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1.
Elife ; 122024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38994733

RESUMO

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , ATPases Vacuolares Próton-Translocadoras , Caenorhabditis elegans/genética , Animais , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Divisão Celular Assimétrica , Apoptose , Epigênese Genética , Nucleossomos/metabolismo
2.
Life Sci Alliance ; 7(10)2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38991729

RESUMO

Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Drosophila melanogaster , Desenvolvimento Embrionário , Animais , Masculino , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Cromatina/metabolismo , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas Embrionárias/metabolismo , Células Germinativas Embrionárias/citologia , Células Germinativas/metabolismo , Epigênese Genética , Feminino , Nucleossomos/metabolismo , Nucleossomos/genética , Análise de Célula Única/métodos
3.
Nat Commun ; 15(1): 5187, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38992002

RESUMO

The histone H2A variant H2A.W occupies transposons and thus prevents access to them in Arabidopsis thaliana. H2A.W is deposited by the chromatin remodeler DDM1, which also promotes the accessibility of chromatin writers to heterochromatin by an unknown mechanism. To shed light on this question, we solve the cryo-EM structures of nucleosomes containing H2A and H2A.W, and the DDM1-H2A.W nucleosome complex. These structures show that the DNA end flexibility of the H2A nucleosome is higher than that of the H2A.W nucleosome. In the DDM1-H2A.W nucleosome complex, DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA and increases the DNA end flexibility of H2A.W nucleosomes. Based on these biochemical and structural results, we propose that DDM1 counters the low accessibility caused by nucleosomes containing H2A.W to enable the maintenance of repressive epigenetic marks on transposons and prevent their activity.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Montagem e Desmontagem da Cromatina , Microscopia Crioeletrônica , Histonas , Nucleossomos , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Nucleossomos/química , Histonas/metabolismo , Histonas/genética , Histonas/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ligação Proteica , Modelos Moleculares , DNA de Plantas/metabolismo , DNA de Plantas/genética
4.
Subcell Biochem ; 104: 101-117, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38963485

RESUMO

Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity.


Assuntos
Histona-Lisina N-Metiltransferase , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Nucleossomos/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Histonas/química , Histonas/genética , Microscopia Crioeletrônica/métodos
5.
Sci Adv ; 10(27): eadm9740, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38959309

RESUMO

Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.


Assuntos
Drosophila melanogaster , Nuclease do Micrococo , Nucleossomos , Saccharomyces cerevisiae , Nucleossomos/metabolismo , Nucleossomos/genética , Animais , Nuclease do Micrococo/metabolismo , Drosophila melanogaster/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina , Genoma , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Cromatina/genética , Cromatina/metabolismo , Análise de Sequência de DNA/métodos
6.
PLoS Genet ; 20(7): e1011345, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38985845

RESUMO

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Células Germinativas , Nucleossomos , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Animais , Caenorhabditis elegans/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Cromatina/metabolismo , Transcrição Gênica , Regulação da Expressão Gênica/genética , Heterocromatina/genética , Heterocromatina/metabolismo , RNA de Interação com Piwi
7.
Proc Natl Acad Sci U S A ; 121(28): e2319772121, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38968124

RESUMO

Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design.


Assuntos
RNA Polimerases Dirigidas por DNA , Nucleossomos , Transcrição Gênica , Nucleossomos/metabolismo , Nucleossomos/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , DNA/metabolismo , DNA/química , DNA/genética , Cromatina/metabolismo , Cromatina/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
8.
Methods Mol Biol ; 2819: 357-379, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028515

RESUMO

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.Microscale thermophoresis (MST) is a biophysical technique for the study of biomolecular interactions. It has seen increasing application in recent years thanks to its solution-based nature, rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events.Here, we describe the use of MST in the study of chromatin interactions. The emphasis lies on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB, the sequential binding of core histone complexes to histone chaperone APLF, the impact of the nucleosomal context on the recognition of histone modifications, and the binding of a viral peptide to the nucleosome. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples.


