Ben Garcia

Background: Rhabdomyosarcoma (RMS) is an aggressive pediatric solid tumor with poor survival and new targeted therapies are needed. Aims: Using an integrative proteogenomic quantitative approach, we sought to identify candidate immunotherapeutic targets then validate top targets in RMS preclinical models. Methods: We first performed plasma membrane enrichment followed by mass spectrometry (MS) to define the surfaceome of 7 fusion-positive (FP) and 14 fusion-negative (FN) RMS patient-derived xenograft models. Surface proteins were scored by IMMUNOTAR, our custom pipeline which extracts and integrates quantitative features from diverse proteogenomic and annotation datasets to provide a score for each protein, using mean-average-precision for score optimization. Cell surface expression of IMMUNOTAR-prioritized proteins was validated using flow cytometry, focusing on targets with …

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and …

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 (Gx6K) 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 …

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.

Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease disrupting lung health throughout the life of an individual and that is increasing in incidence. The TGF-β superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that TGFbr2 is critical for alveolar epithelial (AT1) cell fate maintenance and function. Loss of TGFbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analyses reveal the necessity of TGFbr2 expression in AT1 cells for extracellular matrix production. Moreover, TGF-β signaling regulates integrin …

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.

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 …

(Cell 186, 305–326. e1–e14; January 19, 2023) Our paper used a system called “ICE”(inducible changes to the epigenome) to study whether a loss of epigenetic information leads to aging and whether the expression of a subset of Yamanka factors can reverse these age-associated changes, as tests of the Information Theory of Aging. We provide additional information about our experimental design and reference previous, relevant papers from our group and others that had not been cited in the original submission. We apologize for any confusion that may have arisen due to this information not being available in the original published paper. In our Correction, we add details about the transgenic construct design, tamoxifen administration, and temporal and spatial control of I-PpoI in the Results, Discussion, and STAR Methods sections. Our I-PpoI papers looking at the genotoxic stress response in DNA-damage-sensitive cell types have been referenced. The article has now been corrected online, and the corrected texts are provided below. In the Results section, the original sentence read:“To test our hypothesis in vivo, we performed whole-body I-PpoI expression in 4-to 6-month-old mice for 3 weeks (Figure 1L).” The corrected sentence now reads:“To test our hypothesis in vivo, we performed whole-body I-PpoI expression in 4-to 6-month-old mice for 3 weeks by providing TAM in the diet, rather than TAM intraperitoneal injections, to limit the amount of I-PpoI expressed so genotoxic stress would not arise (Figure 1L).” In the Discussion section, the original sentence read:“… In this paper, we show that non-mutagenic DSB repair…” The corrected …

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 …

Loss of epigenetic information as a cause of mammalian aging Loss of epigenetic information as a cause of mammalian aging Cell. 2024 Feb 29;187(5):1312-1313. doi: 10.1016/j.cell.2024.01.049. Authors Jae-Hyun Yang, Motoshi Hayano, Patrick T Griffin, João A Amorim, Michael S Bonkowski, John K Apostolides, Elias L Salfati, Marco Blanchette, Elizabeth M Munding, Mital Bhakta, Yap Ching Chew, Wei Guo, Xiaojing Yang, Sun Maybury-Lewis, Xiao Tian, Jaime M Ross, Giuseppe Coppotelli, Margarita V Meer, Ryan Rogers-Hammond, Daniel L Vera, Yuancheng Ryan Lu, Jeffrey W Pippin, Michael L Creswell, Zhixun Dou, Caiyue Xu, Sarah J Mitchell, Abhirup Das, Brendan L O'Connell, Sachin Thakur, Alice E Kane, Qiao Su, Yasuaki Mohri, Emi K Nishimura, Laura Schaevitz, Neha Garg, Ana-Maria Balta, Meghan A Rego, Meredith Gregory-Ksander, Tatjana C Jakobs, Lei Zhong, Hiroko Wakimoto, Jihad El Andari, …

Dysregulation of histone acetylation in the brain has emerged as a major contributor to human Alzheimer's disease (AD). The mechanisms by which these protective or risk-conferring epigenetic marks are established and maintained are under intense investigation. ACSS2 (Acetyl-CoA Synthetase 2) is a key metabolic enzyme that is chromatin-associated in neurons. ACSS2 is recruited to specific promoters and generates a local pool of acetyl-CoA from acetate, thereby fueling histone acetylation and driving the expression of neuronal genes that regulate learning and memory. Here, we examine the contribution of ACSS2-mediated histone acetylation to AD-related molecular and behavioral outcomes. Using a mouse model of human pathological AD-Tau injection, we show that loss of ACSS2 exacerbates Tau-related memory impairments, while dietary supplementation of acetate rescues learning in an ACSS2-dependent manner. Combining state-of-the-art proteomic and genomic approaches, we demonstrate that this effect is accompanied by ACSS2-dependent incorporation of acetate into hippocampal histone acetylation, which facilitates gene expression programs related to learning. Further, we identify Cajal-Retzius neurons as a critical hippocampal neuronal population affected, exhibiting the largest epigenetic and transcriptional dysregulation. Overall, these results reveal ACSS2 as a key neuroprotective metabolic enzyme, dysregulation of which might play an important role in the etiology of human AD, and guide the development of future therapies for AD and related dementia.

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression. These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues, , , , –. One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation,. As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen, –, 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 …

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 …

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 …

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.

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 …

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 …

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning …

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 …

The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed …