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1.
Nat Commun ; 14(1): 287, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653380

RESUMO

Plasma cell-free DNA (cfDNA) are small molecules generated through a non-random fragmentation procedure. Despite commendable translational values in cancer liquid biopsy, however, the biology of cfDNA, especially the principles of cfDNA fragmentation, remains largely elusive. Through orientation-aware analyses of cfDNA fragmentation patterns against the nucleosome structure and integration with multidimensional functional genomics data, here we report a DNA methylation - nuclease preference - cutting end - size distribution axis, demonstrating the role of DNA methylation as a functional molecular regulator of cfDNA fragmentation. Hence, low-level DNA methylation could increase nucleosome accessibility and alter the cutting activities of nucleases during DNA fragmentation, which further leads to variation in cutting sites and size distribution of cfDNA. We further develop a cfDNA ending preference-based metric for cancer diagnosis, whose performance has been validated by multiple pan-cancer datasets. Our work sheds light on the molecular basis of cfDNA fragmentation towards broader applications in cancer liquid biopsy.


Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Nucleossomos/genética , Metilação de DNA/genética , Fragmentação do DNA , Neoplasias/genética , Biomarcadores Tumorais/genética
3.
J Ovarian Res ; 16(1): 11, 2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36641505

RESUMO

BACKGROUND: Cell-free DNA (cfDNA) is emerging as a potential biomarker for the detection of ovarian cancer (OC). Recently, we reported a method based upon cfDNA whole-genome sequencing data including the nucleosome distribution (nucleosome footprinting NF), terminal signature sequence (motif), DNA fragmentation (fragment), and copy number variation (CNV).In the present study, we explored whether multiomics early screening technology in cfDNA can be applied for early screening of ovarian cancer. METHODS: Fifty-nine patients with OC and 100 healthy controls were included in this prospective study. Cell-free DNA was extracted from plasma and analyzed by low-pass whole-genome sequencing. Genomic features were obtained for all samples of the cohort, including copy number variation (CNV), 5'-end motifs, fragmentation profiles, and nucleosome footprinting (NF). An integrated scoring system termed the OC score was developed based on the performance of these four features. RESULTS: All four features showed diagnostic potential for OC. Based on the unique genome features of cfDNA, the OC score has high accuracy in distinguishing OC patients from healthy controls (AUC 97.7%; sensitivity 94.7%; specificity 98.0%) as a new comprehensive diagnostic method for OC. The OC score showed a gradual trend from healthy controls to OC patients with different stages, especially for early OC monitoring of concern, which achieved a satisfactory sensitivity (85.7%) at a high specificity. CONCLUSIONS: This is the first study evaluating the potential of cell-free DNA for the diagnosis of primary OC using multidimensional early screening technology. We present a promising method to increase the accuracy of prediction in patients with OC.


Assuntos
Ácidos Nucleicos Livres , Neoplasias Ovarianas , Humanos , Feminino , Variações do Número de Cópias de DNA , Estudos Prospectivos , Nucleossomos/genética , Biomarcadores Tumorais/genética , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética
4.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674455

RESUMO

Liquid biopsies have emerged as a minimally invasive cancer detection and monitoring method, which could identify cancer-related alterations in nucleosome or histone levels and modifications in blood, saliva, and urine. Histones, the core component of the nucleosome, are essential for chromatin compaction and gene expression modulation. Increasing evidence suggests that circulating histones and histone complexes, originating from cell death or immune cell activation, could act as promising biomarkers for cancer detection and management. In this review, we provide an overview of circulating histones as a powerful liquid biopsy approach and methods for their detection. We highlight current knowledge on circulating histones in hematologic malignancies and solid cancer, with a focus on their role in cancer dissemination, monitoring, and tumorigenesis. Last, we describe recently developed strategies to identify cancer tissue-of-origin in blood plasma based on nucleosome positioning, inferred from nucleosomal DNA fragmentation footprint, which is independent of the genetic landscape.


