproliferative activity against DLBCL cells with higher potency than the HDAC8 selective inhibitor PCI-34051. As a selective HDAC8 degrader that avoids pan-HDAC inhibition, YX862 represents a valuable tool for exploring the biological and therapeutic potential of HDAC8.

Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids ( e.g. , Glu and Asp) at the protein N -terminus or side chain. Systemic removal of arginylation after ATE1 knockout (KO) in mouse models resulted in heart defects leading to embryonic lethality. The biological importance of arginylation has motivated the discovery of arginylation sites on proteins using bottom-up approaches. While bottom-up proteomics is powerful in localizing peptide arginylation, it lacks the ability to quantify proteoforms at the protein level. Here we developed a top-down proteomics workflow for characterizing and quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), we have inserted an R residue after the signaling peptide (AA1-17). Upon overexpression in ATE1 KO cells, CALR and R-CALR were purified by affinity purification and analyzed by LCMS in positive mode. Both proteoforms showed charge states ranging from 27-68 with charge 58 as the most intense charge state. Their MS2 spectra from electron-activated dissociation (EAD) showed preferential fragmentation at the protein N -terminals which yielded sufficient c ions facilitating precise localization of the arginylation sites. The calcium-binding domain (CBD) gave minimum characteristic ions possibly due to the abundant presence of >100 D and E residues. Ultraviolet photodissociation (UVPD) compared with EAD and ETD significantly improved the sequence coverage of CBD. This method can identify and quantify CALR arginylation at absence, endogenous (low), and high levels. To our knowledge, our work is the first application of top-down proteomics in characterizing post-translational arginylation in vitro and in vivo .

Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis 1,2 . However, whether analogous pathologic epigenetic mechanisms act directly on the ribosome to advance oncogenesis is unclear. Here we find that trimethylation of the core ribosomal protein L40 (rpL40) at lysine 22 (rpL40K22me3) by the lysine methyltransferase SMYD5 regulates mRNA translation output to promote malignant progression of gastric adenocarcinoma (GAC) with lethal peritoneal ascites. A biochemical-proteomics strategy identifies the monoubiquitin fusion protein partner rpL40 (ref. 3 ) as the principal physiological substrate of SMYD5 across diverse samples. Inhibiting the SMYD5-rpL40K22me3 axis in GAC cell lines reprogrammes protein synthesis to attenuate oncogenic gene expression signatures. SMYD5 and rpL40K22me3 are upregulated in samples from patients with GAC and negatively correlate with clinical outcomes. SMYD5 ablation in vivo in familial and sporadic mouse models of malignant GAC blocks metastatic disease, including peritoneal carcinomatosis. Suppressing SMYD5 methylation of rpL40 inhibits human cancer cell and patient-derived GAC xenograft growth and renders them hypersensitive to inhibitors of PI3K and mTOR. Finally, combining SMYD5 depletion with PI3K-mTOR inhibition and chimeric antigen receptor T cell administration cures an otherwise lethal in vivo mouse model of aggressive GAC-derived peritoneal carcinomatosis. Together, our work uncovers a ribosome-based epigenetic mechanism that facilitates the evolution of malignant GAC and proposes SMYD5 targeting as part of a potential combination therapy to treat this cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4) 2 tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4) 2 tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear., Methods: We utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate Acly in cardiomyocytes of MyH6-Cas9 mice. In vivo, positron emission tomography and ex vivo stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level., Results: Here, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. Ex vivo isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate., Conclusions: This study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype., (© 2024. The Author(s).)

The eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nanoLC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts., Competing Interests: CONFLICTS OF INTEREST The authors have no financial conflicts of interest to disclose.

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general activity-based arginylation profiling (ABAP) platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates ( ex vivo ) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. ABAP has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 μg sample input, with 229 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

Gene expression is tightly controlled during animal development to allow the formation of specialized cell types. Our understanding of how animals evolved this exquisite regulatory control remains elusive, but evidence suggests that changes in chromatin-based mechanisms may have contributed. To investigate this possibility, here we examine chromatin-based gene regulatory features in the closest relatives of animals, choanoflagellates. Using Salpingoeca rosetta as a model system, we examined chromatin accessibility and histone modifications at the genome scale and compared these features to gene expression. We first observed that accessible regions of chromatin are primarily associated with gene promoters and found no evidence of distal gene regulatory elements resembling the enhancers that animals deploy to regulate developmental gene expression. Remarkably, a histone modification deposited by polycomb repressive complex 2, histone H3 lysine 27 trimethylation (H3K27me3), appeared to function similarly in S. rosetta to its role in animals, because this modification decorated genes with cell type-specific expression. Additionally, H3K27me3 marked transposons, retaining what appears to be an ancestral role in regulating these elements. We further uncovered a putative new bivalent chromatin state at cell type-specific genes that consists of H3K27me3 and histone H3 lysine 4 mono-methylation (H3K4me1). Together, our discoveries support the scenario that gene-associated histone modification states that underpin development emerged before the evolution of animal multicellularity.

Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks., Competing Interests: Declaration of interests E.J.B. serves on the scientific advisory board of Aprea Therapeutics, and he consults for and holds equity in this company., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Proneural gliomas are brain tumors characterized by enrichment of oligodendrocyte progenitor cell (OPC) transcripts and genetic alterations. In this study we sought to identify transcriptional and epigenetic differences between OPCs with Trp53 deletion and PDGF-BB overexpression (BB-p53n), which form tumors when transplanted in mouse brains, and those carrying only p53 deletion (p53n), which do not. We used unbiased histone proteomics and RNA-seq analysis on these two genetically modified OPC populations and detected higher levels of H3K27me3 in BB-p53n compared to p53n OPCs. The BB-p53n OPC were characterized by higher levels of transcripts related to proliferation and lower levels of those related to differentiation. Pharmacological inhibition of histone H3K27 trimethylation in BB-p53n OPC reduced cell cycle transcripts and increased the expression of differentiation markers. These data suggest that PDGF-BB overexpression in p53 null OPC results in histone post-translational modifications and consequent transcriptional changes favoring proliferation while halting differentiation, thereby promoting the early stages of transformation.

To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX&#95;001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in Mycobacterium tuberculosis . This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal in vitro against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as M. tuberculosis strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to in vitro killing of NTM, ENTX&#95;001 demonstrates the rescue of infected macrophages from necrotic death by Mycobacteroides abscessus and Mycobacterium avium . Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as Mycobacteroides abscessus , Mycobacterium avium , Mycobacterium intracellulare, as well as Mycobacterium tuberculosis . One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics., Competing Interests: All Endolytix employees may hold stock and/or stock options.

Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric contraction. While patient studies have defined the genetic landscape of DCM, genetics are not currently used in patient care, and patients receive similar treatments regardless of the underlying mutation. It has been suggested that a precision medicine approach based on the molecular mechanism of the underlying mutation could improve outcomes; however, realizing this approach has been challenging due to difficulties linking genotype and phenotype and then leveraging this information to identify therapeutic approaches. Here, we used multiscale experimental and computational approaches to test whether knowledge of molecular mechanism could be harnessed to connect genotype, phenotype, and drug response for a DCM mutation in troponin T, deletion of K210. Previously, we showed that at the molecular scale, the mutation reduces thin filament activation. Here, we used computational modeling of this molecular defect to predict that the mutant will reduce cellular and tissue contractility, and we validated this prediction in human cardiomyocytes and engineered heart tissues. We then used our knowledge of molecular mechanism to computationally model the effects of a small molecule that can activate the thin filament. We demonstrate experimentally that the modeling correctly predicts that the small molecule can partially rescue systolic dysfunction at the expense of diastolic function. Taken together, our results demonstrate how molecular mechanism can be harnessed to connect genotype and phenotype and inspire strategies to optimize mechanism-based therapeutics for DCM., Competing Interests: Conflict of interest statement: All experiments were conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression 1 . These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues 2-7 . One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation 3,8 . As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen 9-11 , we aimed to define how microbes impact the host intestinal chromatin landscape, mainly in female mice. Here we show that in addition to acetylation, intestinal epithelial cells from the caecum and distal mouse intestine also harbour high levels of butyrylation and propionylation on lysines 9 and 27 of histone H3. We demonstrate that these acylations are regulated by the microbiota and that histone butyrylation is additionally regulated by the metabolite tributyrin. Tributyrin-regulated gene programmes are correlated with histone butyrylation, which is associated with active gene-regulatory elements and levels of gene expression. Together, our study uncovers a regulatory layer of how the microbiota and metabolites influence the intestinal epithelium through chromatin, demonstrating a physiological setting in which histone acylations are dynamically regulated and associated with gene regulation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx 6 K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes., (© 2024. The Author(s).)

Early-life stress increases sensitivity to subsequent stress, which has been observed among humans, other animals, at the level of cellular activity, and at the level of gene expression. However, the molecular mechanisms underlying such long-lasting sensitivity are poorly understood. We tested the hypothesis that persistent changes in transcription and transcriptional potential were maintained at the level of the epigenome, through changes in chromatin. We used a combination of bottom-up mass spectrometry, viral-mediated epigenome-editing, behavioral quantification, and RNA-sequencing in a mouse model of early-life stress, focusing on the ventral tegmental area (VTA), a brain region critically implicated in motivation, reward learning, stress response, and mood and drug disorders. We find that early-life stress in mice alters histone dynamics in VTA and that a majority of these modifications are associated with an open chromatin state that would predict active, primed, or poised gene expression, including enriched histone-3 lysine-4 methylation and the H3K4 monomethylase Setd7. Mimicking ELS through over-expression of Setd7 and enrichment of H3K4me1 in VTA recapitulates ELS-induced behavioral and transcriptional hypersensitivity to future stress. These findings enrich our understanding of the epigenetic mechanisms linking early-life environmental experiences to long-term alterations in stress reactivity within the brain's reward circuitry, with implications for understanding and potentially treating mood and anxiety disorders in humans.

Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA), which could increase sensitivity and simplify workflows, has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate its use, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed that GlycanDIA can distinguish glycan composition and isomers from N -glycans, O -glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile N -glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N -glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone., Competing Interests: Declaration of interests J. Mieczkowski is a co-founder of and shareholder in Genegoggle., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Regular exercise has been described to modify both the diversity and the relative abundance of certain bacterial taxa. To our knowledge, the effect of a cycling stage race, which entails extreme physiological and metabolic demands, on the gut microbiota composition and its metabolic activity has not been analysed., Objective: The aim of this cohort study was to analyse the dynamics of faecal microbiota composition and short-chain fatty acids (SCFAs) content of professional cyclists over a Grand Tour and their relationship with performance and dietary intake., Methods: 16 professional cyclists competing in La Vuelta 2019 were recruited. Faecal samples were collected at four time points: the day before the first stage (A); after 9 stages (B); after 15 stages (C); and on the last stage (D). Faecal microbiota populations and SCFA content were analysed using 16S rRNA sequencing and gas chromatography, respectively. A principal component analysis (PCA) followed by Generalised Estimating Equation (GEE) models were carried out to explore the dynamics of microbiota and SCFAs and their relationship with performance., Results: Bifidobacteriaceae , Coriobacteriaceae , Erysipelotrichaceae , and Sutterellaceae dynamics showed a strong final performance predictive value (r = 0.83, ranking, and r = 0.81, accumulated time). Positive correlations were observed between Coriobacteriaceae with acetate (r = 0.530) and isovalerate (r = 0.664) and between Bifidobacteriaceae with isobutyrate (r = 0.682). No relationship was observed between SCFAs and performance. The abundance of Erysipelotrichaceae at the beginning of La Vuelta was directly related to the previous intake of complex-carbohydrate-rich foods (r = 0.956), while during the competition, the abundance of Bifidobacteriaceae was negatively affected by the intake of simple carbohydrates from supplements (r = -0.650)., Conclusions: An ecological perspective represents more realistically the relationship between gut microbiota composition and performance compared to single-taxon approaches. The composition and periodisation of diet and supplementation during a Grand Tour, particularly carbohydrates, could be designed to modulate gut microbiota composition to allow better performance.

Protein translational control is critical for ensuring that the fetus develops correctly and that necessary organs and tissues are formed and functional. We developed an in utero method to quantify tissue-specific protein dynamics by monitoring amino acid incorporation into the proteome after pulse injection. Fetuses of pregnant mice were injected with isotopically labeled lysine and arginine via the vitelline vein at various embyonic days, and organs and tissues were harvested. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates were calculated between 3.81E-5 and 0.424 h -1 . We observed similar protein turnover profiles for analyzed organs (e.g., liver vs. brain); however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates, which correlated with known physiological changes during mouse development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sirtuins, a class of highly conserved histone/protein deacetylases, are heavily implicated in senescence and aging. The regulation of sirtuin proteins is tightly controlled both transcriptionally and translationally and via localization within the cell. While Sirtiun proteins are implicated with aging, how their levels are regulated during aging across cell types and eliciting tissue specific age-related cellular changes is unclear. Here, we demonstrate that SIRT7 is targeted for degradation during senescence and liver aging. To uncover the significance of SIRT7 loss, we performed proteomics analysis and identified a new SIRT7 interactor, the HMG box protein NUCKS1. We found that the NUCKS1 transcription factor is recruited onto chromatin during senescence and this is mediated by SIRT7 loss. Further, depletion of NUCKS1 delayed senescence upon DNA damage leading to reduction of inflammatory gene expression. Examination of NUCKS1 transcriptional regulation during senescence revealed gene targets of transcription factors NFKB1, RELA, and CEBPβ. Consistently, in both Sirt7 KO mouse liver and in naturally aged livers, Nucks1 was recruited to chromatin. Further, Nucks1 was bound at promoters and enhancers of age-related genes, including transcription factor Rela, and, moreover, these bound sites had increased accessibility during aging. Overall, our results uncover NUCKS1 as a novel interactor of SIRT7, and show that loss of SIRT7 during senescence and liver aging promotes NUCKS1 chromatin binding to regulate metabolic and inflammatory genes.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Proteolysis targeting chimera (PROTAC) is a state-of-the-art technology for ablating undruggable targets. A PROTAC degrader achieves targeted protein degradation (TPD) through the simultaneous binding of a protein of interest (POI) and an E3 ligase to form a ternary complex. A nanofibril-based PROTAC strategy to form a polynary (E3) m  : PROTAC : (POI) n complex has not been reported in the TPD field up to this point. A recent innovation shows that a POI ligand and E3 ligase ligand don't have to be within a fused degrader molecule. Instead, they can be recruited to cellular proximity by a self-assembly-driving peptide and click chemistry. The resulting nanofibrils can recruit multiple POI and E3 ligase molecules to form a polynary complex as a degradation center. The so-called Nano-PROTAC provides a novel approach for TPD in cancer therapy., (© 2023 Wiley-VCH GmbH.)

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.