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1.
J Vis Exp ; (175)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34605807

RESUMO

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.


Assuntos
Cromatina , Drosophila , Animais , Núcleo Celular , Drosophila/genética , Drosophila melanogaster/genética , Genômica
2.
Curr Protoc ; 1(10): e266, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34644460

RESUMO

Genome-wide mapping of transcription factors and chromatin regulators is important to distinguish their direct from indirect effects on gene transcription or chromatin function. Novel approaches for studying their genomic localization under native conditions, such us cleavage under target and release using nuclease (CUT&RUN), offer higher resolution and lower sequencing costs than classical chromatin immunoprecipitation (ChIP) assays, and require fewer cells but they still depend on the availability of high-quality antibodies. Here, we describe detailed and robust protocols for greenCUT&RUN, which is a generic CUT&RUN-based approach for mapping the genome-wide localization of green fluorescent protein (GFP)-tagged factors in intact mammalian cells. The greenCUT&RUN method makes use of a micrococcal nuclease (MNase) coupled to a high affinity nanobody against GFP, which exploits the accessibility of multiple surfaces of the GFP tag, thus eliminating issues of antibody variability and availability. We also provide efficient protocols for the expression and purification of two different GFP nanobodies, which recognize non-overlapping GFP epitopes and can be combined for a further gain in sensitivity and accuracy. Compared to traditional CUT&RUN, genomic localization by greenCUT&RUN reduces handling time and experimental variability. GreenCUT&RUN is a versatile, robust, and universal procedure for surveying the genome-wide localization of GFP-tagged versions of proteins that drive key transcriptional programs and regulate chromatin function. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Standard greenCUT&RUN for GFP-tagged proteins in mammalian cells Alternate Protocol: High-Ca++ /low-salt greenCUT&RUN for GFP-tagged histone proteins in mammalian cells Support Protocol: Expression and purification of GFP nanobody-MNase fusion proteins for greenCUT&RUN.


Assuntos
Cromatina , Fatores de Transcrição , Animais , Cromatina/genética , Imunoprecipitação da Cromatina , Genômica , Proteínas de Fluorescência Verde/genética
3.
BMC Genomics ; 22(1): 751, 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34666684

RESUMO

BACKGROUND: The transcription of developmental regulatory genes is often controlled by multiple cis-regulatory elements. The identification and functional characterization of distal regulatory elements remains challenging, even in tractable model organisms like sea urchins. RESULTS: We evaluate the use of chromatin accessibility, transcription and RNA Polymerase II for their ability to predict enhancer activity of genomic regions in sea urchin embryos. ATAC-seq, PRO-seq, and Pol II ChIP-seq from early and late blastula embryos are manually contrasted with experimental cis-regulatory analyses available in sea urchin embryos, with particular attention to common developmental regulatory elements known to have enhancer and silencer functions differentially deployed among embryonic territories. Using the three functional genomic data types, machine learning models are trained and tested to classify and quantitatively predict the enhancer activity of several hundred genomic regions previously validated with reporter constructs in vivo. CONCLUSIONS: Overall, chromatin accessibility and transcription have substantial power for predicting enhancer activity. For promoter-overlapping cis-regulatory elements in particular, the distribution of Pol II is the best predictor of enhancer activity in blastula embryos. Furthermore, ATAC- and PRO-seq predictive value is stage dependent for the promoter-overlapping subset. This suggests that the sequence of regulatory mechanisms leading to transcriptional activation have distinct relevance at different levels of the developmental gene regulatory hierarchy deployed during embryogenesis.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Sequências Reguladoras de Ácido Nucleico , Animais , Cromatina/genética , Regiões Promotoras Genéticas , Ouriços-do-Mar/genética
4.
Nat Commun ; 12(1): 5732, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593797

RESUMO

Although alterations in chromatin structure are known to exist in tumors, how these alterations relate to molecular phenotypes in cancer remains to be demonstrated. Multi-omics profiling of human tumors can provide insight into how alterations in chromatin structure are propagated through the pathway of gene expression to result in malignant protein expression. We applied multi-omics profiling of chromatin accessibility, RNA abundance, and protein abundance to 36 human thyroid cancer primary tumors, metastases, and patient-match normal tissue. Through quantification of chromatin accessibility associated with active transcription units and global protein expression, we identify a local chromatin structure that is highly correlated with coordinated RNA and protein expression. In particular, we identify enhancers located within gene-bodies as predictive of correlated RNA and protein expression, that is independent of overall transcriptional activity. To demonstrate the generalizability of these findings we also identify similar results in an independent cohort of human breast cancers. Taken together, these analyses suggest that local enhancers, rather than distal enhancers, are likely most predictive of cancer gene expression phenotypes. This allows for identification of potential targets for cancer therapeutic approaches and reinforces the utility of multi-omics profiling as a methodology to understand human disease.


