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ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Nucleossomos/química , Nucleossomos/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Código de Barras de DNA Taxonômico , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/genética , Subunidades Proteicas/metabolismoRESUMO
In eukaryotes, chromatin remodeling and post-translational modifications (PTMs) shape the local chromatin landscape to establish permissive and repressive regions within the genome, orchestrating transcription, replication, and DNA repair in concert with other epigenetic mechanisms. Though cellular nutrient signaling encompasses a huge number of pathways, recent attention has turned to the hypothesis that the metabolic state of the cell is communicated to the genome through the type and concentration of metabolites in the nucleus that are cofactors for chromatin-modifying enzymes. Importantly, both epigenetic and metabolic dysregulation are hallmarks of a range of diseases, and this metabolism-chromatin axis may yield a well of new therapeutic targets. In this Perspective, we highlight emerging themes in the inter-regulation of the genome and metabolism via chromatin, including nonenzymatic histone modifications arising from chemically reactive metabolites, the expansion of PTM diversity from cofactor-promiscuous chromatin-modifying enzymes, and evidence for the existence and importance of subnucleocytoplasmic metabolite pools.
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Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/genética , Cromatina/metabolismo , Eucariotos/metabolismo , Redes e Vias Metabólicas , Processamento de Proteína Pós-Traducional/fisiologia , Dano ao DNA , Reparo do DNA , Enzimas/metabolismo , Epigênese Genética , Histonas/metabolismo , HumanosRESUMO
A lysine-to-methionine mutation at lysine 27 of histone 3 (H3K27M) has been shown to promote oncogenesis in a subset of pediatric gliomas. While there is evidence that this "oncohistone" mutation acts by inhibiting the histone methyltransferase PRC2, the details of this proposed mechanism nevertheless continue to be debated. Recent evidence suggests that PRC2 must simultaneously bind both H3K27M and H3K27me3 to experience competitive inhibition of its methyltransferase activity. In this work, we used PRC2 inhibitor treatments in a transgenic H3K27M cell line to validate this dependence in a cellular context. We further used designer chromatin inhibitors to probe the geometric constraints of PRC2 engagement of H3K27M and H3K27me3 in a biochemical setting. We found that PRC2 binds to a bivalent inhibitor unit consisting of an H3K27M and an H3K27me3 nucleosome and exhibits a distance dependence in its affinity for such an inhibitor, which favors closer proximity of the 2 nucleosomes within a chromatin array. Together, our data precisely delineate fundamental aspects of the H3K27M inhibitor and support a model wherein PRC2 becomes trapped at H3K27M-H3K27me3 boundaries.
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Histonas/genética , Complexo Repressor Polycomb 2/fisiologia , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , Histona Metiltransferases/antagonistas & inibidores , Histona Metiltransferases/química , Histonas/química , Histonas/fisiologia , Humanos , Modelos Moleculares , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismoRESUMO
Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2), the enzyme that catalyzes monomethylation, dimethylation, and trimethylation of lysine 27 on histone H3 (H3K27). Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of developmentally important genes. Intriguingly, H3K27me3 is mutually exclusive with H3K36 trimethylation on the same histone tail. Disruptions in this cross-talk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of how PRC2 "senses" H3K36 methylation are unclear. We demonstrate that PRC2 is activated in cis by the unmodified side chain of H3K36, and that this activation results in a fivefold increase in the kcat of its enzymatic activity catalyzing H3K27 methylation compared with activity on a substrate methylated at H3K36. Using a photo-cross-linking MS strategy and histone methyltransferase activity assays on PRC2 mutants, we find that EZH2 contains a specific sensing pocket for the H3K36 methylation state that allows the complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. We also present evidence that this process may be disrupted in some cases of Weaver syndrome.
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Proteína Potenciadora do Homólogo 2 de Zeste , Histonas , Sítios de Ligação/genética , Proteína Potenciadora do Homólogo 2 de Zeste/química , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Recent studies report serine ADP-ribosylation on nucleosomes during the DNA damage response. We unveil histone H3 serine 10 as the primary acceptor residue for chromatin ADP-ribosylation and find that specific histone acetylation marks block this activity. Our results provide a molecular explanation for the well-documented phenomenon of rapid deacetylation at DNA damage sites and support the combinatorial application of PARP and HDAC inhibitors for the treatment of PARP-dependent cancers.
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ADP-Ribosilação , Cromatina/metabolismo , Dano ao DNA , Histonas/metabolismo , Acetilação , Clonagem Molecular , DNA/genética , DNA/metabolismo , Histonas/genética , Humanos , Células Tumorais CultivadasRESUMO
Polycomb Repressive Complex 2 (PRC2) catalyzes mono-, di-, and trimethylation of lysine 27 on histone H3 (H3K27me1-3) to control expression of genes important for differentiation and maintenance of cell identity. PRC2 activity is regulated by a number of different inputs, including allosteric activation by its product, H3K27me3. This positive feedback loop is thought to be important for the establishment of large domains of condensed heterochromatin. In addition to other chromatin modifications, ancillary subunits of PRC2, foremost JARID2, affect the rate of H3K27 methylation. Many gaps remain in our understanding of how PRC2 integrates these various signals to determine where and when to deposit H3K27 methyl marks. In this study, we utilize designer chromatin substrates to demonstrate that propagation of H3K27 methylation by the PRC2 core complex has geometrically defined preferences that are overridden by the presence of JARID2. Our studies also show that phosphorylation of JARID2 can partially regulate its ability to stimulate PRC2 activity. Collectively, these biochemical insights further our understanding of the mechanisms that govern PRC2 activity, and highlight a role for JARID2 in de novo deposition of H3K27me3-containing repressive domains.
