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1.
Eur J Histochem ; 66(1)2022 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-35212500

RESUMO

The spread technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and chromatin in an isolated and distended conformation. The final step of staining the spread chromatin is of critical importance because it can strongly influence the interpretation of the results. We evaluated different staining techniques and the most part of them provided a good result. Specifically, well contrasted micrographs were obtained when staining with H3PW12O40 (PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate staining. Quite good contrast of the spread DNA could be achieved also by using Osmium Ammine; while no or few contrast of nucleic acids was observed by staining with KMnO4 and H3PMo12O40 (PMA) respectively.


Assuntos
Ácidos Nucleicos , Cromatina , Corantes , Microscopia Eletrônica , Coloração e Rotulagem
2.
Methods Mol Biol ; 2418: 203-221, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35119668

RESUMO

Estrogen regulates transcription through two nuclear receptors, ERα and ERß, in a tissue and cellular-dependent manner. Both the receptors bind estrogen and activate transcription through direct or indirect interactions with DNA. Revealing their interactions with the chromatin is key to understanding their transcriptional activities and their biological functions. Chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful technique to map protein-DNA interactions at precise genomic locations. The genome-wide binding of ERα has been extensively studied. Similar studies of ERß, however, have been more difficult, in part due to a lack of endogenous expression in cell lines and lack of specific antibodies. In this chapter, we provide an optimized stepwise ChIP protocol for a well-validated ERß antibody, which is applicable for ChIP-Seq analysis of cell lines with exogenous expression of ERß.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Receptor beta de Estrogênio , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Estrogênios
3.
Epigenomics ; 14(5): 243-259, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35184600

RESUMO

Introduction: Genome-wide association studies (GWAS) have identified numerous stroke-associated SNPs. To understand how SNPs affect gene expression related to increased stroke risk, we studied epigenetic landscapes surrounding 26 common, validated stroke-associated loci. Methods: We mapped the SNPs to linkage disequilibrium (LD) blocks and examined H3K27ac, H3K4me1, H3K9ac, and H3K4me3 histone marks and transcription-factor binding-sites in pathologically relevant cell types (hematopoietic and vascular cells). Hi-C data were used to identify topologically associated domains (TADs) encompassing the LD blocks and overlapping genes. Results: Fibroblasts, smooth muscle, and endothelial cells showed significant enrichment for enhancer-associated marks within stroke-associated LD blocks. Genes within encompassing TADs reflected vessel homeostasis, cellular turnover, and enzymatic activity. Conclusions: Stroke-associated genetic variants confer risk predominantly through vascular cells rather than hematopoietic cell types.


Previous studies have found several variations in the DNA sequence (known as single nucleotide polymorphisms) linked to higher stroke risk. But the mechanisms behind how they increase risk is unknown. One hypothesis is that they affect non-coding DNA elements (i.e., epigenetics), which in turn drive abnormal changes in gene expression leading to increased stroke risk. To investigate this potential mechanism, we mined publicly available, cell-type specific databases. We searched for overlap between the regions with polymorphisms and regions where DNA transcription machinery bind (i.e., enhancers, transcription factor binding sites). We found that fibroblasts and smooth muscle cells (cells in vessel walls) had more of these DNA elements in regions associated with stroke risk. Bioinformatics analyses of genes that could be affected by changes in these elements were linked to stroke-related mechanisms.


Assuntos
Cromatina , Estudo de Associação Genômica Ampla , Cromatina/genética , Células Endoteliais , Elementos Facilitadores Genéticos , Haplótipos , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único
4.
J Vis Exp ; (182)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35532241

RESUMO

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility. With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 °C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.


Assuntos
Infertilidade , Espermatozoides , Biomarcadores , Cromatina , Citometria de Fluxo/métodos , Humanos , Masculino , Metaloproteinases da Matriz
5.
J Vis Exp ; (182)2022 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-35532268

RESUMO

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.


