RESUMO
We present a highly sensitive and selective chemical labeling and capture approach for genome-wide profiling of 5-hydroxylmethylcytosine (5hmC) using DNA isolated from â¼1,000 cells (nano-hmC-Seal). Using this technology, we assessed 5hmC occupancy and dynamics across different stages of hematopoietic differentiation. Nano-hmC-Seal profiling of purified Tet2-mutant acute myeloid leukemia (AML) murine stem cells allowed us to identify leukemia-specific, differentially hydroxymethylated regions that harbor known and candidate disease-specific target genes with differential 5hmC peaks compared to normal stem cells. The change of 5hmC patterns in AML strongly correlates with differential gene expression, demonstrating the importance of dynamic alterations of 5hmC in regulating transcription in AML. Together, covalent 5hmC labeling offers an effective approach to study and detect DNA methylation dynamics in in vivo disease models and in limited clinical samples.
Assuntos
5-Metilcitosina/análogos & derivados , Metilação de DNA , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Promielocítica Aguda/genética , 5-Metilcitosina/metabolismo , Animais , Células Cultivadas , Biologia Computacional , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Dioxigenases , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Estudo de Associação Genômica Ampla , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Mutação , Nanotecnologia , Proteínas Proto-Oncogênicas/genética , Fatores de Tempo , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as 'transcription bubbles'. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.
Assuntos
Aldeídos/química , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Animais , Butanonas , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Transcrição GênicaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Plants, as sessile organisms, deploy transcriptional dynamics for adapting to extreme growth conditions such as cold stress. Emerging evidence suggests that chromatin architecture contributes to transcriptional regulation. However, the relationship between chromatin architectural dynamics and transcriptional reprogramming in response to cold stress remains unclear. Here, we apply a chemical-crosslinking assisted proximity capture (CAP-C) method to elucidate the fine-scale chromatin landscape, revealing chromatin interactions within gene bodies closely associated with RNA polymerase II (Pol II) densities across initiation, pausing, and termination sites. We observe dynamic changes in chromatin interactions alongside Pol II activity alterations during cold stress, suggesting local chromatin dynamics may regulate Pol II activity. Notably, cold stress does not affect large-scale chromatin conformations. We further identify a comprehensive promoter-promoter interaction (PPI) network across the genome, potentially facilitating co-regulation of gene expression in response to cold stress. Our study deepens the understanding of chromatin conformation-associated gene regulation in plant response to cold.
Assuntos
Arabidopsis , Cromatina , Cromatina/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Regiões Promotoras Genéticas/genética , Transcrição GênicaRESUMO
Sialic acid analogues containing a unique chemical functionality or chemical reporter have been metabolically incorporated into sialylated glycans. This process, termed metabolic glycan labeling, has emerged as a powerful tool for studying sialylation as well as other types of glycosylation. Currently, this technique can install only a single functionality. Here we describe a strategy for dual labeling of sialylated glycans using a new class of bifunctional sialic acid analogues containing two distinct chemical reporters at the N-acyl and C9 positions. These bifunctional unnatural sialic acids were metabolically incorporated into cellular glycans, where the two chemical reporters exerted their distinct functions. This approach expands the capability of metabolic glycan labeling to probe sialylation and glycan-protein interactions.
