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1.
Nat Commun ; 11(1): 4402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879318

RESUMO

Genome-wide association studies have identified genetic variation contributing to complex disease risk. However, assigning causal genes and mechanisms has been more challenging because disease-associated variants are often found in distal regulatory regions with cell-type specific behaviours. Here, we collect ATAC-seq, Hi-C, Capture Hi-C and nuclear RNA-seq data in stimulated CD4+ T cells over 24 h, to identify functional enhancers regulating gene expression. We characterise changes in DNA interaction and activity dynamics that correlate with changes in gene expression, and find that the strongest correlations are observed within 200 kb of promoters. Using rheumatoid arthritis as an example of T cell mediated disease, we demonstrate interactions of expression quantitative trait loci with target genes, and confirm assigned genes or show complex interactions for 20% of disease associated loci, including FOXO1, which we confirm using CRISPR/Cas9.


Assuntos
Artrite Reumatoide/genética , Linfócitos T CD4-Positivos/metabolismo , Cromatina , Proteína Forkhead Box O1/genética , Doenças Autoimunes/genética , Linfócitos T CD4-Positivos/citologia , Cromatina/química , Cromatina/genética , Elementos Facilitadores Genéticos , Proteína Forkhead Box O1/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Cultura Primária de Células , Regiões Promotoras Genéticas , Locos de Características Quantitativas
2.
PLoS One ; 15(8): e0238322, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866178

RESUMO

Space-filling curves have been used for decades to study the folding principles of globular proteins, compact polymers, and chromatin. Formally, space-filling curves trace a single circuit through a set of points (x,y,z); informally, they correspond to a polymer melt. Although not quite a melt, the folding principles of Human chromatin are likened to the Hilbert curve: a type of space-filling curve. Hilbert-like curves in general make biologically compelling models of chromatin; in particular, they lack knots which facilitates chromatin folding, unfolding, and easy access to genes. Knot complexity has been intensely studied with the aid of Alexander polynomials; however, the approach does not generalize well to cases of more than one chromosome. Crossing complexity is an understudied alternative better suited for quantifying entanglement between chromosomes. Do Hilbert-like configurations limit crossing complexity between chromosomes? How does crossing complexity for Hilbert-like configurations compare to equilibrium configurations? To address these questions, we extend the Mansfield algorithm to enable sampling of Hilbert-like space filling curves on a simple cubic lattice. We use the extended algorithm to generate equilibrium, intermediate, and Hilbert-like configurational ensembles and compute crossing complexity between curves (chromosomes) in each configurational snapshot. Our main results are twofold: (a) Hilbert-like configurations limit entanglement between chromosomes and (b) Hilbert-like configurations do not limit entanglement in a model of S-phase DNA. Our second result is particularly surprising yet easily rationalized with a geometric argument. We explore ergodicity of the extended algorithm and discuss our results in the context of more sophisticated models of chromatin.


Assuntos
DNA/química , DNA/genética , Fase S/genética , Algoritmos , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Humanos , Polímeros/química
3.
Nat Commun ; 11(1): 4902, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994402

RESUMO

Living cells and tissues experience various complex modes of forces that are important in physiology and disease. However, how different force modes impact gene expression is elusive. Here we apply local forces of different modes via a magnetic bead bound to the integrins on a cell and quantified cell stiffness, chromatin deformation, and DHFR (dihydrofolate reductase) gene transcription. In-plane stresses result in lower cell stiffness than out-of-plane stresses that lead to bead rolling along the cell long axis (i.e., alignment of actin stress fibers) or at different angles (90° or 45°). However, chromatin stretching and ensuing DHFR gene upregulation by the in-plane mode are similar to those induced by the 45° stress mode. Disrupting stress fibers abolishes differences in cell stiffness, chromatin stretching, and DHFR gene upregulation under different force modes and inhibiting myosin II decreases cell stiffness, chromatin deformation, and gene upregulation. Theoretical modeling using discrete anisotropic stress fibers recapitulates experimental results and reveals underlying mechanisms of force-mode dependence. Our findings suggest that forces impact biological responses of living cells such as gene transcription via previously underappreciated means.


