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1.
Postepy Biochem ; 65(3): 202-211, 2019 10 01.
Artigo em Polonês | MEDLINE | ID: mdl-31643167

RESUMO

Advances in high resolution microscopy techniques and development of high throughput DNA analyses allow to reconsider the views concerning bacterial chromosome (nucleoid). Recent reports show that nucleoid exhibits a hierarchical organization, similarly to the eukaryotic chromatin. However, bacterial chromosome undergoes constant modifications and topological rearrangements due to the ongoing DNA replication, transcription and translation processes. Organization of dynamic and highly compacted nucleoid structure depends on physical factors acting on chromosome molecule inside small cell compartment, and is a consequence of action of many different DNA-binding proteins. The main goal of this review is to present the recent reports on bacterial chromatin structure and to elucidate the physical and molecular factors influencing its intracellular organization.


Assuntos
Bactérias/genética , Cromatina/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Bactérias/metabolismo , Cromatina/química , Cromatina/genética , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , Replicação do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo
2.
Genes Dev ; 33(17-18): 1117-1135, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31481536

RESUMO

T-cell development in mammals is a model for lineage choice and differentiation from multipotent stem cells. Although T-cell fate choice is promoted by signaling in the thymus through one dominant pathway, the Notch pathway, it entails a complex set of gene regulatory network and chromatin state changes even before the cells begin to express their signature feature, the clonal-specific T-cell receptors (TCRs) for antigen. This review distinguishes three developmental modules for T-cell development, which correspond to cell type specification, TCR expression and selection, and the assignment of cells to different effector types. The first is based on transcriptional regulatory network events, the second is dominated by somatic gene rearrangement and mutation and cell selection, and the third corresponds to establishing a poised state of latent regulator priming through an unknown mechanism. Interestingly, in different lineages, the third module can be deployed at variable times relative to the completion of the first two modules. This review focuses on the gene regulatory network and chromatin-based kinetic constraints that determine activities of transcription factors TCF1, GATA3, PU.1, Bcl11b, Runx1, and E proteins in the primary establishment of T-cell identity.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Linfócitos T/citologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Cromatina/metabolismo , Redes Reguladoras de Genes , Hematopoese , Linfócitos T/metabolismo , Timo/citologia , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 10(1): 3072, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296853

RESUMO

Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Nucleossomos/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Animais , Fator de Ligação a CCAAT/genética , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias , Técnicas de Silenciamento de Genes , Camundongos , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo
4.
Nat Commun ; 10(1): 3049, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296872

RESUMO

The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.


Assuntos
Ectoderma/embriologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Placa Neural/embriologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Cromatina/metabolismo , Regulação para Baixo , Ectoderma/metabolismo , Embrião não Mamífero , Elementos Facilitadores Genéticos/genética , Epiderme/embriologia , Epiderme/metabolismo , Técnicas de Inativação de Genes , Modelos Animais , Placa Neural/metabolismo , Fosfoproteínas/genética , Ligação Proteica/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transativadores/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
5.
Cell Mol Life Sci ; 76(21): 4291-4307, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31302750

RESUMO

In the presence of chromatin bridges, mammalian cells delay completion of cytokinesis (abscission) to prevent chromatin breakage or tetraploidization by regression of the cleavage furrow. This abscission delay is called "the abscission checkpoint" and is dependent on Aurora B kinase. Furthermore, cells stabilize the narrow cytoplasmic canal between the two daughter cells until the DNA bridges are resolved. Impaired abscission checkpoint signaling or unstable intercellular canals can lead to accumulation of DNA damage, aneuploidy, or generation of polyploid cells which are associated with tumourigenesis. However, the molecular mechanisms involved have only recently started to emerge. In this review, we focus on the molecular pathways of the abscission checkpoint and describe newly identified triggers, Aurora B-regulators and effector proteins in abscission checkpoint signaling. We also describe mechanisms that control intercellular bridge stabilization, DNA bridge resolution, or abscission checkpoint silencing upon satisfaction, and discuss how abscission checkpoint proteins can be targeted to potentially improve cancer therapy.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Citocinese/fisiologia , Animais , Aurora Quinase B/metabolismo , Aurora Quinase B/fisiologia , Genes cdc/fisiologia , Humanos , Transdução de Sinais
6.
Adv Exp Med Biol ; 1166: 47-56, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31301045

