PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression.

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.

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA.

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 (acp3U) 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.

An integrated structural model of the DNA damage-responsive H3K4me3 binding WDR76:SPIN1 complex with the nucleosome.

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.

Bottom-up Histone Post-translational Modification Analysis using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Tandem Mass Spectrometry.

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/K0) 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, me1-3) 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.

Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation.

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.

A multiomic atlas of the aging hippocampus reveals molecular changes in response to environmental enrichment.

Aging involves the deterioration of organismal function, leading to the emergence of multiple pathologies. Environmental stimuli, including lifestyle, can influence the trajectory of this process and may be used as tools in the pursuit of healthy aging. To evaluate the role of epigenetic mechanisms in this context, we have generated bulk tissue and single cell multi-omic maps of the male mouse dorsal hippocampus in young and old animals exposed to environmental stimulation in the form of enriched environments. We present a molecular atlas of the aging process, highlighting two distinct axes, related to inflammation and to the dysregulation of mRNA metabolism, at the functional RNA and protein level. Additionally, we report the alteration of heterochromatin domains, including the loss of bivalent chromatin and the uncovering of a heterochromatin-switch phenomenon whereby constitutive heterochromatin loss is partially mitigated through gains in facultative heterochromatin. Notably, we observed the multi-omic reversal of a great number of aging-associated alterations in the context of environmental enrichment, which was particularly linked to glial and oligodendrocyte pathways. In conclusion, our work describes the epigenomic landscape of environmental stimulation in the context of aging and reveals how lifestyle intervention can lead to the multi-layered reversal of aging-associated decline.

SMYD5 methylation of rpL40 links ribosomal output to gastric cancer.

Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis1,2. 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.

Identifying Sample Provenance From SEM/EDS Automated Particle Analysis via Few-Shot Learning Coupled With Similarity Graph Clustering.

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.

Discovery of a Highly Potent and Selective HDAC8 Degrader: Advancing the Functional Understanding and Therapeutic Potential of HDAC8.

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.

ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart.

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.

Lineage-specific proteome remodeling of diverse lung cancer cells by targeted epigenetic inhibitors.

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 drugs. To determine the mechanisms that drive these heterogeneous cellular responses, we quantified chromatin, proteome, and transcriptome remodeling due to histone deacetylase inhibitor (HDACi) -treated cells derived from diverse genetic backgrounds. We utilized high-throughput sample multiplexed proteomics and integrated intelligent data acquisition methods to map proteomes of cancer cell lines in response to HDACi. We determined cell type-specific and ubiquitous cellular responses based on the quantification of 10,621 total proteins. We then established how coordinated remodeling of the proteome, transcriptome and chromatin state of HDACi treated cancer cells revealed convergent (JUN, MAP2K3, CDKN1A) and divergent (CCND3, ASF1B, BRD7) molecular phenotypes. HDACi-regulated proteins differ greatly across cell lines owing to heterogeneous molecular states of these cell lines. Finally, we demonstrated that HDACi treatment drove a highly cell-type specific response that may in part be explained by cell line-specific off-target drug engagement.

Next-generation sequencing reveals that miR-16-5p, miR-19a-3p, miR-451a, and miR-25-3p cargo in plasma extracellular vesicles differentiates sedentary young males from athletes.

A sedentary lifestyle and Olympic participation are contrary risk factors for global mortality and incidence of cancer and cardiovascular disease. Extracellular vesicle miRNAs have been described to respond to exercise. No molecular characterization of young male sedentary people versus athletes is available; so, our aim was to identify the extracellular vesicle miRNA profile of chronically trained young endurance and resistance male athletes compared to their sedentary counterparts. A descriptive case-control design was used with 16 sedentary young men, 16 Olympic male endurance athletes, and 16 Olympic male resistance athletes. Next-generation sequencing and RT-qPCR and external and internal validation were performed in order to analyze extracellular vesicle miRNA profiles. Endurance and resistance athletes had significant lower levels of miR-16-5p, miR-19a-3p, and miR-451a compared to sedentary people. Taking all together, exercise-trained miRNA profile in extracellular vesicles provides a differential signature of athletes irrespective of the type of exercise compared to sedentary people. Besides, miR-25-3p levels were specifically lower in endurance athletes which defines its role as a specific responder in this type of athletes. In silico analysis of this profile suggests a role in adaptive energy metabolism in this context that needs to be experimentally validated. Therefore, this study provides for the first time basal levels of circulating miRNA in extracellular vesicles emerge as relevant players in intertissue communication in response to chronic exercise exposure in young elite male athletes.

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling.

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.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation.

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.

Chromatin profiling identifies putative dual roles for H3K27me3 in regulating transposons and cell type-specific genes in choanoflagellates.

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.

An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS.

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.

Structure of the Hir histone chaperone complex.

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.

Improved detection of DNA replication fork-associated proteins.

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.

PDGF-BB overexpression in p53 null oligodendrocyte progenitors increases H3K27me3 and induces transcriptional changes which favor proliferation.

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.

Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T.

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.