Assuntos
Polissacarídeos/metabolismo , Ácidos Siálicos/metabolismo , Configuração de Carboidratos , Células HeLa , Humanos , Polissacarídeos/química , Ácidos Siálicos/síntese química , Ácidos Siálicos/química , Propriedades de SuperfícieRESUMO
Azacitidine (AZA) is a DNA methyltransferase inhibitor and epigenetic modulator that can be an effective agent in combination with chemotherapy for patients with high-risk acute myeloid leukemia (AML). However, biological factors driving the therapeutic response of such hypomethylating agent (HMA)-based therapies remain unknown. Herein, the transcriptome and/or genome-wide 5-hydroxymethylcytosine (5hmC) is characterized for 41 patients with high-risk AML from a phase 1 clinical trial treated with AZA epigenetic priming followed by high-dose cytarabine and mitoxantrone (AZA-HiDAC-Mito). Digital cytometry reveals that responders have elevated Granulocyte-macrophage-progenitor-like (GMP-like) malignant cells displaying an active cell cycle program. Moreover, the enrichment of natural killer (NK) cells predicts a favorable outcome in patients receiving AZA-HiDAC-Mito therapy or other AZA-based therapies. Comparing 5hmC profiles before and after five-day treatment of AZA shows that AZA exposure induces dose-dependent 5hmC changes, in which the magnitude correlates with overall survival (p = 0.015). An extreme gradient boosting (XGBoost) machine learning model is developed to predict the treatment response based on 5hmC levels of 11 genes, achieving an area under the curve (AUC) of 0.860. These results suggest that cellular composition markedly impacts the treatment response, and showcase the prospect of 5hmC signatures in predicting the outcomes of HMA-based therapies in AML.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/uso terapêutico , Azacitidina/efeitos adversos , Citarabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Ensaios Clínicos Fase I como AssuntoRESUMO
Determining the spatial organization of chromatin in cells mainly relies on crosslinking-based chromosome conformation capture techniques, but resolution and signal-to-noise ratio of these approaches is limited by interference from DNA-bound proteins. Here we introduce chemical-crosslinking assisted proximity capture (CAP-C), a method that uses multifunctional chemical crosslinkers with defined sizes to capture chromatin contacts. CAP-C generates chromatin contact maps at subkilobase (sub-kb) resolution with low background noise. We applied CAP-C to formaldehyde prefixed mouse embryonic stem cells (mESCs) and investigated loop domains (median size of 200 kb) and nonloop domains (median size of 9 kb). Transcription inhibition caused a greater loss of contacts in nonloop domains than loop domains. We uncovered conserved, transcription-state-dependent chromatin compartmentalization at high resolution that is shared from Drosophila to human, and a transcription-initiation-dependent nuclear subcompartment that brings multiple nonloop domains in close proximity. We also showed that CAP-C could be used to detect native chromatin conformation without formaldehyde prefixing.
Assuntos
Cromatina/metabolismo , Reagentes de Ligações Cruzadas/química , DNA/metabolismo , Transcrição Gênica , Animais , Fator de Ligação a CCCTC/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Inibidores Enzimáticos/farmacologia , Genoma , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genéticaRESUMO
The 5-methylcytosines (5mC) have been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the role of 5-hydroxymethylcytosines (5hmC) that are generated from 5mC through active demethylation, in lymphomagenesis is unknown. We profiled genome-wide 5hmC in circulating cell-free DNA (cfDNA) from 73 newly diagnosed patients with DLBCL and FL. We identified 294 differentially modified genes between DLBCL and FL. The differential 5hmC in the DLBCL/FL-differentiating genes co-localized with enhancer marks H3K4me1 and H3K27ac. A four-gene panel (CNN2, HMG20B, ACRBP, IZUMO1) robustly represented the overall 5hmC modification pattern that distinguished FL from DLBCL with an area under curve of 88.5% in the testing set. The median 5hmC modification levels in signature genes showed potential for separating patients for risk of all-cause mortality. This study provides evidence that genome-wide 5hmC profiles in cfDNA differ between DLBCL and FL and could be exploited as a non-invasive approach.