RESUMO

Compared to other cells, sperm undergo dramatic remodeling of their chromatin during late spermiogenesis in which approximately 95% of histones are removed and replaced with protamines. Despite this large-scale remodeling, key developmental genes, some miRNA genes, and imprinted genes retain their association with histone. The developmental genes have a unique epigenetic signature, termed bivalency, that poises the genes for embryonic activation. Anomalies in that epigenetic poising signature, either in the form of DNA methylation aberrations, improper protamination, or altered histone modifications, are associated with infertility and reduced embryogenesis capability. Additionally, some small noncoding RNAs are retained, while others are actively added to the sperm and appear to affect embryogenesis. Therefore, initial studies have begun to formulate pathways by which the sperm epigenome can be used as a diagnostic tool in the clinic. While in their infancy, these assays likely portend improved diagnostics and added information for patients and clinicians. Recent studies also highlight the possibility that the sperm epigenome can be used to evaluate lifestyle and environmental risks to the patient and potentially to the offspring.


Assuntos
Epigênese Genética , Técnicas de Reprodução Assistida , Espermatozoides , Cromatina/metabolismo , Meio Ambiente , Humanos , Estilo de Vida , Masculino , Fatores de Risco , Espermatogênese
7.
Nat Commun ; 10(1): 2954, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273204

RESUMO

PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). Upon activation, PARP-1 synthesizes a structurally complex polymer composed of ADP-ribose units that facilitates local chromatin relaxation and the recruitment of DNA repair factors. Here, we identify a function for PARP-1 in DNA DSB resection. Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. We show that loss of PARP-1 and hyperresection are associated with loss of Ku, 53BP1 and RIF1 resection inhibitors from the break site. DNA curtains analysis show that EXO1-mediated resection is blocked by PARP-1. Furthermore, PARP-1 abrogation leads to increased DNA resection tracks and an increase of homologous recombination in cellulo. Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. Hence, our work has direct implications for the clinical use and effectiveness of PARP inhibition, which is prescribed for the treatment of various malignancies.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cromatina/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Recombinação Homóloga/genética , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
8.
Nature ; 571(7766): 489-499, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31341302

RESUMO

Epigenetic research has accelerated rapidly in the twenty-first century, generating justified excitement and hope, but also a degree of hype. Here we review how the field has evolved over the last few decades and reflect on some of the recent advances that are changing our understanding of biology. We discuss the interplay between epigenetics and DNA sequence variation as well as the implications of epigenetics for cellular memory and plasticity. We consider the effects of the environment and both intergenerational and transgenerational epigenetic inheritance on biology, disease and evolution. Finally, we present some new frontiers in epigenetics with implications for human health.


Assuntos
Doença/genética , Epigênese Genética/genética , Epigenômica/tendências , Interação Gene-Ambiente , Envelhecimento/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Variação Genética/genética , Humanos , Neoplasias/genética
9.
Nat Commun ; 10(1): 2908, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266948

RESUMO

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity.


Assuntos
Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Pluripotentes/química , Ligação Proteica , Domínios Proteicos , Especificidade da Espécie
10.
Nat Commun ; 10(1): 3004, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285436

RESUMO

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.


Assuntos
Elementos Facilitadores Genéticos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Rabdomiossarcoma/genética , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Rabdomiossarcoma/patologia , Análise de Sequência de RNA , Transcrição Genética/efeitos dos fármacos
11.
Nat Biotechnol ; 37(8): 916-924, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31235917

RESUMO

Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (105 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named 'droplet single-cell assay for transposase-accessible chromatin using sequencing' (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform.


Assuntos
Cromatina/química , Epigenômica/métodos , Microfluídica/métodos , Análise de Célula Única/métodos , Animais , Encéfalo/citologia , Linhagem Celular , Sobrevivência Celular , Cromatina/metabolismo , Técnicas de Química Combinatória , Desoxirribonucleases/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Camundongos
12.
Nature ; 571(7765): 408-412, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31243370

RESUMO

Mutations in the transcription factor FOXA1 define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown1-9. Here, by annotating the landscape of FOXA1 mutations from 3,086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (around 50% of all mutations) and the highly conserved DNA-contact residue R219 (around 5% of all mutations). Wing2 mutations are detected in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumours with neuroendocrine histology. Interrogation of the biological properties of wild-type FOXA1 and fourteen FOXA1 mutants reveals gain of function in mouse prostate organoid proliferation assays. Twelve of these mutants, as well as wild-type FOXA1, promoted an exaggerated pro-luminal differentiation program, whereas two different R219 mutants blocked luminal differentiation and activated a mesenchymal and neuroendocrine transcriptional program. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type FOXA1 and representative Wing2 and R219 mutants revealed marked, mutant-specific changes in open chromatin at thousands of genomic loci and exposed sites of FOXA1 binding and associated increases in gene expression. Of note, ATAC-seq peaks in cells expressing R219 mutants lacked the canonical core FOXA1-binding motifs (GTAAAC/T) but were enriched for a related, non-canonical motif (GTAAAG/A), which was preferentially activated by R219-mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its pioneering function and perturb normal luminal epithelial differentiation programs, providing further support for the role of lineage plasticity in cancer progression.


