scholarly journals Multiple parameters shape the 3D chromatin structure of single nuclei

2022 ◽  
Author(s):  
Markus Goetz ◽  
Olivier Messina ◽  
Sergio Espinola ◽  
Jean-Bernard Fiche ◽  
Marcelo Nollmann
Keyword(s):  
Spatial Organization ◽  
Chromatin Condensation ◽  
Large Cell ◽  
Conformational Space ◽  
Chromatin Conformation ◽  
Multiple Parameters ◽  
Topologically Associating Domains ◽  
Active Transcription ◽  
3D Chromatin Structure ◽  
Drosophila Embryos

The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei displayed strong insulation even before TADs emerged. Moreover, active transcription within a TAD led to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affected insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

1995 ◽  
Vol 53 ◽  
pp. 764-765
Author(s):  
W.F. Marshall ◽  
A.F. Dernburg ◽  
B. Harmon ◽  
J.W. Sedat
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Condensation ◽  
Three Dimensional ◽  
Physiological Role ◽  
Nuclear Surface ◽  
Intact Cells ◽  
Molecular Determinants ◽  
Surface Harmonic ◽  
Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

Laser scanning confocal microscopic analysis of cytoskeletal and nuclear reorganization in live Drosophila embryos

1993 ◽  
Vol 51 ◽  
pp. 174-175
Author(s):  
William Theurkauf
Keyword(s):  
Laser Scanning ◽  
Microscopic Analysis ◽  
Large Cell ◽  
Early Embryo ◽  
Drosophila Embryo ◽  
Cortical Actin ◽  
Nuclear Reorganization ◽  
Cytoskeletal Elements ◽  
Microtubule Structure ◽  
Drosophila Embryos

Cell division in eucaryotes depends on coordinated changes in nuclear and cytoskeletal components. In Drosophila melanogaster embryos, the first 13 nuclear divisions occur without cytokinesis. During the final four divisions, nuclei divide in a uniform monolayer at the surface of the embryo. These surface divisions are accompanied by dramatic changes in cortical actin and microtubule structure (Karr and Alberts, 1986), and inhibitor studies indicate that these changes are essential to orderly mitosis (Zalokar and Erk, 1976). Because the early embryo is syncytial, fluorescent probes introduced by microinjection are incorporated in structures associated with all of the nuclei in the blastoderm. In addition, the nuclei divide synchronously every 10 to 20 min. These characteristics make the syncytial blastoderm embryo an excellent system for the analysis of mitotic reorganization of both nuclear and cytoskeletal elements. However, the Drosophila embryo is a large cell, and resolution of cytoskeletal filaments and nuclear structure is hampered by out-of focus signal.

scholarly journals OnTAD: hierarchical domain structure reveals the divergence of activity among TADs and boundaries

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Lin An ◽  
Tao Yang ◽  
Jiahao Yang ◽  
Johannes Nuebler ◽  
Guanjue Xiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Domain Structure ◽  
High Frequency ◽  
Spatial Organization ◽  
Structural Units ◽  
Regulatory Interactions ◽  
Link Type ◽  
Topologically Associating Domains

AbstractThe spatial organization of chromatin in the nucleus has been implicated in regulating gene expression. Maps of high-frequency interactions between different segments of chromatin have revealed topologically associating domains (TADs), within which most of the regulatory interactions are thought to occur. TADs are not homogeneous structural units but appear to be organized into a hierarchy. We present OnTAD, an optimized nested TAD caller from Hi-C data, to identify hierarchical TADs. OnTAD reveals new biological insights into the role of different TAD levels, boundary usage in gene regulation, the loop extrusion model, and compartmental domains. OnTAD is available at https://github.com/anlin00007/OnTAD.

scholarly journals Heat shock results in cell cycle delay and synchronisation of mitotic domains in cellularised Drosophila melanogaster embryos

1993 ◽  
Vol 105 (3) ◽  
pp. 711-720 ◽  
Author(s):  
G. Maldonado-Codina ◽  
S. Llamazares ◽  
D.M. Glover
Keyword(s):  
Cell Cycle ◽  
Heat Shock ◽  
Chromatin Condensation ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Shock ◽  
Cell Cycle Delay ◽  
Minute Period ◽  
G2 Phase ◽  
Drosophila Embryos

