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1.
Cell ; 152(3): 642-54, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23333102

RESUMO

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Epigênese Genética , Interação Gene-Ambiente , Estudo de Associação Genômica Ampla , Núcleo Celular , Senescência Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Especificidade de Órgãos
2.
Proc Natl Acad Sci U S A ; 121(34): e2402998121, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39133838

RESUMO

Significant racial disparities exist between Black and White patients with uterine serous carcinoma (USC). While the reasons for these disparities are unclear, several studies have demonstrated significantly different rates of driver mutations between racial groups, including TP53. However, limited research has investigated the transcriptional differences of tumors or the composition of the tumor microenvironment (TME) between these groups. Here, we report the single-nuclei RNA-sequencing profiles of primary USC tumors from diverse racial backgrounds. We find that there are significant differences between the tumors of Black and White patients. Tumors from Black patients exhibited higher expression of specific genes associated with aggressiveness, such as PAX8, and axon guidance and synaptic signaling pathways. We also demonstrated that T cell populations are reduced in the tumor tissue compared to matched benign, while anti-inflammatory macrophage populations are retained within the TME. Furthermore, we investigated the connection between PAX8 overexpression and immunosuppression in USC through regulation of several cytokines and chemokines. Notably, we show that PAX8 activity can influence macrophage gene expression and protein secretion. These studies provide a detailed understanding of the USC transcriptome and TME, and identify differences in tumor biology from patients of different racial backgrounds.


Assuntos
Fator de Transcrição PAX8 , Transdução de Sinais , Microambiente Tumoral , Neoplasias Uterinas , Humanos , Feminino , Microambiente Tumoral/imunologia , Microambiente Tumoral/genética , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Fator de Transcrição PAX8/genética , Fator de Transcrição PAX8/metabolismo , Transdução de Sinais/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/imunologia , Regulação Neoplásica da Expressão Gênica , População Branca/genética , Análise de Célula Única , Pessoa de Meia-Idade
3.
Proc Natl Acad Sci U S A ; 117(45): 28068-28079, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33097661

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Proteína-Arginina N-Metiltransferases , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Desenvolvimento de Medicamentos , Técnicas de Inativação de Genes , Humanos , Camundongos Nus , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
4.
Nature ; 539(7628): 242-247, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27830782

RESUMO

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.


Assuntos
Evolução Molecular , Proteínas Musculares/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Animais , Sequência de Bases , Osso e Ossos/metabolismo , Dendritos/metabolismo , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Fatores de Transcrição MEF2/metabolismo , Macaca mulatta , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculos/metabolismo , Neocórtex/citologia , Neurônios/citologia , Especificidade de Órgãos , Especificidade da Espécie , Fatores de Transcrição/genética
5.
Hum Mutat ; 42(10): 1208-1214, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34153138

RESUMO

Genome-wide association studies have identified SNPs associated with glioma risk on 9p21.3, but biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 9p21.3 affects activity of an enhancer, causing altered expression of nearby genes. We considered all SNPs in linkage disequilibrium with the 9p21.3 sentinel SNP rs634537 that mapped to putative enhancers. An enhancer containing rs1537372 exhibited allele-specific effects on luciferase activity. Deletion of this enhancer in GBM cell lines correlated with decreased expression of CDKN2B-AS1. Expression quantitative trait loci analysis using non-diseased brain samples showed rs1537372 to be a consistently significant eQTL for CDKN2B-AS1. Additionally, our analysis of Hi-C data generated in neural progenitor cells showed that the bait region containing rs1537372 interacted with the CDKN2B-AS1 promoter. These data suggest rs1537372, a SNP at the 9p21.3 risk locus, is a functional variant that modulates expression of CDKN2B-AS1.