Assuntos
Neoplasias da Mama/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Mama/patologia , Sequenciamento de Cromatina por Imunoprecipitação , Estudos de Coortes , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos , Epigênese Genética , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Regiões Promotoras Genéticas , Proteômica , RNA/metabolismo , RNA-Seq , Câncer Papilífero da Tireoide/patologia , Câncer Papilífero da Tireoide/cirurgia , Glândula Tireoide/patologia , Glândula Tireoide/cirurgia , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgia , Tireoidectomia , Fatores de Transcrição/metabolismo
5.
Nat Commun ; 12(1): 5756, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599163

RESUMO

The discovery that overexpressing one or a few critical transcription factors can switch cell state suggests that gene regulatory networks are relatively simple. In contrast, genome-wide association studies (GWAS) point to complex phenotypes being determined by hundreds of loci that rarely encode transcription factors and which individually have small effects. Here, we use computer simulations and a simple fitting-free polymer model of chromosomes to show that spatial correlations arising from 3D genome organisation naturally lead to stochastic and bursty transcription as well as complex small-world regulatory networks (where the transcriptional activity of each genomic region subtly affects almost all others). These effects require factors to be present at sub-saturating levels; increasing levels dramatically simplifies networks as more transcription units are pressed into use. Consequently, results from GWAS can be reconciled with those involving overexpression. We apply this pan-genomic model to predict patterns of transcriptional activity in whole human chromosomes, and, as an example, the effects of the deletion causing the diGeorge syndrome.


Assuntos
Redes Reguladoras de Genes , Genoma Humano , Modelos Genéticos , Fatores de Transcrição/metabolismo , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos/química , Cromossomos Humanos/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Polímeros/química , Polímeros/metabolismo , Locos de Características Quantitativas , Transcrição Genética
6.
Nat Commun ; 12(1): 5775, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599169

RESUMO

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.


Assuntos
Carcinoma Neuroendócrino/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Fatores de Transcrição/genética
7.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3061-3070, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622617

RESUMO

The study of distinct genes, chromosomes and the spatio-temporal relationships between them is of great significance in genetics, developmental biology and biomedicine. CRISPR/Cas9 has become the most widely used gene editing tool due to its excellent targeting ability. Recently, researchers have developed a series of advanced live cell imaging techniques based on the nuclease-inactivated mutant of Cas9 (dCas9), providing rapid and convenient tools for high-resolution imaging of specific sites in the chromatin and genome. This review summarizes the advances of CRISPR/dCas9 system in live cell imaging from three aspects, including the strategies of cell delivery, optimization of the fluorescence signals, as well as orthogonal and multicolor imaging. Furthermore, we shed light on the development trends and prospects of this field.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Cromatina , Endonucleases
8.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3310-3322, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622638

RESUMO

The effect of altering the promoter region of ubiquitous chromatin-opening element (UCOE) and matrix attachment region (MAR) on stable and efficient expression of genes was investigated. Four different promoters were tested, namely, oct4 containing an enhancer region, sox2 having a CpG island, nanog having no regulatory elements, and CMV containing a CpG island and an enhancer region. Eight reporter plasmids were constructed: pOCT4-UCOE, pOCT4-MAR, pSOX2-UCOE, pSOX2-MAR, pNANOG-UCOE, pNANOG-MAR, pCMV-UCOE, and pCMV-MAR. Stable and efficient expression was observed when UCOE combined with the oct4 promoter, whereas the sox2 was the best promoter suited for MAR. Comparison of the stable clones of oct4-UCOE and sox2-MAR showed that UCOE-regulated expression is more stable and efficient than MAR-regulated expression. When CpG island-containing promoter is linked with UCOE, stable and efficient expression could be observed. These data suggest that an enhancer region in the promoter leads to high, yet unstable expression when combined with UCOE, whereas CpG islands stabilize expression. In conclusion, UCOE and MAR interact with regulatory elements on the promoter by altering the chromatin open state and chromatin loop to regulate gene expression.