Assuntos
Cromatina , Histonas , Epigenômica/métodos , Código das Histonas , Histonas/genética , Histonas/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo
6.
Adv Protein Chem Struct Biol ; 130: 189-243, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35534108

RESUMO

Histone modifying enzymes regulate chromatin architecture through covalent modifications and ultimately control multiple aspects of cellular function. Disruption of histone modification leads to changes in gene expression profiles and may lead to disease. Both small molecule inhibitors and intermediary metabolites have been shown to modulate histone modifying enzyme activity although our ability to identify successful drug candidates or novel metabolic regulators of these enzymes has been limited. Using a combination of large scale in silico screens and in vivo phenotypic analysis, we identified several small molecules and intermediary metabolites with distinctive HME activity. Our approach using unsupervised learning identifies the chemical fingerprints of both small molecules and metabolites that facilitate recognition by the enzymes active sites which can be used as a blueprint to design novel inhibitors. Furthermore, this work supports the idea that histone modifying enzymes sense intermediary metabolites integrating genes, environment and cellular physiology.


Assuntos
Cromatina , Histonas , Montagem e Desmontagem da Cromatina , Desenho de Fármacos , Histonas/metabolismo , Processamento de Proteína Pós-Traducional
7.
JCO Clin Cancer Inform ; 6: e2100156, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35522898

RESUMO

PURPOSE: Allogenic hematopoietic stem-cell transplant (HCT) is a curative therapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Relapse post-HCT is the most common cause of treatment failure and is associated with a poor prognosis. Pathologist-based visual assessment of aspirate images and the manual myeloblast counting have shown to be predictive of relapse post-HCT. However, this approach is time-intensive and subjective. The premise of this study was to explore whether computer-extracted morphology and texture features from myeloblasts' chromatin patterns could help predict relapse and prognosticate relapse-free survival (RFS) after HCT. MATERIALS AND METHODS: In this study, Wright-Giemsa-stained post-HCT aspirate images were collected from 92 patients with AML/MDS who were randomly assigned into a training set (St = 52) and a validation set (Sv = 40). First, a deep learning-based model was developed to segment myeloblasts. A total of 214 texture and shape descriptors were then extracted from the segmented myeloblasts on aspirate slide images. A risk score on the basis of texture features of myeloblast chromatin patterns was generated by using the least absolute shrinkage and selection operator with a Cox regression model. RESULTS: The risk score was associated with RFS in St (hazard ratio = 2.38; 95% CI, 1.4 to 3.95; P = .0008) and Sv (hazard ratio = 1.57; 95% CI, 1.01 to 2.45; P = .044). We also demonstrate that this resulting signature was predictive of AML relapse with an area under the receiver operating characteristic curve of 0.71 within Sv. All the relevant code is available at GitHub. CONCLUSION: The texture features extracted from chromatin patterns of myeloblasts can predict post-HCT relapse and prognosticate RFS of patients with AML/MDS.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Cromatina , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Aprendizado de Máquina , Síndromes Mielodisplásicas/terapia , Recidiva
8.
Methods Mol Biol ; 2477: 129-147, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524116

RESUMO

Mapping the epigenome is key to describe the relationship between chromatin landscapes and the control of DNA-based cellular processes such as transcription. Cleavage under targets and release using nuclease (CUT&RUN) is an in situ chromatin profiling strategy in which controlled cleavage by antibody-targeted Micrococcal Nuclease solubilizes specific protein-DNA complexes for paired-end DNA sequencing. When applied to budding yeast, CUT&RUN profiling yields precise genome-wide maps of histone modifications, histone variants, transcription factors, and ATP-dependent chromatin remodelers, while avoiding cross-linking and solubilization issues associated with the most commonly used chromatin profiling technique Chromatin Immunoprecipitation (ChIP). Furthermore, targeted chromatin complexes cleanly released by CUT&RUN can be used as input for a subsequent native immunoprecipitation step (CUT&RUN.ChIP) to simultaneously map two epitopes in single molecules genome-wide. The intrinsically low background and high resolution of CUT&RUN and CUT&RUN.ChIP allows for identification of transient genomic features such as dynamic nucleosome-remodeling intermediates. Starting from cells, one can perform CUT&RUN or CUT&RUN.ChIP and obtain purified DNA for sequencing library preparation in 2 days.