Assuntos
Histonas , Neoplasias , Humanos , Histonas/metabolismo , Nucleossomos , Cromatina/genética , Neoplasias/diagnóstico , Neoplasias/genética
5.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36675018

RESUMO

Cell-free DNA molecules are released into the plasma via apoptotic or necrotic events and active release mechanisms, which carry the genetic and epigenetic information of its origin tissues. However, cfDNA is the mixture of various cell fragments, and the efficient enrichment of cfDNA fragments with diagnostic value remains a great challenge for application in the clinical setting. Evidence from recent years shows that cfDNA fragmentomics' characteristics differ in normal and diseased individuals without the need to distinguish the source of the cfDNA fragments, which makes it a promising novel biomarker. Moreover, cfDNA fragmentomics can identify tissue origins by inferring epigenetic information. Thus, further insights into the fragmentomics of plasma cfDNA shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In this review, we focus on the cfDNA fragment characteristics and its potential application, such as fragment length, end motifs, jagged ends, preferred end coordinates, as well as nucleosome footprints, open chromatin region, and gene expression inferred by the cfDNA fragmentation pattern across the genome. Furthermore, we summarize the methods for deducing the tissue of origin by cfDNA fragmentomics.


Assuntos
Ácidos Nucleicos Livres , Humanos , Ácidos Nucleicos Livres/genética , Biomarcadores , Cromatina , Nucleossomos/genética
6.
J Mol Biol ; 435(2): 167913, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36495919

RESUMO

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie between the segments of the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. Through in vitro binding and kinetic studies using nucleosomes, we find that while the ARID domain is required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. In addition, the unstructured linker region between the ARID and PHD1 domains interacts with PHD1 and is necessary for nucleosome binding. Our data suggests a model in which the PHD1 domain inhibits DNA recognition by KDM5C. This inhibitory effect is relieved by the H3 tail, enabling recognition of flanking DNA on the nucleosome. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains break this regulation by enhancing DNA binding, resulting in the loss of specificity of substrate chromatin recognition and rendering demethylase activity lower in the presence of flanking DNA. Our findings suggest a model by which specific XLID mutations could alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.


Assuntos
Cromatina , Histona Desmetilases , Retardo Mental Ligado ao Cromossomo X , Humanos , Cromatina/genética , Cromatina/metabolismo , DNA/química , DNA/metabolismo , Histona Desmetilases/química , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Cinética , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Retardo Mental Ligado ao Cromossomo X/genética , Ligação Proteica , Domínios Proteicos
7.
J Mol Biol ; 435(2): 167916, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36495920

RESUMO

Pioneer transcription factors (pTFs) can bind directly to silent chromatin and promote vital transcriptional programs. Here, by integrating high-resolution nuclear magnetic resonance (NMR) spectroscopy with biochemistry, we reveal new structural and mechanistic insights into the interaction of pluripotency pTFs and functional partners Sox2 and Oct4 with nucleosomes. We find that the affinity and conformation of Sox2 for solvent-exposed nucleosome sites depend strongly on their position and DNA sequence. Sox2, which is partially disordered but becomes structured upon DNA binding and bending, forms a super-stable nucleosome complex at superhelical location +5 (SHL+5) with similar affinity and conformation to that with naked DNA. However, at suboptimal internal and end-positioned sites where DNA may be harder to deform, Sox2 favors partially unfolded and more dynamic states that are encoded in its intrinsic flexibility. Importantly, Sox2 structure and DNA bending can be stabilized by synergistic Oct4 binding, but only on adjacent motifs near the nucleosome edge and with the full Oct4 DNA-binding domain. Further mutational studies reveal that strategically impaired Sox2 folding is coupled to reduced DNA bending and inhibits nucleosome binding and Sox2-Oct4 cooperation, while increased nucleosomal DNA flexibility enhances Sox2 association. Together, our findings fit a model where the site-specific DNA bending propensity and structural plasticity of Sox2 govern distinct modes of nucleosome engagement and modulate Sox2-Oct4 synergism. The principles outlined here can potentially guide pTF site selection in the genome and facilitate interaction with other chromatin factors or chromatin opening in vivo.


Assuntos
DNA , Conformação de Ácido Nucleico , Nucleossomos , Fatores de Transcrição SOXB1 , Sequência de Bases , Cromatina , DNA/química , DNA/metabolismo , Nucleossomos/metabolismo , Domínios Proteicos , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Humanos
8.
J Mol Biol ; 435(2): 167917, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36502880