Cells of Drosophila embryos that are subjected to a 37 degrees C temperature shock whilst undergoing the S-phase of cell cycle 14 arrest with their microtubules in an interphase-like state, and with nuclei showing unusual chromatin condensation. They do not recover from this state within a 30 minute period even though extensive gastrulation movements can occur. Cells of embryos heat shocked in G2-phase are delayed in interphase with high levels of cyclins A and B. Within ten minutes recovery from heat shock, cells enter mitosis throughout the embryo. The degradation of the mitotic cyclins A and B in these synchronised mitotic domains does not follow the normal timing, but is delayed. These findings point to a need for caution when interpreting experiments that use the heat shock promoter to study the expression of cell cycle control genes in Drosophila.

scholarly journals Heterochromatin rewiring and domain disruption-mediated chromatin compaction during erythropoiesis

2021 ◽  
Author(s):  
Dong Li ◽  
Fan Wu ◽  
Shuo Zhou ◽  
Xiaojun Huang ◽  
Hsiang-Ying Sherry Lee
Keyword(s):  
Molecular Mechanisms ◽  
Chromatin Condensation ◽  
Nuclear Periphery ◽  
Chromatin Modification ◽  
Three Dimensional ◽  
Molecular Characteristics ◽  
Structural Factors ◽  
Erythroid Progenitors ◽  
Chromatin Compaction ◽  
Active Transcription

Development of mammalian red blood cells involves progressive chromatin compaction and subsequent enucleation in terminal stages of differentiation, but the molecular mechanisms underlying the three-dimensional chromatin reorganization and compaction remains obscure. Here, we systematically analyze the distinct features of higher-order chromatin in purified populations of primary human erythroblasts. Our results reveal that while heterochromatin regions undergo substantial compression, select transcription competent regions with active transcription signature are preferentially maintained to achieve a highly-compacted yet functional chromatin state in terminal erythropoiesis, which is about 20-30% of the nuclear volume compared to that of erythroid progenitors. While the partition of euchromatic and heterochromatic regions (compartment A and B) remain mostly unchanged, H3K9me3 marks relocalize to the nuclear periphery and a significant number of H3K9me3 long-range interactions are formed in the three-dimensional rewiring during terminal erythroid chromatin condensation. Moreover, ~63% of the topologically associating domain (TAD) boundaries are disrupted, while certain TADs with active chromatin modification are selectively maintained during terminal erythropoiesis. The most well-maintained TADs are enriched for chromatin structural factors CTCF and SMC3, as well as factors and marks of the active transcription state. Finally, we demonstrate that the erythroid master regulator GATA1 involves in safeguarding select essential chromatin domains during terminal erythropoiesis. Our study therefore delineate the molecular characteristics of a development-driven chromatin compaction process, which reveals transcription competence as a key determinant of the select domain maintenance to ensure appropriate gene expression during immense chromatin compaction.

scholarly journals rDNA nascent transcripts promote a unique spatial organization during mouse early development

2021 ◽  
Author(s):  
Martine Chebrout ◽  
Maimouna Coura Kone ◽  
Habib U. Jan ◽  
Marie Cournut ◽  
Martine Letheule ◽  
...  
Keyword(s):  
Early Development ◽  
Spatial Organization ◽  
Ribosomal Genes ◽  
Cell Stage ◽  
Major Satellite ◽  
Cell Cycles ◽  
Embryonic Genome ◽  
Active Transcription ◽  
Polymerase I ◽  
Nascent Transcripts

AbstractDuring the first cell cycles of the early development, the chromatin of the embryo is highly reprogrammed alongside that embryonic genome starts its own transcription. The spatial organization of the genome is a major process that contributes to regulating gene transcription in time and space, however, it is poorly studied in the context of early embryos. To study the cause and effect link between transcription and spatial organization in embryos, we focused on the ribosomal genes, that are first silent and begin to transcribe during the 2-cell stage in mouse. We demonstrated that ribosomal sequences are spatially organized in a very peculiar manner from the 2-cell to the 16-cell stage with transcription and processing of ribosomal RNAs excluding mutually. Using drugs inhibiting the RNA polymerase I, we show that this organization, totally different from somatic cells, depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors major satellite sequences transcription after the 4-cell stage.

scholarly journals Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Mustafa Mir ◽  
Michael R Stadler ◽  
Stephan A Ortiz ◽  
Colleen E Hannon ◽  
Melissa M Harrison ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Molecule ◽  
High Speed ◽  
Light Sheet ◽  
Editorial Note ◽  
Regulation Of Transcription ◽  
Light Sheet Microscopy ◽  
4D Imaging ◽  
Active Transcription ◽  
Drosophila Embryos