Assuntos
Epigenoma , Saccharomycetales , Cromatina , Imunoprecipitação da Cromatina , DNA/genética , Endonucleases/genética , Nuclease do Micrococo , Nucleossomos/genética , Saccharomycetales/genética
9.
Methods Mol Biol ; 2477: 149-175, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524117

RESUMO

Chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) is a powerful method to identify protein interactions, and has long been used to gain insights into regulatory networks in relevant fungal species as well as many other organisms. In this chapter, we discuss a similar technique called ChIP-SICAP (chromatin immunoprecipitation with selective isolation of chromatin-associated proteins) that overcomes many of the traditional limitations of ChIP-MS, and describe a protocol that allows ChIP-SICAP to be applied to Candida albicans and other yeasts. Notably, the technique design permits stringent washing to remove contaminating proteins and antibodies before subsequent mass spectrometry processing, allows for genome-wide mapping of the bait protein by ChIP-seq after ChIP-SICAP from the same sample through a DNA recovery process, and specifically purifies and identifies proteins associating with chromatin. In the future, ChIP-SICAP will provide the yeast genomics research community an additional method to explore the complex dynamics of the gene-regulatory networks modulating morphology, metabolism and response to stress.


Assuntos
Candida albicans , Leveduras , Candida albicans/genética , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Redes Reguladoras de Genes , Leveduras/genética
10.
BMC Biol ; 20(1): 99, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35524220

RESUMO

BACKGROUND: The three-dimensional (3D) architecture of the genome has a highly ordered and hierarchical nature, which influences the regulation of essential nuclear processes at the basis of gene expression, such as gene transcription. While the hierarchical organization of heterochromatin and euchromatin can underlie differences in gene expression that determine evolutionary differences among species, the way 3D genome architecture is affected by evolutionary forces within major lineages remains unclear. Here, we report a comprehensive comparison of 3D genomes, using high resolution Hi-C data in fibroblast cells of fish, chickens, and 10 mammalian species. RESULTS: This analysis shows a correlation between genome size and chromosome length that affects chromosome territory (CT) organization in the upper hierarchy of genome architecture, whereas lower hierarchical features, including local transcriptional availability of DNA, are selected through the evolution of vertebrates. Furthermore, conservation of topologically associating domains (TADs) appears strongly associated with the modularity of expression profiles across species. Additionally, LINE and SINE transposable elements likely contribute to heterochromatin and euchromatin organization, respectively, during the evolution of genome architecture. CONCLUSIONS: Our analysis uncovers organizational features that appear to determine the conservation and transcriptional regulation of functional genes across species. These findings can guide ongoing investigations of genome evolution by extending our understanding of the mechanisms shaping genome architecture.


Assuntos
Cromatina , Heterocromatina , Animais , Galinhas/genética , Elementos de DNA Transponíveis , Eucromatina/genética , Heterocromatina/genética , Mamíferos/genética , Vertebrados/genética
11.
Epigenetics Chromatin ; 15(1): 14, 2022 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-35526078

RESUMO

In eukaryotes, DNA is packaged into chromatin, which presents significant barriers to transcription. Non-histone chromatin proteins such as the Heterochromatin Protein 1 (HP1) proteins are critical regulators of transcription, contributing to gene regulation through a variety of molecular mechanisms. HP1 proteins are highly conserved, and many eukaryotic genomes contain multiple HP1 genes. Given the presence of multiple HP1 family members within a genome, HP1 proteins can have unique as well as shared functions. Here, we review the mechanisms by which HP1 proteins contribute to the regulation of transcription. Focusing on the Drosophila melanogaster HP1 proteins, we examine the role of these proteins in regulating the transcription of genes, transposable elements, and piRNA clusters. In D. melanogaster, as in other species, HP1 proteins can act as transcriptional repressors and activators. The available data reveal that the precise impact of HP1 proteins on gene expression is highly context dependent, on the specific HP1 protein involved, on its protein partners present, and on the specific chromatin context the interaction occurs in. As a group, HP1 proteins utilize a variety of mechanisms to contribute to transcriptional regulation, including both transcriptional (i.e. chromatin-based) and post-transcriptional (i.e. RNA-based) processes. Despite extensive studies of this important protein family, open questions regarding their functions in gene regulation remain, specifically regarding the role of hetero- versus homodimerization and post-translational modifications of HP1 proteins.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo
12.
Commun Biol ; 5(1): 362, 2022 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-35501457

