RESUMO
Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.
Assuntos
Nucléolo Celular/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Sondas Moleculares/química , Domínios Proteicos , Proteínas Repressoras/metabolismo , Metilação , Mieloma Múltiplo/metabolismo , Nucleossomos/metabolismoRESUMO
Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized 'codes' that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the 'histone code' concept and interrogate it at the nucleosome level.
Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , Código das Histonas , Cromatina , Processamento de Proteína Pós-Traducional , Peptídeos/metabolismoRESUMO
In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Nucleossomos , Metilação , AcetilaçãoRESUMO
BACKGROUND: Damage-associated molecular patterns (DAMPs) stimulate endothelial syndecan-1 shedding and neutrophil extracellular traps (NET) formation. The role of NETs in trauma and trauma-induced hypercoagulability is unknown. We hypothesized that trauma patients with accelerated thrombin generation would have increased NETosis and syndecan-1 levels. METHODS: In this pilot study, we analyzed 50 citrated plasma samples from 30 trauma patients at 0âh (nâ=â22) and 6âh (nâ=â28) from time of injury (TOI) and 21 samples from healthy volunteers, for a total of 71 samples included in analysis. Thrombin generation was quantified using calibrated automated thrombogram (CAT) and reported as lag time (LT), peak height (PH), and time to peak (ttPeak). Nucleosome calibrated (H3NUC) and free histone standardized (H3Free) ELISAs were used to quantify NETs. Syndecan-1 levels were quantified by ELISA. Results are presented as median [interquartile range] and Spearman rank correlations. RESULTS: Plasma levels of H3NUC were increased in trauma patients as compared with healthy volunteers both at 0âh (89.8âng/mL [35.4, 180.3]; 18.1âng/mL [7.8, 37.4], Pâ=â0.002) and at 6âh (86.5âng/mL [19.2, 612.6]; 18.1âng/mL [7.8, 37.4], Pâ=â0.003) from TOI. H3Free levels were increased in trauma patients at 0âh (5.74âng/mL [3.19, 8.76]; 1.61âng/mL [0.66, 3.50], Pâ=â0.002) and 6âh (5.52âng/mL [1.46, 11.37]; 1.61âng/mL [0.66, 3.50], Pâ=â0.006). Syndecan-1 levels were greater in trauma patients (4.53âng/mL [3.28, 6.28]; 2.40âng/mL [1.66, 3.20], Pâ<â0.001) only at 6âh from TOI. H3Free and syndecan-1 levels positively correlated both at 0âh (0.376, Pâ=â0.013) and 6âh (0.583, Pâ<â0.001) from TOI. H3NUC levels and syndecan-1 levels were positively correlated at 6âh from TOI (0.293, Pâ=â0.041). TtPeak correlated inversely to H3 NUC (-0.358, Pâ=â0.012) and syndecan-1 levels (-0.298, Pâ=â0.038) at 6âh from TOI. CONCLUSIONS: Our pilot study demonstrates that trauma patients have increased NETosis, measured by H3NUC and H3Free levels, increased syndecan-1 shedding, and accelerated thrombin generation kinetics early after injury.
Assuntos
Armadilhas Extracelulares/fisiologia , Sindecana-1/sangue , Trombina/metabolismo , Ferimentos e Lesões/sangue , Adulto , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Fatores de Tempo , Ferimentos e Lesões/complicaçõesRESUMO
BACKGROUND: Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. OBJECTIVES: We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. METHODS: We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. RESULTS: We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. CONCLUSIONS: Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.
Assuntos
Armadilhas Extracelulares , Histonas , Bioensaio , Humanos , Nucleossomos , Plasma , Processamento de Proteína Pós-TraducionalRESUMO
Cationic gold(I) complexes containing 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (1), [AuL(1)](n+) (where L is Cl(-), Br(-), SCN(-), PEt(3), PPh(3), or 1), derived from a class of analogous platinum(II) antitumor agents, have been synthesized. Unlike platinum, gold does not form permanent adducts with DNA, and its complexes are 2 orders of magnitude less cytotoxic in non-small-cell lung cancer cells than the most active platinum-based agent. Instead, several gold analogues show submicromolar and selective antimicrobial activity against Mycobacterium tuberculosis.