RESUMO

In addition to the stage of transcriptional initiation, the production of mRNAs is regulated during elongation. Accordingly, the synthesis of mRNAs by RNA polymerase II (RNAPII) in the chromatin context is modulated by various transcript elongation factors. TFIIS is an elongation factor that stimulates the transcript cleavage activity of RNAPII to reactivate stalled elongation complexes at barriers to transcription including nucleosomes. Since Arabidopsis tfIIs mutants grow normally under standard conditions, we have exposed them to heat stress (HS), revealing that tfIIs plants are highly sensitive to elevated temperatures. Transcriptomic analyses demonstrate that particularly HS-induced genes are expressed at lower levels in tfIIs than in wildtype. Mapping the distribution of elongating RNAPII uncovered that in tfIIs plants RNAPII accumulates at the +1 nucleosome of genes that are upregulated upon HS. The promoter-proximal RNAPII accumulation in tfIIs under HS conditions conforms to that observed upon inhibition of the RNAPII transcript cleavage activity. Further analysis of the RNAPII accumulation downstream of transcriptional start sites illustrated that RNAPII stalling occurs at +1 nucleosomes that are depleted in the histone variant H2A.Z upon HS. Therefore, assistance of early transcript elongation by TFIIS is required for reprogramming gene expression to establish plant thermotolerance.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Resposta ao Choque Térmico , Nucleossomos , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Resposta ao Choque Térmico/genética , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo
9.
J Am Chem Soc ; 145(1): 706-716, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36573457

RESUMO

Inspired by the conventional use of ethanol to induce DNA precipitation, ethanol condensation has been applied as a routine method to dynamically tune "bond" lengths (i.e., the surface-to-surface distances between adjacent nanoparticles that are linked by DNA) and thermal stabilities of colloidal crystals involving DNA-linked nanoparticles. However, the underlying mechanism of how the DNA bond that links gold nanoparticles changes in this class of colloidal crystals in response to ethanol remains unclear. Here, we conducted a series of all-atom molecular dynamic (MD) simulations to explore the free energy landscape for DNA condensation and decondensation. Our simulations confirm that DNA condensation is energetically much more favorable under 80% ethanol conditions than in pure water, as a result of ethanol's role in enhancing electrostatic interactions between oppositely charged species. Moreover, the condensed DNA adopts B-form in pure water and A-form in 80% ethanol, which indicates that the higher-order transition does not affect DNA's conformational preferences. We further propose a nucleosome-like supercoiled model for the DNA condensed state, and we show that the DNA end-to-end distance derived from this model matches the experimentally measured DNA bond length of about 3 nm in the fully condensed state for DNA where the measured length is 16 nm in water. Overall, this study provides an atomistic understanding of the mechanism underlying ethanol-induced condensation and water-induced decondensation, while our proposed nucleosome-like model allows the design of new strategies for interpreting experimental studies of DNA condensation.


Assuntos
Nanopartículas Metálicas , Nucleossomos , Etanol/química , Ouro , DNA/química , Água/química
10.
Proc Natl Acad Sci U S A ; 120(1): e2216611120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36574674

RESUMO

Small molecules that bind in the minor groove of DNA are in clinical use as antibiotics and antitumor drugs. Two members of this class of molecules, netropsin and chromomycin, are shown here to displace DNA from the nucleosome and promote transfer of the histone octamer to an acceptor protein. The effects of these groove-binding molecules are exploited to address an outstanding problem in the mechanism of the RSC chromatin remodeling complex. RSC and other remodeling complexes are DNA translocases, acting near the center of the nucleosomal DNA, but translocation is apparently impossible because DNA cannot slide across the histone surface in the nucleosome. Netropsin and chromomycin promote the release of DNA from the histone surface, enhance the formation of a RSC-nucleosome complex, and synergize with RSC in chromatin remodeling. These findings are in keeping with an involvement of bulge translocation in chromatin remodeling.


Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Histonas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Montagem e Desmontagem da Cromatina , Netropsina/metabolismo , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina
11.
J Phys Chem B ; 127(1): 37-44, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36537668

RESUMO

Force manipulation on the biological entities from living cells to protein molecules has revealed many mechanical details of cell biology from resolving folding and unfolding pathways to finding molecular interaction forces. A nucleosome is the basic repeating unit of chromatin where the histone octamer is wrapped by DNA, important for gene stability and regulation. How the inner side of the DNA gets accessed by other DNA binding molecules has been a puzzle that has been intensively studied and debated, important to epigenetics, gene stability, and regulations. Here we report our observation of spontaneous ruptures of human nucleosomes under pico-Newton (pN) compressive force. The amplitude of the compressive force, a squeezing rather than pulling force, involved in our experiment is tens of pN, which can be thermally available by biological force fluctuation at room temperature and under physiological conditions. This kind of structural rupture can loosen up the DNA around the histone, which in turn makes the DNA accessible to transcription and epigenetic modifications.