RESUMO

Deficiency of the immune checkpoint lymphocyte activation gene-3 (LAG3) protein is significantly associated with both elevated HDL-cholesterol (HDL-C) and myocardial infarction risk. We determined the association of genetic variants within ±500 kb of LAG3 with plasma LAG3 and defined LAG3-associated plasma proteins with HDL-C and clinical outcomes. Whole genome sequencing and plasma proteomics were obtained from the Multi-Ethnic Study of Atherosclerosis (MESA) and the Framingham Heart Study (FHS) cohorts as part of the Trans-Omics for Precision Medicine program. In situ Hi-C chromatin capture was performed in EBV-transformed cell lines isolated from four MESA participants. Genetic association analyses were performed in MESA using multivariate regression models, with validation in FHS. A LAG3-associated protein network was tested for association with HDL-C, coronary heart disease, and all-cause mortality. We identify an association between the LAG3 rs3782735 variant and plasma LAG3 protein. Proteomics analysis reveals 183 proteins significantly associated with LAG3 with four proteins associated with HDL-C. Four proteins discovered for association with all-cause mortality in FHS shows nominal associations in MESA. Chromatin capture analysis reveals significant cis interactions between LAG3 and C1S, LRIG3, TNFRSF1A, and trans interactions between LAG3 and B2M. A LAG3-associated protein network has significant associations with HDL-C and mortality.


Assuntos
Aterosclerose , Medicina de Precisão , HDL-Colesterol , Cromatina , Humanos , Ativação Linfocitária , Proteínas de Membrana
13.
BMC Genomics ; 23(1): 337, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35501690

RESUMO

BACKGROUND: The cohesin complex is essential for proper chromosome structure and gene expression. Defects in cohesin subunits and regulators cause changes in cohesin complex dynamics and thereby alter three-dimensional genome organization. However, the molecular mechanisms that drive cohesin localization and function remain poorly understood. RESULTS: In this study, we observe that loss of WIZ causes changes to cohesin localization that are distinct from loss of the known WIZ binding partner G9a. Whereas loss of WIZ uniformly increases cohesin levels on chromatin at known binding sites and leads to new, ectopic cohesin binding sites, loss of G9a does not. Ectopic cohesin binding on chromatin after the loss of WIZ occurs at regions that are enriched for activating histone modifications and transcription factors motifs. Furthermore, loss of WIZ causes changes in cohesin localization that are distinct from those observed by loss of WAPL, the canonical cohesin unloading factor. CONCLUSIONS: The evidence presented here suggests that WIZ can function independently from its previously identified role with G9a and GLP in heterochromatin formation. Furthermore, while WIZ limits the levels and localization pattern of cohesin across the genome, it appears to function independently of WAPL-mediated cohesin unloading.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo
14.
BMC Genomics ; 23(1): 338, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35501711