Assuntos
Cromatina , Histonas , Nucleossomos , Ligação Proteica , Cromatina/metabolismo , Cromatina/genética , Nucleossomos/metabolismo , Histonas/metabolismo , DNA/metabolismo , DNA/química , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Chaperonas de Histonas/metabolismo
9.
Postepy Biochem ; 70(1): 39-40, 2024 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-39016223
10.
Bioconjug Chem ; 35(7): 944-953, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38954775

RESUMO

The chemical synthesis of homogeneously ubiquitylated histones is a powerful approach to decipher histone ubiquitylation-dependent epigenetic regulation. Among the various methods, α-halogen ketone-mediated conjugation chemistry has recently been an attractive strategy to generate single-monoubiquitylated histones for biochemical and structural studies. Herein, we report the use of this strategy to prepare not only dual- and even triple-monoubiquitylated histones but also diubiquitin-modified histones. We were surprised to find that the synthetic efficiencies of multi-monoubiquitylated histones were comparable to those of single-monoubiquitylated ones, suggesting that this strategy is highly tolerant to the number of ubiquitin monomers installed onto histones. The facile generation of a series of single-, dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate the influence of ubiquitylation patterns on the binding of DNA methyltransferase 1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific conjugation chemistry to generate chemically defined histones for epigenetic studies.


Assuntos
Histonas , Cetonas , Ubiquitinação , Histonas/química , Histonas/metabolismo , Histonas/síntese química , Cetonas/química , Ubiquitina/química , Humanos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/química , Nucleossomos/química , Nucleossomos/metabolismo
11.
Epigenetics Chromatin ; 17(1): 19, 2024 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-38825690

RESUMO

BACKGROUND: Over the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function. RESULTS: We report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes. CONCLUSIONS: These data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.


Assuntos
Proteína Centromérica A , Histonas , Nucleossomos , Processamento de Proteína Pós-Traducional , Humanos , Proteína Centromérica A/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Células HeLa , Cinetocoros/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Ligação Proteica
12.
Sci Adv ; 10(23): eadn5175, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38838138

RESUMO

Inheritance of epigenetic information is critical for maintaining cell identity. The transfer of parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications on histone proteins, represents a crucial yet poorly understood step in the inheritance of epigenetic information. Here, we show the lagging strand DNA polymerase, Pol δ, interacts directly with H3-H4 and that the interaction between Pol δ and the sliding clamp PCNA regulates parental histone transfer to lagging strands, most likely independent of their roles in DNA synthesis. When combined, mutations at Pol δ and Mcm2 that compromise parental histone transfer result in a greater reduction in nucleosome occupancy at nascent chromatin than mutations in either alone. Last, PCNA contributes to nucleosome positioning on nascent chromatin. On the basis of these results, we suggest that the PCNA-Pol δ complex couples lagging strand DNA synthesis to parental H3-H4 transfer, facilitating epigenetic inheritance.


Assuntos
DNA Polimerase III , Replicação do DNA , Epigênese Genética , Histonas , Antígeno Nuclear de Célula em Proliferação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Histonas/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase III/genética , Nucleossomos/metabolismo , Nucleossomos/genética , DNA/metabolismo , Humanos , Ligação Proteica , Mutação , Cromatina/metabolismo , Cromatina/genética
13.
J Phys Chem B ; 128(24): 5803-5813, 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38860885

RESUMO

Centromeric chromatin is a subset of chromatin structure and governs chromosome segregation. The centromere is composed of both CENP-A nucleosomes (CENP-Anuc) and H3 nucleosomes (H3nuc) and is enriched with alpha-satellite (α-sat) DNA repeats. These CENP-Anuc have a different structure than H3nuc, decreasing the base pairs (bp) of wrapped DNA from 147 bp for H3nuc to 121 bp for CENP-Anuc. All these factors can contribute to centromere function. We investigated the interaction of H3nuc and CENP-Anuc with NF-κB, a crucial transcription factor in regulating immune response and inflammation. We utilized atomic force microscopy (AFM) to characterize complexes of both types of nucleosomes with NF-κB. We found that NF-κB unravels H3nuc, removing more than 20 bp of DNA, and that NF-κB binds to the nucleosomal core. Similar results were obtained for the truncated variant of NF-κB comprised only of the Rel homology domain and missing the transcription activation domain (TAD), suggesting that RelATAD is not critical in unraveling H3nuc. By contrast, NF-κB did not bind to or unravel CENP-Anuc. These findings with different affinities for two types of nucleosomes to NF-κB may have implications for understanding the mechanisms of gene expression in bulk and centromere chromatin.