Assuntos
Cromatina/química , Fibras de Estresse/química , Tetra-Hidrofolato Desidrogenase/genética , Transcrição Genética/fisiologia , Regulação para Cima/fisiologia , Animais , Anisotropia , Fenômenos Biomecânicos/genética , Células CHO , Cromatina/metabolismo , Cricetulus , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Microscopia Intravital , Microscopia de Fluorescência , Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Estresse Mecânico , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
4.
Nat Commun ; 11(1): 4747, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958761

RESUMO

Chromosome structure at the multi-nucleosomal level has remained ambiguous in spite of its central role in epigenetic regulation and genome dynamics. Recent investigations of chromatin architecture portray diverse modes of interaction within and between nucleosome chains, but how this is realized at the atomic level is unclear. Here we present near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone. As opposed to adopting folded helical '30 nm' structures, the fibres instead assume open zigzag conformations that are interdigitated with one another. Zigzag conformations obviate extreme bending of the linker DNA, while linker DNA size (nucleosome repeat length) dictates fibre configuration and thus fibre-fibre packing, which is supported by variable linker histone binding. This suggests that nucleosome chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.


Assuntos
Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Bases , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica
5.
PLoS Comput Biol ; 16(9): e1008173, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946435

RESUMO

Single-cell Hi-C (scHi-C) interrogates genome-wide chromatin interaction in individual cells, allowing us to gain insights into 3D genome organization. However, the extremely sparse nature of scHi-C data poses a significant barrier to analysis, limiting our ability to tease out hidden biological information. In this work, we approach this problem by applying topic modeling to scHi-C data. Topic modeling is well-suited for discovering latent topics in a collection of discrete data. For our analysis, we generate nine different single-cell combinatorial indexed Hi-C (sci-Hi-C) libraries from five human cell lines (GM12878, H1Esc, HFF, IMR90, and HAP1), consisting over 19,000 cells. We demonstrate that topic modeling is able to successfully capture cell type differences from sci-Hi-C data in the form of "chromatin topics." We further show enrichment of particular compartment structures associated with locus pairs in these topics.


Assuntos
Cromatina , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Linhagem Celular , Cromatina/química , Cromatina/genética , Análise por Conglomerados , Biblioteca Gênica , Humanos , Processamento de Linguagem Natural
6.
Nat Commun ; 11(1): 3839, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737294

RESUMO

Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteína BRCA2/genética , Cromatina/química , Proteínas de Ligação a DNA/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Reparo de DNA por Recombinação , ATPases Associadas a Diversas Atividades Celulares/deficiência , Animais , Apoptose/genética , Proteína BRCA2/deficiência , Divisão Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismo , Células-Tronco Neurais/citologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
7.
PLoS One ; 15(7): e0236666, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735574

RESUMO

Deciphering long-range chromatin interactions is critical for understanding temporal and tissue-specific gene expression regulated by cis- and trans-acting factors. By combining the chromosome conformation capture (3C) and biotinylated dCas9 system, we previously established a method CAPTURE-3C-seq to unbiasedly identify high-resolution and locus-specific long-range DNA interactions. Here we present the statistical model and a flexible pipeline, C3S, for analysing CAPTURE-3C-seq or similar experimental data from raw sequencing reads to significantly interacting chromatin loci. C3S provides all steps for data processing, quality control and result illustration. It can automatically define the bin size based on the binding peak of the dCas9-targeted regions. Furthermore, it supports the analysis of intra- and inter-chromosomal interactions for different mammalian cell types. We successfully applied C3S across multiple datasets in human K562 cells and mouse embryonic stem cells (mESC) for detecting known and new chromatin interactions at multiple scales. Integrative and topological analysis of the interacted loci at the human ß-globin gene cluster provides new insights into mechanisms in developmental gene regulation and network structure in local chromosomal architecture. Furthermore, computational results in mESCs reveal a role for chromatin interacting loops between enhancers and promoters in regulating alternative transcripts of the pluripotency gene OCT4.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Cromatina/química , Cromatina/metabolismo , Modelos Moleculares , Cromatina/genética , Ligação Proteica , Conformação Proteica
8.
BMC Bioinformatics ; 21(1): 317, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32689977