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An elevated level of circulating cell-free DNA (cfDNA) has been associated with tumor bulk and poor prognosis in diffuse large B-cell lymphoma (DLBCL), but the tumor-specific molecular alterations in cfDNA with prognostic significance remain unclear. We investigated the association between 5-hydroxymethylcytosines (5hmC), a mark of active demethylation and gene activation, in cfDNA from blood plasma and prognosis in newly diagnosed DLBCL patients. We used 5hmC-Seal, a highly sensitive chemical labeling technique, to profile genome-wide 5hmC in plasma cfDNA from 48 DLBCL patients at the University of Chicago Medical Center between 2010 and 2013. Patients were followed through 31 December 2017. We found a distinct genomic distribution of 5hmC in cfDNA marking tissue-specific enhancers, consistent with their putative roles in gene regulation. The 5hmC profiles in cfDNA differed by cell of origin and were associated with clinical prognostic factors, including stage and the International Prognostic Index. We developed a 29 gene-based weighted prognostic score (wp-score) for predicting event-free survival (EFS) and overall survival (OS) by applying the elastic net regularization on the Cox proportional-hazards model. The wp-scores outperformed (eg, prognostic accuracy, sensitivity, specificity) established prognostic factors in predicting EFS and OS. In multivariate Cox models, patients with high wp-scores had worse EFS (hazard ratio, 9.17; 95% confidence interval, 2.01-41.89; P = .004) compared with those in the low-risk group. Our findings suggest that the 5hmC signatures in cfDNA at the time of diagnosis are associated with clinical outcomes and may provide a novel minimally invasive prognostic approach for DLBCL.
Assuntos
5-Metilcitosina/análogos & derivados , Ácidos Nucleicos Livres/metabolismo , Linfoma Difuso de Grandes Células B/genética , 5-Metilcitosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Linfoma Difuso de Grandes Células B/patologia , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Adulto JovemRESUMO
Neu5Ac, Neu5Gc, and KDN are three forms of sialic acids in vertebrates that possess distinct biological functions. Herein, we report the synthesis and metabolic incorporation of the 9-azido analogues of three sialic acid forms in mammalian cells. The incorporated sialic acid analogues enable fluorescent imaging of cell-surface sialoglycans and proteomic profiling of sialoglycoproteins. Furthermore, we apply them to metabolically engineer cell surfaces with sialoglycans terminated with distinct sialic acids or their 9-azido analogues. The remodeled cells expressing specific cell-surface sialoglycoforms show distinct binding affinity toward subtilase cytotoxin (SubAB), a toxin secreted by Shiga toxigenic Escherichia coli. The 9-azido analogues of sialic acid forms developed in this work provide a versatile tool for metabolic remodeling of cell-surface properties and modulating pathogen-host interactions.
Assuntos
Azidas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ácidos Siálicos/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Células CHO , Engenharia Celular/métodos , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetulus , Proteínas de Escherichia coli/metabolismo , Humanos , Glicoproteínas de Membrana/química , Proteômica , Sialoglicoproteínas/química , Subtilisinas/metabolismo , Células VeroRESUMO
Most low GC Gram-positive bacteria possess an essential walKR two-component system (TCS) for signal transduction involved in regulating cell wall homoeostasis. Despite the well-established intracellular regulatory mechanism, the role of this TCS in extracellular signal recognition and factors that modulate the activity of this TCS remain largely unknown. Here we identify the extracellular receptor of the kinase 'WalK' (erWalK) as a key hub for bridging extracellular signal input and intracellular kinase activity modulation in Staphylococcus aureus. Characterization of the crystal structure of erWalK revealed a canonical Per-Arnt-Sim (PAS) domain for signal sensing. Single amino-acid mutation of potential signal-transduction residues resulted in severely impaired function of WalKR. A small molecule derived from structure-based virtual screening against erWalK is capable of selectively activating the walKR TCS. The molecular level characterization of erWalK will not only facilitate exploration of natural signal(s) but also provide a template for rational design of erWalK inhibitors.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transdução de Sinais , Staphylococcus aureus/metabolismo , Benzofenonas/farmacologia , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Mutação/genética , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus/genética , Staphylococcus aureus/ultraestrutura , Transcrição Gênica/efeitos dos fármacosRESUMO
Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.