Assuntos
Glioma , RNA Longo não Codificante , Elementos Facilitadores Genéticos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Glioma/genética , Humanos , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética
6.
Hum Mutat ; 42(1): 77-88, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33169458

RESUMO

Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with glioma risk on 20q13.33, but the biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 20q13.33 impacted the activity of an enhancer, leading to an altered expression of nearby genes. To identify candidate functional SNPs, we identified all SNPs in linkage disequilibrium with the risk-associated SNP rs2297440 that mapped to putative enhancers. Putative enhancers containing candidate functional SNPs were tested for allele-specific effects in luciferase enhancer activity assays against glioblastoma multiforme (GBM) cell lines. An enhancer containing SNP rs3761124 exhibited allele-specific effects on activity. Deletion of this enhancer by CRISPR-Cas9 editing in GBM cell lines correlated with an altered expression of multiple genes, including STMN3, RTEL1, RTEL1-TNFRSF6B, GMEB2, and SRMS. Expression quantitative trait loci (eQTL) analyses using nondiseased brain samples, isocitrate dehydrogenase 1 (IDH1) wild-type glioma, and neurodevelopmental tissues showed STMN3 to be a consistent significant eQTL with rs3761124. RTEL1 and GMEB2 were also significant eQTLs in the context of early CNS development and/or in IDH1 wild-type glioma. We provide evidence that rs3761124 is a functional variant on 20q13.33 related to glioma/GBM risk that modulates the expression of STMN3 and potentially other genes across diverse cellular contexts.


Assuntos
Estudo de Associação Genômica Ampla , Glioma , Alelos , Predisposição Genética para Doença , Glioma/genética , Glioma/metabolismo , Humanos , Polimorfismo de Nucleotídeo Único
7.
Nat Methods ; 14(7): 710-712, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28581493

RESUMO

CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Códon de Terminação/genética , Inativação Gênica , Códon sem Sentido , Regulação da Expressão Gênica , Marcação de Genes/métodos , Vetores Genéticos , Células HEK293 , Humanos , Plasmídeos
8.
Nature ; 489(7414): 155-9, 2012 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-22820254

RESUMO

The identification of somatic activating mutations in JAK2 (refs 1­4) and in the thrombopoietin receptor gene (MPL) in most patients with myeloproliferative neoplasm (MPN) led to the clinical development of JAK2 kinase inhibitors. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms but does not significantly decrease or eliminate the MPN clone in most patients with MPN. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic inhibition of JAK2. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK­STAT signalling and with heterodimerization between activated JAK2 and JAK1 or TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible: JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, in murine models and in patients treated with JAK2 inhibitors. RNA interference and pharmacological studies show that JAK2-inhibitor-persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.


Assuntos
Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Multimerização Proteica , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Granulócitos/efeitos dos fármacos , Granulócitos/enzimologia , Granulócitos/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Janus Quinase 1/biossíntese , Janus Quinase 1/deficiência , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/deficiência , Janus Quinase 2/genética , Camundongos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Fosforilação , Biossíntese de Proteínas , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , TYK2 Quinase/biossíntese , TYK2 Quinase/deficiência , TYK2 Quinase/genética , TYK2 Quinase/metabolismo
9.
PLoS Genet ; 11(2): e1005001, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25658338

RESUMO

Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe) support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes; 2) the distance between the genes is within 30kb; 3) the 5' genes are actively transcribing; and 4) the chimeras tend to have the second-to-last exon in the 5' genes joined to the second exon in the 3' genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.


Assuntos
Perfilação da Expressão Gênica , Fusão Gênica , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Sequência de Bases , Fator de Ligação a CCCTC , Fusão Celular , Linhagem Celular Tumoral , Éxons , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Masculino , Neoplasias da Próstata/patologia , Splicing de RNA/genética , Análise de Sequência de RNA
10.
PLoS Pathog ; 11(11): e1005278, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26565973

RESUMO

Chemical and nutrient signaling are fundamental for all cellular processes, including interactions between the mammalian host and the microbiota, which have a significant impact on health and disease. Ethanolamine is an essential component of cell membranes and has profound signaling activity within mammalian cells by modulating inflammatory responses and intestinal physiology. Here, we describe a virulence-regulating pathway in which the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) exploits ethanolamine signaling to recognize and adapt to distinct niches within the host. The bacterial transcription factor EutR promotes ethanolamine metabolism in the intestine, which enables S. Typhimurium to establish infection. Subsequently, EutR directly activates expression of the Salmonella pathogenicity island 2 in the intramacrophage environment, and thus augments intramacrophage survival. Moreover, EutR is critical for robust dissemination during mammalian infection. Our findings reveal that S. Typhimurium co-opts ethanolamine as a signal to coordinate metabolism and then virulence. Because the ability to sense ethanolamine is a conserved trait among pathogenic and commensal bacteria, our work indicates that ethanolamine signaling may be a key step in the localized adaptation of bacteria within their mammalian hosts.