Assuntos
Cromatina , Regulação da Expressão Gênica , Cromatina/genética , Ilhas de CpG/genética , Expressão Gênica , Regiões Promotoras Genéticas/genética
9.
Nat Commun ; 12(1): 5548, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545082

RESUMO

Isoniazid (INH) is a first-line anti-tuberculosis drug used for nearly 70 years. However, the mechanism underlying the side effects of INH has remained elusive. Here, we report that INH and its metabolites induce a post-translational modification (PTM) of histones, lysine isonicotinylation (Kinic), also called 4-picolinylation, in cells and mice. INH promotes the biosynthesis of isonicotinyl-CoA (Inic-CoA), a co-factor of intracellular isonicotinylation. Mass spectrometry reveals 26 Kinic sites in histones in HepG2 cells. Acetyltransferases CREB-binding protein (CBP) and P300 catalyse histone Kinic, while histone deacetylase HDAC3 functions as a deisonicotinylase. Notably, MNase sensitivity assay and RNA-seq analysis show that histone Kinic relaxes chromatin structure and promotes gene transcription. INH-mediated histone Kinic upregulates PIK3R1 gene expression and activates the PI3K/Akt/mTOR signalling pathway in liver cancer cells, linking INH to tumourigenicity in the liver. We demonstrate that Kinic is a histone acylation mark with a pyridine ring, which may have broad biological effects. Therefore, INH-induced isonicotinylation potentially accounts for the side effects in patients taking INH long-term for anti-tuberculosis therapy, and this modification may increase the risk of cancer in humans.


Assuntos
Antituberculosos/farmacologia , Código das Histonas , Isoniazida/farmacologia , Ácidos Isonicotínicos/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , Coenzima A/metabolismo , Células HeLa , Células Hep G2 , Histona Desacetilases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Ácidos Isonicotínicos/química , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo
10.
Nat Commun ; 12(1): 5533, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545091

RESUMO

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly in mice, Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. In Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold, indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression with unique spatial modularity in the limb, and are necessary for Lmx1b-mediated limb dorsalization. These enhancers, being conserved across vertebrates (including coelacanth, but not other fish species), and required for normal locomotion, provide a unique opportunity to study the role of dorsalization in the fin to limb transition. We also report on two NPS patient families with normal LMX1B coding sequence, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.


Assuntos
Extremidades/fisiopatologia , Proteínas com Homeodomínio LIM/metabolismo , Síndrome da Unha-Patela/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Galinhas , Cromatina/metabolismo , Feminino , Deleção de Genes , Genes Reporter , Homozigoto , Humanos , Masculino , Camundongos , Especificidade de Órgãos , Linhagem , Fenótipo
11.
Nat Commun ; 12(1): 5285, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489442

RESUMO

The mammalian DNA methylome is formed by two antagonizing processes, methylation by DNA methyltransferases (DNMT) and demethylation by ten-eleven translocation (TET) dioxygenases. Although the dynamics of either methylation or demethylation have been intensively studied in the past decade, the direct effects of their interaction on gene expression remain elusive. Here, we quantify the concurrence of DNA methylation and demethylation by the percentage of unmethylated CpGs within a partially methylated read from bisulfite sequencing. After verifying 'methylation concurrence' by its strong association with the co-localization of DNMT and TET enzymes, we observe that methylation concurrence is strongly correlated with gene expression. Notably, elevated methylation concurrence in tumors is associated with the repression of 40~60% of tumor suppressor genes, which cannot be explained by promoter hypermethylation alone. Furthermore, methylation concurrence can be used to stratify large undermethylated regions with negligible differences in average methylation into two subgroups with distinct chromatin accessibility and gene regulation patterns. Together, methylation concurrence represents a unique methylation metric important for transcription regulation and is distinct from conventional metrics, such as average methylation and methylation variation.


Assuntos
Metilação de DNA , Metilases de Modificação do DNA/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Genoma , Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Transcrição Genética , Animais , Cromatina/química , Cromatina/metabolismo , Ilhas de CpG , DNA/genética , DNA/metabolismo , Metilases de Modificação do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ontologia Genética , Histonas/genética , Histonas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Anotação de Sequência Molecular , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo
12.
Nat Commun ; 12(1): 5307, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489465

RESUMO

Prostate cancer is heterogeneous and patients would benefit from methods that stratify those who are likely to respond to systemic therapy. Here, we employ single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identify pre-existing and treatment-persistent cell subpopulations that possess regenerative potential when subjected to treatment. We find distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent cells are able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal as yet unrecognized molecular predictors of treatment response. This suggests that the application of single-cell methods with high analytical resolution in pre-clinical models may powerfully inform clinical decision-making.


Assuntos
Cromatina/química , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Transcriptoma , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Nitrilas/uso terapêutico , Feniltioidantoína/uso terapêutico , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Análise de Sobrevida , Sequenciamento Completo do Exoma
13.
Nat Commun ; 12(1): 5228, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471111

RESUMO

EpiScanpy is a toolkit for the analysis of single-cell epigenomic data, namely single-cell DNA methylation and single-cell ATAC-seq data. To address the modality specific challenges from epigenomics data, epiScanpy quantifies the epigenome using multiple feature space constructions and builds a nearest neighbour graph using epigenomic distance between cells. EpiScanpy makes the many existing scRNA-seq workflows from scanpy available to large-scale single-cell data from other -omics modalities, including methods for common clustering, dimension reduction, cell type identification and trajectory learning techniques, as well as an atlas integration tool for scATAC-seq datasets. The toolkit also features numerous useful downstream functions, such as differential methylation and differential openness calling, mapping epigenomic features of interest to their nearest gene, or constructing gene activity matrices using chromatin openness. We successfully benchmark epiScanpy against other scATAC-seq analysis tools and show its outperformance at discriminating cell types.