RESUMO

BACKGROUND: The binding sites of transcription factors (TFs) and the localisation of histone modifications in the human genome can be quantified by the chromatin immunoprecipitation assay coupled with next-generation sequencing (ChIP-seq). The resulting chromatin feature data has been successfully adopted for genome-wide enhancer identification by several unsupervised and supervised machine learning methods. However, the current methods predict different numbers and different sets of enhancers for the same cell type and do not utilise the pattern of the ChIP-seq coverage profiles efficiently. RESULTS: In this work, we propose a PRobabilistic Enhancer PRedictIoN Tool (PREPRINT) that assumes characteristic coverage patterns of chromatin features at enhancers and employs a statistical model to account for their variability. PREPRINT defines probabilistic distance measures to quantify the similarity of the genomic query regions and the characteristic coverage patterns. The probabilistic scores of the enhancer and non-enhancer samples are utilised to train a kernel-based classifier. The performance of the method is demonstrated on ENCODE data for two cell lines. The predicted enhancers are computationally validated based on the transcriptional regulatory protein binding sites and compared to the predictions obtained by state-of-the-art methods. CONCLUSION: PREPRINT performs favorably to the state-of-the-art methods, especially when requiring the methods to predict a larger set of enhancers. PREPRINT generalises successfully to data from cell type not utilised for training, and often the PREPRINT performs better than the previous methods. The PREPRINT enhancers are less sensitive to the choice of prediction threshold. PREPRINT identifies biologically validated enhancers not predicted by the competing methods. The enhancers predicted by PREPRINT can aid the genome interpretation in functional genomics and clinical studies.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Genoma Humano , Genômica/métodos , Histonas/genética , Modelos Estatísticos , Fatores de Transcrição/metabolismo , Cromatina/química , Cromatina/metabolismo , Código das Histonas , Histonas/química , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional
9.
Nat Commun ; 11(1): 3289, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620890

RESUMO

The spatial organization of chromosomes has key functional roles, yet how chromosomes fold remains poorly understood at the single-molecule level. Here, we employ models of polymer physics to investigate DNA loci in human HCT116 and IMR90 wild-type and cohesin depleted cells. Model predictions on single-molecule structures are validated against single-cell imaging data, providing evidence that chromosomal architecture is controlled by a thermodynamics mechanism of polymer phase separation whereby chromatin self-assembles in segregated globules by combinatorial interactions of chromatin factors that include CTCF and cohesin. The thermodynamics degeneracy of single-molecule conformations results in broad structural and temporal variability of TAD-like contact patterns. Globules establish stable environments where specific contacts are highly favored over stochastic encounters. Cohesin depletion reverses phase separation into randomly folded states, erasing average interaction patterns. Overall, globule phase separation appears to be a robust yet reversible mechanism of chromatin organization where stochasticity and specificity coexist.


Assuntos
Cromatina/química , Conformação Molecular , Fenômenos Físicos , Polímeros/química , Análise de Célula Única/métodos , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Células HCT116 , Humanos , Ligação Proteica , Processos Estocásticos , Termodinâmica
10.
Nat Commun ; 11(1): 3428, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647330

RESUMO

Accurately predicting chromatin loops from genome-wide interaction matrices such as Hi-C data is critical to deepening our understanding of proper gene regulation. Current approaches are mainly focused on searching for statistically enriched dots on a genome-wide map. However, given the availability of orthogonal data types such as ChIA-PET, HiChIP, Capture Hi-C, and high-throughput imaging, a supervised learning approach could facilitate the discovery of a comprehensive set of chromatin interactions. Here, we present Peakachu, a Random Forest classification framework that predicts chromatin loops from genome-wide contact maps. We compare Peakachu with current enrichment-based approaches, and find that Peakachu identifies a unique set of short-range interactions. We show that our models perform well in different platforms, across different sequencing depths, and across different species. We apply this framework to predict chromatin loops in 56 Hi-C datasets, and release the results at the 3D Genome Browser.


Assuntos
Cromatina/química , Genoma , Aprendizado de Máquina Supervisionado , Bases de Dados Genéticas , Humanos , Células K562 , Especificidade de Órgãos , Análise de Sequência de DNA , Especificidade da Espécie
11.
Nat Commun ; 11(1): 3391, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636369

RESUMO

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.