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Heterocromatina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Biocatálise , Heterocromatina/química , Humanos , Cinética , Complexo Repressor Polycomb 2/químicaRESUMO
Glycerides are of interest to the areas of food science and medicine because they are the main component of fat. From a chemical sensing perspective, glycerides are challenging analytes because they are structurally similar to one another and lack diversity in terms of functional groups. Furthermore, because animal and plant fat consists of a number of stereo- and regioisomeric acylglycerols, their components remain challenging analytes for chromatographic and mass spectrometric determination, particularly the quantitation of species in mixtures. In this study, we demonstrated the use of an array of cross-reactive serum albumins and fluorescent indicators with chemometric analysis to differentiate a panel of mono-, di-, and triglycerides. Due to the difficulties in identifying the regio- and stereochemistry of the unsaturated glycerides, a sample pretreatment consisting of olefin cross-metathesis with an allyl fluorescein species was used before array analysis. Using this simple assay, we successfully discriminated 20 glycerides via principal component analysis and linear discriminant analysis (PCA and LDA, respectively), including stereo- and regioisomeric pairs. The resulting chemometric patterns were used as a training space for which the structural characteristics of unknown glycerides were identified. In addition, by using our array to perform a standard addition analysis on a mixture of triglycerides and using a method introduced herein, we demonstrated the ability to quantitate glyceride components in a mixture.
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Glicerídeos/química , Algoritmos , Alcenos/química , Animais , Diabetes Mellitus Tipo 2/metabolismo , Corantes Fluorescentes/química , Humanos , Metabolismo dos Lipídeos , Espectrometria de Massas , Obesidade/metabolismo , Análise de Componente Principal , Proteínas/química , Albumina Sérica/química , Estereoisomerismo , Triglicerídeos/químicaRESUMO
By mimicking the mammalian senses of taste and smell, artificial arrays of cross-reactive receptors have found use in a variety of sensing applications. Pattern recognition algorithms allow these arrays to be used for discriminating analytes and even for predicting the identity of unknown analytes. Furthermore, in selecting a signaling method for these assays, the choice of optical detection is particularly desirable due to its high sensitivity and the associated convenient instrumentation. This tutorial review provides a brief introduction to array sensing using optical detection and chemometrics. While differential sensing approaches have been used for a number of applications, this review focuses on progress towards the detection of chemical and biological hazards.
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Técnicas Biossensoriais , Análise em Microsséries , Imagem Óptica , Algoritmos , Bactérias/isolamento & purificação , Substâncias para a Guerra Química/análise , Substâncias Perigosas/análise , Resíduos Industriais/análise , Reconhecimento Automatizado de Padrão , Compostos Orgânicos Voláteis/análiseRESUMO
Background: SPARK launched in 2016 to build a US cohort of autistic individuals and their family members. Enrollment includes online consent to share data and optional consent to provide saliva for genomic analysis. SPARK's recruitment strategies include social media and support of a nation-wide network of clinical sites. This study evaluates SPARK's recruitment strategies to enroll a core study population. Methods: Individuals who joined between January 31, 2018, and May 29, 2019 were included in the analysis. Data include sociodemographic characteristics, clinical site referral, the website URL used to join, how the participant heard about SPARK, enrollment completion (online registration, study consents, and returning saliva sample), and completion of the baseline questionnaire. Logistic regressions were performed to evaluate the odds of core participant status (completing enrollment and baseline questionnaire) by recruitment strategy. Results: In total, 31,715 individuals joined during the study period, including 40% through a clinical site. Overall, 88% completed online registration, 46% returned saliva, and 38% were core participants. Those referred by a clinical site were almost twice as likely to be core participants. Those who directly visited the SPARK website or performed a Google search were more likely to be core participants than those who joined through social media. Discussion: Being a core participant may be associated with the "personal" connection and support provided by a clinical site and/or site staff, as well as greater motivation to seek research opportunities. Findings from this study underscore the value of adopting a multimodal recruitment approach that combines social media and a physical presence.
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Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA production and consumption are highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. In this work, we engineer an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). We biochemically characterize the sensor and demonstrate its selectivity for acetyl-CoA over other CoA species. We then deploy the biosensor in E. coli and HeLa cells to demonstrate its utility in living cells. In E. coli, we show that the biosensor enables detection of rapid changes in acetyl-CoA levels. In human cells, we show that the biosensor enables subcellular detection and reveals the compartmentalization of acetyl-CoA metabolism.