Assuntos
Histonas , Nucleossomos , Humanos , Histonas/química , Cromatina , DNA/química , Epigênese Genética
12.
Nat Struct Mol Biol ; 30(1): 31-37, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36536103

RESUMO

To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPß. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns. The DNA binding domain of PU.1 binds mononucleosomes, but requires an additional intrinsically disordered domain to bind and open compacted chromatin. The canonical mammalian SWI/SNF (cBAF) remodeler was unable to act upon two forms of locally open chromatin unless cBAF was enabled by a separate transactivation domain of PU.1. cBAF potentiates the PU.1 DNA binding domain to weakly open chromatin in the absence of the PU.1 disordered domain. Our findings reveal a hierarchy by which chromatin is opened and show that pioneer factors can provide specificity for action by nucleosome remodelers.


Assuntos
Cromatina , Nucleossomos , Animais , Fatores de Transcrição/metabolismo , DNA , Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina , Mamíferos/genética
13.
J Transl Med ; 20(1): 557, 2022 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-36463222

RESUMO

BACKGROUND: Lymph node metastasis (LNM) is one of the most important factors affecting the prognosis of breast cancer. The accurate evaluation of lymph node status is useful to predict the outcomes of patients and guide the choice of cancer treatment. However, there is still lack of a low-cost non-invasive method to assess the status of axillary lymph node (ALN). Gene expression signature has been used to assess lymph node metastasis status of breast cancer. In addition, nucleosome footprint of cell-free DNA (cfDNA) carries gene expression information of its original tissues, so it may be used to evaluate the axillary lymph node status in breast cancer. METHODS: In this study, we found that the cfDNA nucleosome footprints between the ALN-positive patients and ALN-negative patients showed different patterns by implementing whole-genome sequencing (WGS) to detect 15 ALN-positive and 15 ALN-negative patients. In order to further evaluate its potential for assessing ALN status, we developed a classifier with multiple machine learning models by using 330 WGS data of cfDNA from 162 ALN-positive and 168 ALN-negative samples to distinguish these two types of patients. RESULTS: We found that the promoter profiling between the ALN-positive patients and ALN-negative patients showed distinct patterns. In addition, we observed 1071 genes with differential promoter coverage and their functions were closely related to tumorigenesis. We found that the predictive classifier based on promoter profiling with a support vector machine model, named PPCNM, produced the largest area under the curve of 0.897 (95% confidence interval 0.86-0.93). CONCLUSIONS: These results indicate that promoter profiling can be used to distinguish ALN-positive patients from ALN-negative patients, which may be helpful to guide the choice of cancer treatment.


Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , Humanos , Feminino , Neoplasias da Mama/genética , Metástase Linfática/genética , Nucleossomos , Linfonodos , Ácidos Nucleicos Livres/genética
14.
Nat Commun ; 13(1): 7475, 2022 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-36463275

RESUMO

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we develop Griffin, a framework for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing data. Griffin employs a GC correction procedure tailored to variable cfDNA fragment sizes, which generates a better representation of chromatin accessibility and improves the accuracy of cancer detection and tumor subtype classification. We demonstrate estrogen receptor subtyping from cfDNA in metastatic breast cancer. We predict estrogen receptor subtype in 139 patients with at least 5% detectable circulating tumor DNA with an area under the receive operator characteristic curve (AUC) of 0.89 and validate performance in independent cohorts (AUC = 0.96). In summary, Griffin is a framework for accurate tumor subtyping and can be generalizable to other cancer types for precision oncology applications.


Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Ácidos Nucleicos Livres/genética , Nucleossomos/genética , Neoplasias/diagnóstico , Neoplasias/genética , Receptores de Estrogênio , Medicina de Precisão
15.
Mol Cell ; 82(23): 4401-4402, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36459981

RESUMO

Not only does Marseillevirus bear the name of the city where it was identified, it also encompasses its values and what makes Marseille a wonderful city. Marseillevirus is unique and intriguing. As such, Bryson et al. in this issue of Molecular Cell reveal how virion-associated Marseillevirus DNA is packed with nucleosomes.