Assuntos
Diferenciação Celular/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Mutação , Fenótipo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem da Célula , Cromatina/genética , Cromatina/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Motivos de Nucleotídeos , Organoides/citologia , Organoides/metabolismo
13.
Nature ; 571(7765): 413-418, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31243372

RESUMO

ABTRACT: Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland1,2. FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours3-8. However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive9-11 and oncogenic12-14 roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and-through TLE3 inactivation-promote metastasis driven by the WNT pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element-herein denoted FOXA1 mastermind (FOXMIND)-to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies.


Assuntos
Fator 3-alfa Nuclear de Hepatócito/genética , Mutação/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Fator 3-alfa Nuclear de Hepatócito/química , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Modelos Moleculares , Metástase Neoplásica/genética , Domínios Proteicos , Receptores Androgênicos/metabolismo , Via de Sinalização Wnt
14.
Nat Plants ; 5(6): 616-625, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182849

RESUMO

The lipid-derived hormone jasmonate (JA) regulates plant immunity and adaptive growth by triggering a genome-wide transcriptional programme. In Arabidopsis thaliana, JA-triggered transcriptional programming is largely orchestrated by the master transcription factor MYC2. The function of MYC2 is dependent on its physical interaction with the MED25 subunit of the Mediator transcriptional co-activator complex. Here we report the identification of JA enhancers (JAEs) through profiling the occupancy pattern of MYC2 and MED25. JA regulates the dynamic chromatin looping between JAEs and their promoters in a MED25-dependent manner, while MYC2 auto-regulates itself through JAEs. Interestingly, the JAE of the MYC2 locus (named ME2) positively regulates MYC2 expression during short-term JA responses but negatively regulates it during constant JA responses. We demonstrate that new gene editing tools open up new avenues to elucidate the in vivo function of enhancers. Our work provides a paradigm for functional study of plant enhancers in the regulation of specific physiological processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclopentanos/metabolismo , Proteínas Nucleares/metabolismo , Oxilipinas/metabolismo , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Arseniato Redutases/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
15.
Nat Genet ; 51(6): 1060-1066, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31152164

RESUMO

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.


Assuntos
Neoplasias da Mama/genética , Imunoprecipitação da Cromatina , Cromatina/genética , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Cromatina/metabolismo , Biologia Computacional/métodos , Epigênese Genética , Feminino , Histonas/metabolismo , Humanos , Técnicas Analíticas Microfluídicas , Análise de Célula Única/métodos , Células Estromais , Fluxo de Trabalho
16.
Genome Biol ; 20(1): 115, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159833

RESUMO

BACKGROUND: Hox transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. While the different Hox family members show clear functional specificity in vivo, they all show similar binding specificity in vitro and a satisfactory understanding of in vivo Hox target selectivity is still lacking. RESULTS: Using transient transfection in Kc167 cells, we systematically analyze the binding of all eight Drosophila Hox proteins. We find that Hox proteins show considerable binding selectivity in vivo even in the absence of canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility, being highest in less accessible chromatin. Individual Hox proteins exhibit different propensities to bind less accessible chromatin, and high binding selectivity is associated with high-affinity binding regions, leading to a model where Hox proteins derive binding selectivity through affinity-based competition with nucleosomes. Extradenticle/Homothorax cofactors generally facilitate Hox binding, promoting binding to regions in less accessible chromatin but with little effect on the overall selectivity of Hox targeting. These cofactors collaborate with Hox proteins in opening chromatin, in contrast to the pioneer factor, Glial cells missing, which facilitates Hox binding by independently generating accessible chromatin regions. CONCLUSIONS: These studies indicate that chromatin accessibility plays a key role in Hox selectivity. We propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of in vivo genomic targets for different Hox proteins.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Drosophila , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo
17.
Nat Commun ; 10(1): 2626, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201308