Assuntos
Adenosina Difosfato Ribose/química , Cromatina/química , Quebras de DNA de Cadeia Simples , Reparo do DNA , Glicosídeo Hidrolases/genética , Mutação , Linhagem Celular Tumoral , Sobrevivência Celular , Fibroblastos , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Histonas/química , Humanos , Doenças Neurodegenerativas/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
12.
Nat Commun ; 11(1): 3398, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636384

RESUMO

SWI/SNF remodelers play a key role in regulating chromatin architecture and gene expression. Here, we report the cryo-EM structure of the Saccharomyces cerevisiae Swi/Snf complex in a nucleosome-free state. The structure consists of a stable triangular base module and a flexible Arp module. The conserved subunits Swi1 and Swi3 form the backbone of the complex and closely interact with other components. Snf6, which is specific for yeast Swi/Snf complex, stabilizes the binding of the ATPase-containing subunit Snf2 to the base module. Comparison of the yeast Swi/Snf and RSC complexes reveals conserved structural features that govern the assembly and function of these two subfamilies of chromatin remodelers. Our findings complement those from recent structures of the yeast and human chromatin remodelers and provide further insights into the assembly and function of the SWI/SNF remodelers.


Assuntos
Adenosina Trifosfatases/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Humanos , Nucleossomos , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química
13.
BMC Bioinformatics ; 21(1): 272, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611376

RESUMO

BACKGROUND: Chromatin 3D conformation plays important roles in regulating gene or protein functions. High-throughout chromosome conformation capture (3C)-based technologies, such as Hi-C, have been exploited to acquire the contact frequencies among genomic loci at genome-scale. Various computational tools have been proposed to recover the underlying chromatin 3D structures from in situ Hi-C contact map data. As connected residuals in a polymer, neighboring genomic loci have intrinsic mutual dependencies in building a 3D conformation. However, current methods seldom take this feature into account. RESULTS: We present a method called ShNeigh, which combines the classical MDS technique with local dependence of neighboring loci modeled by a Gaussian formula, to infer the best 3D structure from noisy and incomplete contact frequency matrices. We validated ShNeigh by comparing it to two typical distance-based algorithms, ShRec3D and ChromSDE. The comparison results on simulated Hi-C dataset showed that, while keeping the high-speed nature of classical MDS, ShNeigh can recover the true structure better than ShRec3D and ChromSDE. Meanwhile, ShNeigh is more robust to data noise. On the publicly available human GM06990 Hi-C data, we demonstrated that the structures reconstructed by ShNeigh are more reproducible between different restriction enzymes than by ShRec3D and ChromSDE, especially at high resolutions manifested by sparse contact maps, which means ShNeigh is more robust to signal coverage. CONCLUSIONS: Our method can recover stable structures in high noise and sparse signal settings. It can also reconstruct similar structures from Hi-C data obtained using different restriction enzymes. Therefore, our method provides a new direction for enhancing the reconstruction quality of chromatin 3D structures.


Assuntos
Cromatina/química , Genômica/métodos , Algoritmos , Cromossomos/química , Cromossomos/genética , Loci Gênicos , Humanos , Conformação Molecular , Interface Usuário-Computador
14.
Nature ; 584(7820): 244-251, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728217

RESUMO

DNase I hypersensitive sites (DHSs) are generic markers of regulatory DNA1-5 and contain genetic variations associated with diseases and phenotypic traits6-8. We created high-resolution maps of DHSs from 733 human biosamples encompassing 438 cell and tissue types and states, and integrated these to delineate and numerically index approximately 3.6 million DHSs within the human genome sequence, providing a common coordinate system for regulatory DNA. Here we show that these maps highly resolve the cis-regulatory compartment of the human genome, which encodes unexpectedly diverse cell- and tissue-selective regulatory programs at very high density. These programs can be captured comprehensively by a simple vocabulary that enables the assignment to each DHS of a regulatory barcode that encapsulates its tissue manifestations, and global annotation of protein-coding and non-coding RNA genes in a manner orthogonal to gene expression. Finally, we show that sharply resolved DHSs markedly enhance the genetic association and heritability signals of diseases and traits. Rather than being confined to a small number of distal elements or promoters, we find that genetic signals converge on congruently regulated sets of DHSs that decorate entire gene bodies. Together, our results create a universal, extensible coordinate system and vocabulary for human regulatory DNA marked by DHSs, and provide a new global perspective on the architecture of human gene regulation.