RESUMO

BACKGROUND: Gram-negative bacteria are important pathogens in cattle, causing severe infectious diseases, including mastitis. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and crucial mediators of chronic inflammation in cattle. LPS modulations of bovine immune responses have been studied before. However, the single-cell transcriptomic and chromatin accessibility analyses of bovine peripheral blood mononuclear cells (PBMCs) and their responses to LPS stimulation were never reported. RESULTS: We performed single-cell RNA sequencing (scRNA-seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) in bovine PBMCs before and after LPS treatment and demonstrated that seven major cell types, which included CD4 T cells, CD8 T cells, and B cells, monocytes, natural killer cells, innate lymphoid cells, and dendritic cells. Bioinformatic analyses indicated that LPS could increase PBMC cell cycle progression, cellular differentiation, and chromatin accessibility. Gene analyses further showed significant changes in differential expression, transcription factor binding site, gene ontology, and regulatory interactions during the PBMC responses to LPS. Consistent with the findings of previous studies, LPS induced activation of monocytes and dendritic cells, likely through their upregulated TLR4 receptor. NF-κB was observed to be activated by LPS and an increased transcription of an array of pro-inflammatory cytokines, in agreement that NF-κB is an LPS-responsive regulator of innate immune responses. In addition, by integrating LPS-induced differentially expressed genes (DEGs) with large-scale GWAS of 45 complex traits in Holstein, we detected trait-relevant cell types. We found that selected DEGs were significantly associated with immune-relevant health, milk production, and body conformation traits. CONCLUSION: This study provided the first scRNAseq and scATAC-seq data for cattle PBMCs and their responses to the LPS stimulation to the best of our knowledge. These results should also serve as valuable resources for the future study of the bovine immune system and open the door for discoveries about immune cell roles in complex traits like mastitis at single-cell resolution.


Assuntos
Lipopolissacarídeos , Mastite , Animais , Bovinos , Cromatina/genética , Cromatina/metabolismo , Feminino , Humanos , Imunidade Inata , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Linfócitos/metabolismo , NF-kappa B/metabolismo , Transcriptoma
15.
Genome Biol ; 23(1): 109, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35501845

RESUMO

The precise spatiotemporal gene expression is orchestrated by enhancers that lack general sequence features and thus are difficult to be computationally identified. By nascent RNA sequencing combined with epigenome profiling, we detect active transcription of enhancers from the complex bread wheat genome. We find that genes associated with transcriptional enhancers are expressed at significantly higher levels, and enhancer RNA is more precise and robust in predicting enhancer activity compared to chromatin features. We demonstrate that sub-genome-biased enhancer transcription could drive sub-genome-biased gene expression. This study highlights enhancer transcription as a hallmark in regulating gene expression in wheat.


Assuntos
Pão , Triticum , Cromatina/genética , Elementos Facilitadores Genéticos , Transcriptoma , Triticum/genética
16.
Cell Rep ; 39(5): 110769, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35508135

RESUMO

Distinguishing between conserved and divergent regulatory mechanisms is essential for translating preclinical research from mice to humans, yet there is a lack of information about how evolutionary genome rearrangements affect the regulation of the immune response, a rapidly evolving system. The current model is topologically associating domains (TADs) are conserved between species, buffering evolutionary rearrangements and conserving long-range interactions within a TAD. However, we find that TADs frequently span evolutionary translocation and inversion breakpoints near genes with species-specific expression in immune cells, creating unique enhancer-promoter interactions exclusive to the mouse or human genomes. This includes TADs encompassing immune-related transcription factors, cytokines, and receptors. For example, we uncover an evolutionary rearrangement that created a shared LPS-inducible regulatory module between OASL and P2RX7 in human macrophages that is absent in mice. Therefore, evolutionary genome rearrangements disrupt TAD boundaries, enabling sequence-conserved enhancer elements from divergent genomic locations between species to create unique regulatory modules.


Assuntos
Cromatina , Genoma Humano , Animais , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Rearranjo Gênico/genética , Genômica , Humanos , Camundongos
18.
Front Immunol ; 13: 864314, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35514969

