RESUMO
The analysis of cell-free tumor DNA (ctDNA) and proteins in the blood of cancer patients potentiates a new generation of non-invasive diagnostics and treatment monitoring approaches. However, confident detection of these tumor-originating markers is challenging, especially in the context of brain tumors, in which extremely low amounts of these analytes circulate in the patient's plasma. Here, we applied a sensitive single-molecule technology to profile multiple histone modifications on millions of individual nucleosomes from the plasma of Diffuse Midline Glioma (DMG) patients. The system reveals epigenetic patterns that are unique to DMG, significantly differentiating this group of patients from healthy subjects or individuals diagnosed with other cancer types. We further develop a method to directly capture and quantify the tumor-originating oncoproteins, H3-K27M and mutant p53, from the plasma of children diagnosed with DMG. This single-molecule system allows for accurate molecular classification of patients, utilizing less than 1ml of liquid-biopsy material. Furthermore, we show that our simple and rapid detection strategy correlates with MRI measurements and droplet-digital PCR (ddPCR) measurements of ctDNA, highlighting the utility of this approach for non-invasive treatment monitoring of DMG patients. This work underscores the clinical potential of single-molecule-based, multi-parametric assays for DMG diagnosis and treatment monitoring.
RESUMO
The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.
Assuntos
Ácidos Nucleicos Livres , Neoplasias , Nucleossomos , Humanos , Biomarcadores Tumorais , Ácidos Nucleicos Livres/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Proteínas de Neoplasias/genética , Neoplasias/diagnóstico , Neoplasias/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Imagem Individual de MoléculaRESUMO
Cancer-associated mutations in genes encoding histones dramatically reshape chromatin and support tumorigenesis. Lysine to methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, known to abrogate polycomb repressive complex 2 (PRC2) activity. We applied single-molecule systems to image individual nucleosomes and delineate the combinatorial epigenetic patterns associated with H3-K27M expression. We found that chromatin marks on H3-K27M-mutant nucleosomes are dictated both by their incorporation preferences and by intrinsic properties of the mutation. Mutant nucleosomes not only preferentially bind PRC2 but also directly interact with MLL1, leading to genome-wide redistribution of H3K4me3. H3-K27M-mediated deregulation of repressive and active chromatin marks leads to unbalanced "bivalent" chromatin, which may support a poorly differentiated cellular state. This study provides evidence for a direct effect of H3-K27M oncohistone on the MLL1-H3K4me3 pathway and highlights the capability of single-molecule tools to reveal mechanisms of chromatin deregulation in cancer.
Assuntos
Neoplasias Encefálicas , Glioma , Histona-Lisina N-Metiltransferase , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Neoplasias Encefálicas/genética , Criança , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Glioma/genética , Glioma/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/imunologia , Imagem Individual de Molécula/métodos , Proteínas Virais/genética , Anticorpos Antivirais/sangue , Sequência de Bases , COVID-19/sangue , COVID-19/imunologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/normas , Teste Sorológico para COVID-19/normas , Ensaio de Imunoadsorção Enzimática , Humanos , Soros Imunes/química , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Nasofaringe/virologia , Poliproteínas/sangue , Poliproteínas/genética , RNA Viral/sangue , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade , Imagem Individual de Molécula/instrumentação , Proteínas Virais/sangueRESUMO
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
RESUMO
Root endodermis, the innermost cortical layer surrounding the root vasculature, serves as the foremost barrier to water, solutes, and nutrients taken up from soil. Endodermis barrier functionality is achieved via its hydrophobic coating of lignified Casparian strips and the suberin lamellae; nonetheless the regulatory mechanisms underlying endodermis suberization are still elusive. Here, we discovered that the Arabidopsis SUBERMAN (SUB) transcription factor controls the establishment of the root suberin lamellae. Transient expression of SUB in Nicotiana benthamiana leaves resulted in the induction of heterologous suberin genes, the accumulation of suberin-type monomers, and consequent deposition of suberin-like lamellae. We demonstrate that SUB exerts its regulatory roles by transactivating promoters of suberin genes. In Arabidopsis, SUB is expressed in patchy and continuous suberization root endodermal cells, and thus roots with higher or lower expression of SUB display altered suberin polymer deposition patterns and modified composition. While these changes did not interfere with Casparian strip formation they had a substantial effect on root uptake capacity, resulting in varied root and leaf ionomic phenotypes. Gene expression profiling revealed that SUB function impacts transcriptional networks associated with suberin, phenylpropanoids, lignin, and cuticular lipid biosynthesis, as well as root transport activities, hormone signalling, and cell wall modification. Our findings highlight SUB as a regulator of root endodermis suberization during normal development, and its characterization is thus a key step towards dissecting the molecular mechanisms partaking in root endodermal barrier functionalities.