Assuntos
DNA , Nucleossomos , Nucleossomos/genética , DNA/genética , Vírion/genética
16.
PLoS Genet ; 18(12): e1010535, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36508455

RESUMO

Noise in expression of individual genes gives rise to variations in activity of cellular pathways and generates heterogeneity in cellular phenotypes. Phenotypic heterogeneity has important implications for antibiotic persistence, mutation penetrance, cancer growth and therapy resistance. Specific molecular features such as the presence of the TATA box sequence and the promoter nucleosome occupancy have been associated with noise. However, the relative importance of these features in noise regulation is unclear and how well these features can predict noise has not yet been assessed. Here through an integrated statistical model of gene expression noise in yeast we found that the number of regulating transcription factors (TFs) of a gene was a key predictor of noise, whereas presence of the TATA box and the promoter nucleosome occupancy had poor predictive power. With an increase in the number of regulatory TFs, there was a rise in the number of cooperatively binding TFs. In addition, an increased number of regulatory TFs meant more overlaps in TF binding sites, resulting in competition between TFs for binding to the same region of the promoter. Through modeling of TF binding to promoter and application of stochastic simulations, we demonstrated that competition and cooperation among TFs could increase noise. Thus, our work uncovers a process of noise regulation that arises out of the dynamics of gene regulation and is not dependent on any specific transcription factor or specific promoter sequence.


Assuntos
Nucleossomos , Fatores de Transcrição , Nucleossomos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ligação Proteica , Sítios de Ligação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Expressão Gênica
17.
Nat Commun ; 13(1): 7644, 2022 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-36496390

RESUMO

BAF and PBAF are mammalian SWI/SNF family chromatin remodeling complexes that possess multiple histone/DNA-binding subunits and create nucleosome-depleted/free regions for transcription activation. Despite previous structural studies and recent advance of SWI/SNF family complexes, it remains incompletely understood how PBAF-nucleosome complex is organized. Here we determined structure of 13-subunit human PBAF in complex with acetylated nucleosome in ADP-BeF3-bound state. Four PBAF-specific subunits work together with nine BAF/PBAF-shared subunits to generate PBAF-specific modular organization, distinct from that of BAF at various regions. PBAF-nucleosome structure reveals six histone-binding domains and four DNA-binding domains/modules, the majority of which directly bind histone/DNA. This multivalent nucleosome-binding pattern, not observed in previous studies, suggests that PBAF may integrate comprehensive chromatin information to target genomic loci for function. Our study reveals molecular organization of subunits and histone/DNA-binding domains/modules in PBAF-nucleosome complex and provides structural insights into PBAF-mediated nucleosome association complimentary to the recently reported PBAF-nucleosome structure.


Assuntos
Proteínas Cromossômicas não Histona , Nucleossomos , Animais , Humanos , Nucleossomos/genética , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Histonas/genética , Histonas/metabolismo , Montagem e Desmontagem da Cromatina , Mamíferos/genética
18.
Nat Commun ; 13(1): 7698, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36509793

RESUMO

The cohesin complex participates in many structural and functional aspects of genome organization. Cohesin recruitment onto chromosomes requires nucleosome-free DNA and the Scc2-Scc4 cohesin loader complex that catalyzes topological cohesin loading. Additionally, the cohesin loader facilitates promoter nucleosome clearance in a yet unknown way, and it recognizes chromatin receptors such as the RSC chromatin remodeler. Here, we explore the cohesin loader-RSC interaction. Amongst multi-pronged contacts by Scc2 and Scc4, we find that Scc4 contacts a conserved patch on the RSC ATPase motor module. The cohesin loader directly stimulates in vitro nucleosome sliding by RSC, providing an explanation how it facilitates promoter nucleosome clearance. Furthermore, we observe cohesin loader interactions with a wide range of chromatin remodelers. Our results provide mechanistic insight into how the cohesin loader recognizes, as well as influences, the chromatin landscape, with implications for our understanding of human developmental disorders including Cornelia de Lange and Coffin-Siris syndromes.