RESUMO

Pathogenic Th17, featured by their production of pro-inflammatory cytokines, are considered as a key player in most autoimmune diseases. The transcriptome of them is obviously distinct from that of conventional regulatory Th17. However, chromatin accessibility of the two Th17 groups have not been comprehensively compared yet. Here, we found that their chromatin-accessible regions(ChARs) significantly correlated with the expression of related genes, indicating that they might engage in the regulation of these genes. Indeed, pathogenic Th17 specific ChARs (patho-ChARs) exhibited a significant distribution preference in TSS-proximal region. We further filtered the patho-ChARs based on their conservation among mammalians or their concordance with the expression of their related genes. In either situation, the filtered patho-ChARs also showed a preference for TSS-proximal region. Enrichment of expression concordant patho-ChARs related genes suggested that they might involve in the pathogenicity of Th17. Thus, we also examined all ChARs of patho-ChARs related genes, and defined an opening ChAR set according to their changes in the Th17 to Th1 conversion. Interestingly, these opening ChARs displayed a sequential accessibility change from TSS-proximal region to TSS-distal region. Meanwhile, a group of patho-TFs (transcription factors) were identified based on the appearance of their binding motifs in the opening ChARs. Consistently, some of them also displayed a similar preference for binding the TSS-proximal region. Single-cell transcriptome analysis further confirmed that these patho-TFs were involved in the generation of pathogenic Th17. Therefore, our results shed light on a new regulatory mechanism underlying the generation of pathogenic Th17, which is worth to be considered for autoimmune disease therapy.


Assuntos
Doenças Autoimunes , Cromatina , Animais , Cromatina/genética , Cromatina/metabolismo , Mamíferos/genética , Células Th17 , Fatores de Transcrição/metabolismo , Virulência
19.
Commun Biol ; 5(1): 433, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35538147

RESUMO

The DNA sensor cGAS detects cytosolic DNA and instigates type I interferon (IFN) expression. Recent studies find that cGAS also localizes in the nucleus and binds the chromatin. Despite the mechanism controlling nuclear cGAS activation is well elucidated, whether nuclear cGAS participates in DNA sensing is unclear. Here, we report that herpes simplex virus 1 (HSV-1) infection caused the release of cGAS from the chromatin into the nuclear soluble fraction. Like its cytosolic counterpart, the leaked nuclear soluble cGAS also sensed viral DNA, produced cGAMP, and induced mRNA expression of type I IFN and interferon-stimulated genes. Consistently, the nuclear soluble cGAS limited HSV-1 infection. Furthermore, enzyme-deficient mutation (D307A) or cGAS inhibitor RU.251 abolished nuclear cGAS-mediated innate immune responses, suggesting that enzymatic activity is also required for nuclear soluble cGAS. Taken all together, our study demonstrates that nuclear soluble cGAS acts as a nuclear DNA sensor detecting nuclear-replicating DNA viruses.


Assuntos
Infecções por Vírus de DNA , Vírus de DNA , Nucleotidiltransferases , Cromatina , DNA/genética , DNA/metabolismo , Infecções por Vírus de DNA/genética , Infecções por Vírus de DNA/metabolismo , Infecções por Vírus de DNA/virologia , Vírus de DNA/genética , Vírus de DNA/metabolismo , Herpes Simples/genética , Humanos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
20.
J Hematol Oncol ; 15(1): 49, 2022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-35509102

RESUMO

Chromatin has distinct three-dimensional (3D) architectures important in key biological processes, such as cell cycle, replication, differentiation, and transcription regulation. In turn, aberrant 3D structures play a vital role in developing abnormalities and diseases such as cancer. This review discusses key 3D chromatin structures (topologically associating domain, lamina-associated domain, and enhancer-promoter interactions) and corresponding structural protein elements mediating 3D chromatin interactions [CCCTC-binding factor, polycomb group protein, cohesin, and Brother of the Regulator of Imprinted Sites (BORIS) protein] with a highlight of their associations with cancer. We also summarise the recent development of technologies and bioinformatics approaches to study the 3D chromatin interactions in gene expression regulation, including crosslinking and proximity ligation methods in the bulk cell population (ChIA-PET and HiChIP) or single-molecule resolution (ChIA-drop), and methods other than proximity ligation, such as GAM, SPRITE, and super-resolution microscopy techniques.


Assuntos
Cromatina , Neoplasias , Regulação da Expressão Gênica , Humanos , Masculino , Neoplasias/genética , Regiões Promotoras Genéticas
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