RESUMO

Chromatin of mammalian nucleus folds into discrete contact enriched regions such as Topologically Associating Domains (TADs). Folding hierarchy and internal organization of TADs is highly dynamic throughout cellular differentiation, and are correlated with gene activation and silencing. To account for multiple interacting TADs, we developed a parsimonious randomly cross-linked (RCL) polymer model that maps high frequency Hi-C encounters within and between TADs into direct loci interactions using cross-links at a given base-pair resolution. We reconstruct three TADs of the mammalian X chromosome for three stages of differentiation. We compute the radius of gyration of TADs and the encounter probability between genomic segments. We found 1) a synchronous compaction and decompaction of TADs throughout differentiation and 2) high order organization into meta-TADs resulting from weak inter-TAD interactions. Finally, the present framework allows to infer transient properties of the chromatin from steady-state statistics embedded in the Hi-C/5C data.


Assuntos
Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Polímeros/química , Cromossomo X/metabolismo , Animais , Reagentes para Ligações Cruzadas/química , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Moleculares , Conformação de Ácido Nucleico
18.
Nat Commun ; 10(1): 2633, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201330

RESUMO

Long-range chromatin interactions are important for transcriptional regulation of genes, many of which are related to complex agronomics traits. However, the pattern of three-dimensional chromatin interactions remains unclear in plants. Here we report the generation of chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data and the construction of extensive H3K4me3- and H3K27ac-centered chromatin interaction maps in maize. Results show that the interacting patterns between proximal and distal regulatory regions of genes are highly complex and dynamic. Genes with chromatin interactions have higher expression levels than those without interactions. Genes with proximal-proximal interactions prefer to be transcriptionally coordinated. Tissue-specific proximal-distal interactions are associated with tissue-specific expression of genes. Interactions between proximal and distal regulatory regions further interweave into organized network communities that are enriched in specific biological functions. The high-resolution chromatin interaction maps will help to understand the transcription regulation of genes associated with complex agronomic traits of maize.


Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Genética/genética , Zea mays/genética , Cromatina/genética , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Produção Agrícola , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Histonas/genética , Histonas/imunologia , Regiões Promotoras Genéticas , Locos de Características Quantitativas/genética
19.
Nat Commun ; 10(1): 2632, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201335

RESUMO

Chromatin loops connect regulatory elements to their target genes. They serve as bridges between transcriptional regulation and phenotypic variation in mammals. However, spatial organization of regulatory elements and its impact on gene expression in plants remain unclear. Here, we characterize epigenetic features of active promoter proximal regions and candidate distal regulatory elements to construct high-resolution chromatin interaction maps for maize via long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). The maps indicate that chromatin loops are formed between regulatory elements, and that gene pairs between promoter proximal regions tend to be co-expressed. The maps also demonstrated the topological basis of quantitative trait loci which influence gene expression and phenotype. Many promoter proximal regions are involved in chromatin loops with distal regulatory elements, which regulate important agronomic traits. Collectively, these maps provide a high-resolution view of 3D maize genome architecture, and its role in gene expression and phenotypic variation.


Assuntos
Cromatina/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes/genética , Locos de Características Quantitativas/genética , Zea mays/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Produção Agrícola , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Epigenômica/métodos , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Mutação , Fenótipo , Regiões Promotoras Genéticas/genética
20.
Nat Commun ; 10(1): 2771, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235807

RESUMO

Diverse antibody repertoires are generated through remote genomic interactions involving immunoglobulin variable (VH), diversity (DH) and joining (JH) gene segments. How such interactions are orchestrated remains unknown. Here we develop a strategy to track VH-DHJH motion in B-lymphocytes. We find that VH and DHJH segments are trapped in configurations that allow only local motion, such that spatially proximal segments remain in proximity, while spatially remote segments remain remote. Within a subset of cells, however, abrupt changes in VH-DHJH motion are observed, plausibly caused by temporal alterations in chromatin configurations. Comparison of experimental and simulated data suggests that constrained motion is imposed by a network of cross-linked chromatin chains characteristic of a gel phase, yet poised near the sol phase, a solution of independent chromatin chains. These results suggest that chromosome organization near the sol-gel phase transition dictates the timing of genomic interactions to orchestrate gene expression and somatic recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes de Imunoglobulinas/genética , Recombinação V(D)J/fisiologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Cromossomos/genética , Proteínas de Ligação a DNA/deficiência , Genômica , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Transição de Fase
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