Assuntos
Adaptação Biológica , Etanolamina/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium , Transdução de Sinais , Adaptação Biológica/imunologia , Animais , Ilhas Genômicas/imunologia , Humanos , Salmonella typhimurium/patogenicidade , Virulência/genética , Fatores de Virulência/metabolismo
11.
Nature ; 471(7339): 467-72, 2011 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-21430775

RESUMO

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumour genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the data set. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signalling was indicated by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge.


Assuntos
Genoma Humano/genética , Mieloma Múltiplo/genética , Mutação/genética , Sequência de Aminoácidos , Coagulação Sanguínea/genética , Ilhas de CpG/genética , Análise Mutacional de DNA , Reparo do DNA/genética , Éxons/genética , Complexo Multienzimático de Ribonucleases do Exossomo , Genômica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Homeostase/genética , Humanos , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/metabolismo , NF-kappa B/metabolismo , Oncogenes/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas/genética , Conformação Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Processamento Pós-Transcricional do RNA/genética , Ribonucleases/química , Ribonucleases/genética , Transdução de Sinais/genética , Transcrição Gênica/genética
12.
Nucleic Acids Res ; 43(18): e118, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26032770

RESUMO

The CRISPR system has become a powerful biological tool with a wide range of applications. However, improving targeting specificity and accurately predicting potential off-targets remains a significant goal. Here, we introduce a web-based CR: ISPR/Cas9 O: ff-target P: rediction and I: dentification T: ool (CROP-IT) that performs improved off-target binding and cleavage site predictions. Unlike existing prediction programs that solely use DNA sequence information; CROP-IT integrates whole genome level biological information from existing Cas9 binding and cleavage data sets. Utilizing whole-genome chromatin state information from 125 human cell types further enhances its computational prediction power. Comparative analyses on experimentally validated datasets show that CROP-IT outperforms existing computational algorithms in predicting both Cas9 binding as well as cleavage sites. With a user-friendly web-interface, CROP-IT outputs scored and ranked list of potential off-targets that enables improved guide RNA design and more accurate prediction of Cas9 binding or cleavage sites.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Cromatina/metabolismo , Desoxirribonucleases/metabolismo , Software , Algoritmos , Sítios de Ligação , Clivagem do DNA , Humanos , Análise de Sequência de DNA , Análise de Sequência de RNA/métodos
13.
J Cell Biochem ; 116(6): 893-902, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25560433

RESUMO

In mammals, formation of new nephrons ends perinatally due to consumption of mesenchymal progenitor cells. Premature depletion of progenitors due to prematurity or postnatal loss of nephrons due to injury causes chronic kidney disease and hypertension. Intensive efforts are currently invested in designing regenerative strategies to form new nephron progenitors from pluripotent cells, which upon further differentiation provide a potential source of new nephrons. To know if reprogramed renal cells can maintain their identity and fate requires knowledge of the epigenetic states of native nephron progenitors and their progeny. In this article, we summarize current knowledge and gaps in the epigenomic landscape of the developing kidney. We now know that Pax2/PTIP/H3K4 methyltransferase activity provides the initial epigenetic specification signal to the metanephric mesenchyme. During nephrogenesis, the cap mesenchyme housing nephron progenitors is enriched in bivalent chromatin marks; as tubulogenesis proceeds, the tubular epithelium acquires H3K79me2. The latter mark is uniquely induced during epithelial differentiation. Analysis of histone landscapes in clonal metanephric mesenchyme cell lines and in Wilms tumor and normal fetal kidney has revealed that promoters of poised nephrogenesis genes carry bivalent histone signatures in progenitors. Differentiation or stimulation of Wnt signaling promotes resolution of bivalency; this does not occur in Wilms tumor cells consistent with their developmental arrest. The use of small cell number ChIP-Seq should facilitate the characterization of the chromatin landscape of the metanephric mesenchyme and various nephron compartments during nephrogenesis. Only then we will know if stem and somatic cell reprogramming into kidney progenitors recapitulates normal development.