Assuntos
Micrognatismo , Proteínas de Saccharomyces cerevisiae , Humanos , Cromatina , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Nucleossomos , Segregação de Cromossomos
19.
Phys Med Biol ; 68(1)2022 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-36533598

RESUMO

Objective. To develop a metaphase chromosome model representing the complete genome of a human lymphocyte cell to support microscopic Monte Carlo (MMC) simulation-based radiation-induced DNA damage studies.Approach. We first employed coarse-grained polymer physics simulation to obtain a rod-shaped chromatid segment of 730 nm in diameter and 460 nm in height to match Hi-C data. We then voxelized the segment with a voxel size of 11 nm per side and connected the chromatid with 30 types of pre-constructed nucleosomes and 6 types of linker DNAs in base pair (bp) resolutions. Afterward, we piled different numbers of voxelized chromatid segments to create 23 pairs of chromosomes of 1-5µm long. Finally, we arranged the chromosomes at the cell metaphase plate of 5.5µm in radius to create the complete set of metaphase chromosomes. We implemented the model in gMicroMC simulation by denoting the DNA structure in a four-level hierarchical tree: nucleotide pairs, nucleosomes and linker DNAs, chromatid segments, and chromosomes. We applied the model to compute DNA damage under different radiation conditions and compared the results to those obtained with G0/G1 model and experimental measurements. We also performed uncertainty analysis for relevant simulation parameters.Main results. The chromatid segment was successfully voxelized and connected in bps resolution, containing 26.8 mega bps (Mbps) of DNA. With 466 segments, we obtained the metaphase chromosome containing 12.5 Gbps of DNA. Applying it to compute the radiation-induced DNA damage, the obtained results were self-consistent and agreed with experimental measurements. Through the parameter uncertainty study, we found that the DNA damage ratio between metaphase and G0/G1 phase models was not sensitive to the chemical simulation time. The damage was also not sensitive to the specific parameter settings in the polymer physics simulation, as long as the produced metaphase model followed a similar contact map distribution.Significance. Experimental data reveal that ionizing radiation induced DNA damage is cell cycle dependent. Yet, DNA chromosome models, except for the G0/G1 phase, are not available in the state-of-the-art MMC simulation. For the first time, we successfully built a metaphase chromosome model and implemented it into MMC simulation for radiation-induced DNA damage computation.


Assuntos
Dano ao DNA , Nucleossomos , Humanos , Metáfase , Radiação Ionizante , DNA , Polímeros
20.
Epigenetics Chromatin ; 15(1): 41, 2022 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-36544209

RESUMO

BACKGROUND: Regulatory elements such as promoters, enhancers, and insulators interact each other to mediate molecular processes. To capture chromatin interactions of regulatory elements, 3C-derived methods such as Hi-C and Micro-C are developed. Here, we generated and analyzed Hi-C, Micro-C, and promoter capture Micro-C datasets with different sequencing depths to study chromatin interactions of regulatory elements and nucleosome positions in human prostate cancer cells. RESULTS: Compared to Hi-C, Micro-C identifies more high-resolution loops, including ones around structural variants. By evaluating the effect of sequencing depth, we revealed that more than 2 billion reads of Micro-C are needed to detect chromatin interactions at 1 kb resolution. Moreover, we found that deep-sequencing identifies additional long-range loops that are longer than 1 Mb in distance. Furthermore, we found that more than 50% of the loops are involved in insulators while less than 10% of the loops are promoter-enhancer loops. To comprehensively capture chromatin interactions that promoters are involved in, we performed promoter capture Micro-C. Promoter capture Micro-C identifies loops near promoters with a lower amount of sequencing reads. Sequencing of 160 million reads of promoter capture Micro-C resulted in reaching a plateau of identifying loops. However, there was still a subset of promoters that are not involved in loops even after deep-sequencing. By integrating Micro-C with NOMe-seq and ChIP-seq, we found that active promoters involved in loops have a more accessible region with lower levels of DNA methylation and more highly phased nucleosomes, compared to active promoters that are not involved in loops. CONCLUSION: We determined the required sequencing depth for Micro-C and promoter capture Micro-C to generate high-resolution chromatin interaction maps and loops. We also investigated the effect of sequencing coverage of Hi-C, Micro-C, and promoter capture Micro-C on detecting chromatin loops. Our analyses suggest the presence of distinct regulatory element groups, which are differently involved in nucleosome positions and chromatin interactions. This study does not only provide valuable insights on understanding chromatin interactions of regulatory elements, but also present guidelines for designing research projects on chromatin interactions among regulatory elements.


Assuntos
Cromatina , Nucleossomos , Humanos , Cromatina/genética , Nucleossomos/genética , Sequências Reguladoras de Ácido Nucleico , Regiões Promotoras Genéticas , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos
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