Assuntos
Cromatina/genética , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Anotação de Sequência Molecular , Cromatina/química , Cromatina/metabolismo , DNA/química , DNA/genética , Regulação da Expressão Gênica , Genes/genética , Genoma Humano/genética , Humanos , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética
15.
Mol Cell ; 79(3): 488-503.e11, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32585128

RESUMO

Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.


Assuntos
RNA Polimerase III/genética , RNA Polimerase II/genética , RNA Polimerase I/genética , RNA Fúngico/química , Saccharomyces cerevisiae/genética , Elongação da Transcrição Genética , Composição de Bases , Sequência de Bases , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Regulação Fúngica da Expressão Gênica , Ligação Proteica , Dobramento de RNA , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Sítios de Splice de RNA , Processamento de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Termodinâmica
16.
Science ; 368(6498): 1449-1454, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32587015

RESUMO

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imagem Individual de Molécula/métodos , Animais , DNA/química , DNA/genética , Drosophila melanogaster , Humanos , Células K562 , Nucleossomos/química , Regiões Promotoras Genéticas , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Fatores de Transcrição/química
17.
Nat Commun ; 11(1): 2606, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451376

RESUMO

Nucleoporin proteins (Nups) have been proposed to mediate spatial and temporal chromatin organization during gene regulation. Nevertheless, the molecular mechanisms in mammalian cells are not well understood. Here, we report that Nucleoporin 153 (NUP153) interacts with the chromatin architectural proteins, CTCF and cohesin, and mediates their binding across cis-regulatory elements and TAD boundaries in mouse embryonic stem (ES) cells. NUP153 depletion results in altered CTCF and cohesin binding and differential gene expression - specifically at the bivalent developmental genes. To investigate the molecular mechanism, we utilize epidermal growth factor (EGF)-inducible immediate early genes (IEGs). We find that NUP153 controls CTCF and cohesin binding at the cis-regulatory elements and POL II pausing during the basal state. Furthermore, efficient IEG transcription relies on NUP153. We propose that NUP153 links the nuclear pore complex (NPC) to chromatin architecture allowing genes that are poised to respond rapidly to developmental cues to be properly modulated.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Fator de Ligação a CCCTC/química , Proteínas de Ciclo Celular/química , Linhagem Celular , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/química , Genes Precoces , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/metabolismo , Elementos Reguladores de Transcrição
18.
Adv Exp Med Biol ; 1195: 73-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468461

RESUMO

Quantum entanglement has recently been demonstrated in macroscopic structures at the scale of microns. The densely packed chromatin that efficiently stores DNA strands may allow for gene expression through epigenetic modifiers within the close proximity of nearby strands and may also experience gene expression through quantum entanglement of epigenetic modifiers. Such an approach may have an evolutionary advantage in the densely packed realm of chromatin.


Assuntos
Cromatina/química , Cromatina/genética , Expressão Gênica , Epigênese Genética
19.
Nucleic Acids Res ; 48(10): 5407-5425, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356874

RESUMO

Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3' end processing machinery to the vicinity of pA sites.


Assuntos
Histona-Lisina N-Metiltransferase/fisiologia , Metiltransferases/fisiologia , Poliadenilação , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Cromatina/química , Cromatina/efeitos dos fármacos , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histonas , Metiltransferases/genética , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
20.
Nat Commun ; 11(1): 2564, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444798

RESUMO

Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome conformation capture (3C)-based experiments combined with computational modelling are pivotal for unveiling 3D chromosome structure. Here, we introduce TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We apply TADdyn on in situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterize the structural rearrangements that take place upon changes in the transcriptional state of 21 genomic loci of diverse expression dynamics. By measuring various structural and dynamical properties, we find that during gene activation, the transcription starting site contacts with open and active regions in 3D chromatin domains. We propose that these 3D hubs of open and active chromatin may constitute a general feature to trigger and maintain gene transcription.


Assuntos
Linfócitos B/metabolismo , Reprogramação Celular , Cromatina/química , Ativação Transcricional , Animais , Linfócitos B/química , Linfócitos B/citologia , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Camundongos , Células-Tronco Pluripotentes/química , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
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