Assuntos
Rim/citologia , Néfrons/citologia , Animais , Diferenciação Celular/fisiologia , Epigenômica , Feminino , Humanos , Rim/embriologia , Rim/metabolismo , Mesoderma/citologia , Néfrons/metabolismo , Gravidez , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
15.
J Clin Invest ; 134(1)2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38165032

RESUMO

Following a period of slow progress, the completion of genome sequencing and the paradigm shift relative to the cell of origin for high grade serous ovarian cancer (HGSOC) led to a new perspective on the biology and therapeutic solutions for this deadly cancer. Experimental models were revisited to address old questions, and improved tools were generated. Additional pathways emerging as drivers of ovarian tumorigenesis and key dependencies for therapeutic targeting, in particular, VEGF-driven angiogenesis and homologous recombination deficiency, were discovered. Molecular profiling of histological subtypes of ovarian cancer defined distinct genetic events for each entity, enabling the first attempts toward personalized treatment. Armed with this knowledge, HGSOC treatment was revised to include new agents. Among them, PARP inhibitors (PARPis) were shown to induce unprecedented improvement in clinical benefit for selected subsets of patients. Research on mechanisms of resistance to PARPis is beginning to discover vulnerabilities and point to new treatment possibilities. This Review highlights these advances, the remaining challenges, and unsolved problems in the field.


Assuntos
Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Biologia
16.
Nat Commun ; 15(1): 1169, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38326302

RESUMO

Uterine fibroids (UF), that can disrupt normal uterine function and cause significant physical and psychological health problems, are observed in nearly 70% of women of reproductive age. Although heritable genetics is a significant risk factor, specific genetic variations and gene targets causally associated with UF are poorly understood. Here, we performed a meta-analysis on existing fibroid genome-wide association studies (GWAS) and integrated the identified risk loci and potentially causal single nucleotide polymorphisms (SNPs) with epigenomics, transcriptomics, 3D chromatin organization from diverse cell types as well as primary UF patient's samples. This integrative analysis identifies 24 UF-associated risk loci that potentially target 394 genes, of which 168 are differentially expressed in UF tumors. Critically, integrating this data with single-cell gene expression data from UF patients reveales the causal cell types with aberrant expression of these target genes. Lastly, CRISPR-based epigenetic repression (dCas9-KRAB) or activation (dCas9-p300) in a UF disease-relevant cell type further refines and narrows down the potential gene targets. Our findings and the methodological approach indicate the effectiveness of integrating multi-omics data with locus-specific epigenetic editing approaches for identifying gene- and celt type-targets of disease-relevant risk loci.


Assuntos
Estudo de Associação Genômica Ampla , Leiomioma , Humanos , Feminino , Epigenômica , Leiomioma/patologia , Fatores de Risco , Perfilação da Expressão Gênica , Polimorfismo de Nucleotídeo Único
17.
Genome Biol ; 25(1): 77, 2024 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519987

RESUMO

BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. RESULTS: Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. CONCLUSIONS: Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.


Assuntos
Cromatina , Lamina Tipo B , Animais , Lamina Tipo B/genética , Heterocromatina , Hibridização in Situ Fluorescente , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Laminas , Expressão Gênica , Mamíferos/genética
18.
Nat Methods ; 7(8): 615-8, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20622861

RESUMO

Current methods for whole-genome mapping of protein-DNA interactions, performed by coupling chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), require large amounts of starting materials, which precludes their application to rare cell types. Here we combine a high-sensitivity ChIP assay with a new library preparation procedure to map histone modifications in as few as 10,000 cells. We used the technique to characterize mouse hematopoietic progenitors and thereby gain insight into their developmental program.


Assuntos
Mapeamento Cromossômico/métodos , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas , Animais , Imunoprecipitação da Cromatina , Biblioteca Gênica , Histonas/metabolismo , Camundongos , Análise de Sequência de DNA
19.
Nat Methods ; 7(1): 47-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19946276

RESUMO

Chromatin structure and transcription factor localization can be assayed genome-wide by sequencing genomic DNA fractionated by protein occupancy or other properties, but current technologies involve multiple steps that introduce bias and inefficiency. Here we apply a single-molecule approach to directly sequence chromatin immunoprecipitated DNA with minimal sample manipulation. This method is compatible with just 50 pg of DNA and should thus facilitate charting chromatin maps from limited cell populations.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Análise de Sequência de DNA/métodos , Animais , Viés , Cromatina/metabolismo , Perfilação da Expressão Gênica , Genoma/genética , Humanos , Camundongos , Fatores de Transcrição/metabolismo
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