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Introduction: Under enrollment of participants in clinical research is costly and delays study completion to impact public health. Given that research personnel make decisions about which strategies to pursue and participants are the recipients of these efforts, we surveyed research staff (n = 52) and participants (n = 4,144) affiliated with SPARK (Simons Foundation Powering Autism for Knowledge) - the largest study of autism in the U.S. - to understand their perceptions of effective recruitment strategies. Methods: In Study 1, research personnel were asked to report recruitment strategies that they tried for SPARK and to indicate which ones they would and would not repeat/recommend. In Study 2, SPARK participants were asked to indicate all the ways they heard about the study prior to enrollment and which one was most influential in their decisions to enroll. Results: Staff rated speaking with a SPARK-study-team member (36.5%), speaking with a medical provider (19.2%), word of mouth (11.5%), and a live TV news story (11.5%) as the most successful strategies. Participants most often heard about SPARK via social media (47.0%), speaking with a medical provider (23.1%), and an online search (20.1%). Research personnel's and participants' views on effective recruitment strategies often differed, with the exception of speaking with a medical provider. Conclusion: Results suggest that a combination of strategies is likely to be most effective in reaching diverse audiences. Findings have implications for the selection of strategies that meet a study's specific needs, as well as recruitment-strategy "combinations" that may enhance the influence of outreach efforts.
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In eukaryotes, DNA is packaged with histone proteins in a complex known as chromatin. Both the DNA and histone components of chromatin can be chemically modified in a wide variety of ways, resulting in a complex landscape often referred to as the "epigenetic code". These modifications are recognized by effector proteins that remodel chromatin and modulate transcription, translation, and repair of the underlying DNA. In this Review, we examine the development of methods for characterizing proteins that interact with these histone and DNA modifications. "Mark first" approaches utilize chemical, peptide, nucleosome, or oligonucleotide probes to discover interactors of a specific modification. "Reader first" approaches employ arrays of peptides, nucleosomes, or oligonucleotides to profile the binding preferences of interactors. These complementary strategies have greatly enhanced our understanding of how chromatin modifications effect changes in genomic regulation, bringing us ever closer to deciphering this complex language.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Nucleossomos , DNA/metabolismo , GenômicaRESUMO
We report the characterization of the penilumamide biosynthetic cluster from Aspergillus flavipes CNL-338. In vitro reconstitution experiments demonstrated that three nonribosomal peptide synthetases are required for constructing the tripeptide and studies with dissected adenylation domains allowed for the first biochemical characterization of a domain that selects a pterin-derived building block.
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To explore issues surrounding re-consenting youth in longitudinal studies as they reach legal adulthood interviews were conducted with 46 parents plus 13 autistic teens enrolled in the Simons Foundation Powering Autism Research for Knowledge (SPARK) study. Qualitative analysis focused on family sensitivities regarding guardianship decisions, transition concerns, and the re-consenting process. Questions regarding guardianship were difficult for parents unsure of a teen's future status. Mothers were key facilitators of re-consenting for soon-to-be-independent teens. As legal adulthood approached, parents were willing to assist teens with re-consenting but needed support, asking for multiple contacts, transition resources, and explanatory materials from the research team. Most teens were not cognizant of SPARK but willing to continue participation once made aware.
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Because of the COVID-19 pandemic, in-person services for individuals with neurodevelopmental disabilities were disrupted globally, resulting in a transition to remote delivery of services and therapies. For individuals with neurogenetic conditions, reliance on nonclinical caregivers to facilitate all therapies and care was unprecedented. The study aimed to (1) describe caregivers' reported impact on their dependent's services, therapies, medical needs, and impact on themselves as a result of the COVID-19 pandemic and (2) assess the relationship between the extent of disruption of services and the degree of self-reported caregiver burden. Two online questionnaires were completed by caregivers participating in Simons Searchlight in April and May 2020. Surveys were completed by caregivers of children or dependent adults with neurodevelopmental genetic conditions in Simons Searchlight. Caregivers reported that the impact of the COVID-19 pandemic moderately or severely disrupted services, therapies, or medical supports. The majority of caregivers were responsible for providing some aspect of therapy. Caregivers reported "feeling stressed but able to deal with problems as they arise," and reported lower anxiety at follow-up. Caregivers reported that telehealth services were not meeting the needs of those with complex medical needs. Future surveys will assess if and how medical systems, educational programs, therapists, and caregivers adapt to the challenges arising during the COVID-19 pandemic.
Assuntos
COVID-19/psicologia , Sobrecarga do Cuidador/psicologia , Cuidadores/psicologia , Pesquisas sobre Atenção à Saúde/métodos , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Transtornos do Neurodesenvolvimento/terapia , Adolescente , Adulto , Cuidadores/estatística & dados numéricos , Criança , Pré-Escolar , Feminino , Pesquisas sobre Atenção à Saúde/estatística & dados numéricos , Humanos , Masculino , Avaliação das Necessidades , Pandemias , SARS-CoV-2 , Inquéritos e QuestionáriosRESUMO
Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP 'oncohistone-mimic', that dysregulate gene silencing to promote tumorigenesis.