Assuntos
Epigenômica/métodos , Análise de Célula Única/métodos , Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Análise por Conglomerados , Metilação de DNA , Humanos , Análise de Sequência de RNA
14.
Nat Commun ; 12(1): 5380, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508092

RESUMO

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.


Assuntos
Anáfase/genética , Cromatina/metabolismo , Reparo do DNA , Pontos de Checagem da Fase M do Ciclo Celular/genética , Rad51 Recombinase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Segregação de Cromossomos , DNA/biossíntese , Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Humanos , Microscopia Intravital , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo
15.
Nat Immunol ; 22(10): 1327-1340, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34556886

RESUMO

During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE 'seeds' featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions.


Assuntos
Cromatina/imunologia , Imunidade Humoral/imunologia , Transportador 2 de Cátion Orgânico/imunologia , Domínios Proteicos/imunologia , Animais , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Epigenômica/métodos , Feminino , Genômica/métodos , Centro Germinativo/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/imunologia
16.
J Vis Exp ; (174)2021 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-34487116

RESUMO

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.


Assuntos
Dano ao DNA , Endonucleases , Cromatina , Imunoprecipitação da Cromatina , DNA/genética , Reparo do DNA , Endonucleases/genética
17.
Mol Cell ; 81(18): 3708-3730, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34547235

RESUMO

Lipids play crucial roles in signal transduction, contribute to the structural integrity of cellular membranes, and regulate energy metabolism. Questions remain as to which lipid species maintain metabolic homeostasis and which disrupt essential cellular functions, leading to metabolic disorders. Here, we discuss recent advances in understanding lipid metabolism with a focus on catabolism, synthesis, and signaling. Technical advances, including functional genomics, metabolomics, lipidomics, lipid-protein interaction maps, and advances in mass spectrometry, have uncovered new ways to prioritize molecular mechanisms mediating lipid function. By reviewing what is known about the distinct effects of specific lipid species in physiological pathways, we provide a framework for understanding newly identified targets regulating lipid homeostasis with implications for ameliorating metabolic diseases.


Assuntos
Metabolismo dos Lipídeos/fisiologia , Doenças Metabólicas/metabolismo , Transdução de Sinais/fisiologia , Animais , Cromatina/metabolismo , Doença , Metabolismo Energético/fisiologia , Saúde , Homeostase/fisiologia , Humanos , Imunidade/fisiologia , Lipidômica/métodos , Lipídeos/fisiologia , Doenças Metabólicas/fisiopatologia , Metabolômica/métodos , Microbiota/fisiologia
18.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502060

RESUMO

The transcriptome of every cell is orchestrated by the complex network of interaction between transcription factors (TFs) and their binding sites on DNA. Disruption of this network can result in many forms of organism malfunction but also can be the substrate of positive natural selection. However, understanding the specific determinants of each of these individual TF-DNA interactions is a challenging task as it requires integrating the multiple possible mechanisms by which a given TF ends up interacting with a specific genomic region. These mechanisms include DNA motif preferences, which can be determined by nucleotide sequence but also by DNA's shape; post-translational modifications of the TF, such as phosphorylation; and dimerization partners and co-factors, which can mediate multiple forms of direct or indirect cooperative binding. Binding can also be affected by epigenetic modifications of putative target regions, including DNA methylation and nucleosome occupancy. In this review, we describe how all these mechanisms have a role and crosstalk in one specific family of TFs, the basic helix-loop-helix (bHLH), with a very conserved DNA binding domain and a similar DNA preferred motif, the E-box. Here, we compile and discuss a rich catalog of strategies used by bHLH to acquire TF-specific genome-wide landscapes of binding sites.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Humanos , Ligação Proteica , Ativação Transcricional
19.
Nat Commun ; 12(1): 5450, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521827

RESUMO

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.


Assuntos
Carcinogênese/genética , Ciclina D1/genética , Ciclina D2/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Mieloma Múltiplo/genética , Transcriptoma , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Plasmócitos/metabolismo , Plasmócitos/patologia , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Sobrevida
20.
Nat Commun ; 12(1): 5579, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552088

RESUMO

Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid-liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.


Assuntos
Reprogramação Celular , Cromatina/metabolismo , DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , DNA/química , DNA/genética , Metilação de DNA , Humanos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Modelos Moleculares , Mutação , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição SOXB1/genética , Dedos de Zinco/genética
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