Assuntos
Centrômero , Cromatina , NF-kappa B , Nucleossomos , Centrômero/metabolismo , Centrômero/química , Cromatina/metabolismo , Cromatina/química , NF-kappa B/metabolismo , Nucleossomos/metabolismo , Nucleossomos/química , Humanos , Microscopia de Força Atômica , Ligação Proteica , Proteína Centromérica A/metabolismo , Proteína Centromérica A/química , DNA/química , DNA/metabolismo
14.
Proc Natl Acad Sci U S A ; 121(26): e2317911121, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38900792

RESUMO

Euchromatin is an accessible phase of genetic material containing genes that encode proteins with increased expression levels. The structure of euchromatin in vitro has been described as a 30-nm fiber formed from ordered nucleosome arrays. However, recent advances in microscopy have revealed an in vivo euchromatin architecture that is much more disordered, characterized by variable-length linker DNA and sporadic nucleosome clusters. In this work, we develop a theoretical model to elucidate factors contributing to the disordered in vivo architecture of euchromatin. We begin by developing a 1D model of nucleosome positioning that captures the interactions between bound epigenetic reader proteins to predict the distribution of DNA linker lengths between adjacent nucleosomes. We then use the predicted linker lengths to construct 3D chromatin configurations consistent with the physical properties of DNA within the nucleosome array, and we evaluate the distribution of nucleosome cluster sizes in those configurations. Our model reproduces experimental cluster-size distributions, which are dramatically influenced by the local pattern of epigenetic marks and the concentration of reader proteins. Based on our model, we attribute the disordered arrangement of euchromatin to the heterogeneous binding of reader proteins and subsequent short-range interactions between bound reader proteins on adjacent nucleosomes. By replicating experimental results with our physics-based model, we propose a mechanism for euchromatin organization in the nucleus that impacts gene regulation and the maintenance of epigenetic marks.


Assuntos
Epigênese Genética , Eucromatina , Nucleossomos , Nucleossomos/metabolismo , Nucleossomos/genética , Eucromatina/metabolismo , Eucromatina/genética , DNA/metabolismo , DNA/química
15.
Genes (Basel) ; 15(6)2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38927609

RESUMO

MOTIVATION: High-resolution Hi-C data, capable of detecting chromatin features below the level of Topologically Associating Domains (TADs), significantly enhance our understanding of gene regulation. Micro-C, a variant of Hi-C incorporating a micrococcal nuclease (MNase) digestion step to examine interactions between nucleosome pairs, has been developed to overcome the resolution limitations of Hi-C. However, Micro-C experiments pose greater technical challenges compared to Hi-C, owing to the need for precise MNase digestion control and higher-resolution sequencing. Therefore, developing computational methods to derive Micro-C data from existing Hi-C datasets could lead to better usage of a large amount of existing Hi-C data in the scientific community and cost savings. RESULTS: We developed C2c ("high" or upper case C to "micro" or lower case c), a computational tool based on a residual neural network to learn the mapping between Hi-C and Micro-C contact matrices and then predict Micro-C contact matrices based on Hi-C contact matrices. Our evaluation results show that the predicted Micro-C contact matrices reveal more chromatin loops than the input Hi-C contact matrices, and more of the loops detected from predicted Micro-C match the promoter-enhancer interactions. Furthermore, we found that the mutual loops from real and predicted Micro-C better match the ChIA-PET data compared to Hi-C and real Micro-C loops, and the predicted Micro-C leads to more TAD-boundaries detected compared to the Hi-C data. The website URL of C2c can be found in the Data Availability Statement.


Assuntos
Cromatina , Cromatina/genética , Humanos , Biologia Computacional/métodos , Redes Neurais de Computação , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genética , Nucleossomos/genética , Software
16.
Methods Mol Biol ; 2832: 33-46, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38869785

RESUMO

Nucleosome occupancy plays an important role in chromatin compaction, affecting biological processes by hampering the binding of cis-acting elements such as transcription factors, RNA polymerase machinery, and coregulatory. Accessible regions allow for cis-acting elements to bind DNA and regulate transcription. Here, we detail our protocol to profile nucleosome occupancy and chromatin structure dynamics under drought stress at the genome-wide scale using micrococcal nuclease (MNase) digestion. Combining variable MNase concentration treatments and high-throughput sequencing, we investigate the changes in the overall chromatin state using bread wheat samples from an exemplary drought experiment.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Nuclease do Micrococo , Nucleossomos , Estresse Fisiológico , Triticum , Nucleossomos/metabolismo , Nucleossomos/genética , Nuclease do Micrococo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Estresse Fisiológico/genética , Triticum/genética , Triticum/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Cromatina/metabolismo , Cromatina/genética , Plantas/genética , Plantas/metabolismo
17.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38830772

RESUMO

Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin.


Assuntos
Montagem e Desmontagem da Cromatina , DNA , Regulação da Expressão Gênica , Nucleossomos , Transcrição Gênica , Nucleossomos/metabolismo , Nucleossomos/genética , Humanos , Montagem e Desmontagem da Cromatina/genética , DNA/genética , DNA/metabolismo , Cromatina/metabolismo , Cromatina/genética , Genoma Humano , Sequência de Bases
18.
Sci Adv ; 10(23): eadn2955, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38848364

RESUMO

The hierarchical chromatin organization begins with formation of nucleosomes, which fold into chromatin domains punctuated by boundaries and ultimately chromosomes. In a hierarchal organization, lower levels shape higher levels. However, the dependence of higher-order 3D chromatin organization on the nucleosome-level organization has not been studied in cells. We investigated the relationship between nucleosome-level organization and higher-order chromatin organization by perturbing nucleosomes across the genome by deleting Imitation SWItch (ISWI) and Chromodomain Helicase DNA-binding (CHD1) chromatin remodeling factors in budding yeast. We find that changes in nucleosome-level properties are accompanied by changes in 3D chromatin organization. Short-range chromatin contacts up to a few kilo-base pairs decrease, chromatin domains weaken, and boundary strength decreases. Boundary strength scales with accessibility and moderately with width of nucleosome-depleted region. Change in nucleosome positioning seems to alter the stiffness of chromatin, which can affect formation of chromatin contacts. Our results suggest a biomechanical "bottom-up" mechanism by which nucleosome distribution across genome shapes 3D chromatin organization.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Genoma Fúngico , Nucleossomos , Saccharomyces cerevisiae , Nucleossomos/genética , Nucleossomos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Adenosina Trifosfatases
19.
Int J Mol Sci ; 25(12)2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38928493

RESUMO

The incorporation of histone variants has structural ramifications on nucleosome dynamics and stability. Due to their unique sequences, histone variants can alter histone-histone or histone-DNA interactions, impacting the folding of DNA around the histone octamer and the overall higher-order structure of chromatin fibers. These structural modifications alter chromatin compaction and accessibility of DNA by transcription factors and other regulatory proteins to influence gene regulatory processes such as DNA damage and repair, as well as transcriptional activation or repression. Histone variants can also generate a unique interactome composed of histone chaperones and chromatin remodeling complexes. Any of these perturbations can contribute to cellular plasticity and the progression of human diseases. Here, we focus on a frequently overlooked group of histone variants lying within the four human histone gene clusters and their contribution to breast cancer.


Assuntos
Neoplasias da Mama , Histonas , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Histonas/metabolismo , Histonas/genética , Feminino , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Cromatina/genética , Nucleossomos/metabolismo , Família Multigênica
20.
Phys Rev E ; 109(5-1): 054411, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38907407

RESUMO

Genomic regions can acquire heritable epigenetic states through unique histone modifications, which lead to stable gene expression patterns without altering the underlying DNA sequence. However, the relationship between chromatin conformational dynamics and epigenetic stability is poorly understood. In this paper, we propose kinetic models to investigate the dynamic fluctuations of histone modifications and the spatial interactions between nucleosomes. Our model explicitly incorporates the influence of chemical modifications on the structural stability of chromatin and the contribution of chromatin contacts to the cooperative nature of chemical reactions. Through stochastic simulations and analytical theory, we have discovered distinct steady-state outcomes in different kinetic regimes, resembling a dynamical phase transition. Importantly, we have validated that the emergence of this transition, which occurs on biologically relevant timescales, is robust against variations in model design and parameters. Our findings suggest that the viscoelastic properties of chromatin and the timescale at which it transitions from a gel-like to a liquidlike state significantly impact dynamic processes that occur along the one-dimensional DNA sequence.


Assuntos
Cromatina , Histonas , Cromatina/metabolismo , Cromatina/química , Histonas/metabolismo , Histonas/química , Modelos Moleculares , Transição de Fase , Cinética , Nucleossomos/metabolismo , Nucleossomos/química , DNA/metabolismo , DNA/química , Processos Estocásticos
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