The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. Here, we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on our observations, we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

6 ALTERED CHROMATIN CONFORMATION IN BOVINE SPERMATOZOA PERTURBS DYNAMIC DNA METHYLATION IN THE MALE PRONUCLEUS AFTER FERTILIZATION IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 150 ◽  
Author(s):  
M. B. Rahman ◽  
M. M. Kamal ◽  
T. Rijsselaere ◽  
L. Vandaele ◽  
M. Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
De Novo ◽  
Chromatin Condensation ◽  
Sperm Chromatin ◽  
Chromatin Conformation ◽  
Epigenetic Marks ◽  
Time Points ◽  
Male Pronucleus ◽  
De Novo Methylation

Soon after fertilization, mammalian zygotes need proper DNA methylation reprogramming, at which time the epigenetic marks that the oocyte and sperm have acquired during gametogenesis are erased to allow totipotent zygotic development. Aberrant epigenetic marks in the paternal genome are thought to be associated with altered chromatin condensation in spermatozoa of suboptimal quality. We have recently reported that heat stress on bulls during germ cell development, especially at the spermiogenesis stage, altered sperm chromatin condensation. The objective of this study was to investigate dynamic DNA methylation reprogramming in the male pronucleus after fertilization of oocytes with sperm known to have altered chromatin conformation. To evaluate dynamic DNA methylation reprogramming, zygotes collected at 3 different time points [i.e. 12, 18, and 24 h post-insemination (hpi)] were immunocytochemically investigated using an antibody against 5-methylcytosine (5mC). The total fluorescence intensity of the male pronuclei (n = 89, ≥25 in each group) was measured by ImageJ and data were analyzed by ANOVA. The DNA methylation pattern in male pronuclei when oocytes were fertilized with heat-stressed sperm did not change between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. The results of this study indicated that dynamic DNA methylation reprogramming patterns such as DNA demethylation followed by de novo methylation in the male pronucleus soon after fertilization were altered when oocytes were fertilized with heat-stressed sperm. In conclusion, altered sperm chromatin conformation due to heat stress perturbs dynamic DNA methylation reprogramming in the male pronucleus, which may hamper nuclear totipotency and embryo survival.

scholarly journals Topology, structures, and energy landscapes of human chromosomes

2015 ◽  
Vol 112 (19) ◽  
pp. 6062-6067 ◽  
Author(s):  
Bin Zhang ◽  
Peter G. Wolynes
Keyword(s):  
Liquid Crystalline ◽  
Spatial Organization ◽  
Human Chromosomes ◽  
Energy Landscapes ◽  
Interphase Chromosome ◽  
Chromosome Conformation ◽  
Information Theoretic ◽  
Knot Invariants ◽  
Topologically Associating Domains ◽  
And Topology

Chromosome conformation capture experiments provide a rich set of data concerning the spatial organization of the genome. We use these data along with a maximum entropy approach to derive a least-biased effective energy landscape for the chromosome. Simulations of the ensemble of chromosome conformations based on the resulting information theoretic landscape not only accurately reproduce experimental contact probabilities, but also provide a picture of chromosome dynamics and topology. The topology of the simulated chromosomes is probed by computing the distribution of their knot invariants. The simulated chromosome structures are largely free of knots. Topologically associating domains are shown to be crucial for establishing these knotless structures. The simulated chromosome conformations exhibit a tendency to form fibril-like structures like those observed via light microscopy. The topologically associating domains of the interphase chromosome exhibit multistability with varying liquid crystalline ordering that may allow discrete unfolding events and the landscape is locally funneled toward “ideal” chromosome structures that represent hierarchical fibrils of fibrils.

scholarly journals CNEr: a toolkit for exploring extreme noncoding conservation

2019 ◽  
Author(s):  
Ge Tan ◽  
Dimitris Polychronopoulos ◽  
Boris Lenhard
Keyword(s):  
Large Scale ◽  
Fruit Fly ◽  
Tsetse Fly ◽  
Chromatin Conformation ◽  
Developmentally Regulated ◽  
Link Type ◽  
Topologically Associating Domains ◽  
Conserved Noncoding Elements ◽  
Chromatin Biology ◽  
Genome Comparisons

AbstractConserved Noncoding Elements (CNEs) are elements exhibiting extreme noncoding conservation in Metazoan genomes. They cluster around developmental genes and act as long-range enhancers, yet nothing that we know about their function explains the observed conservation levels. Clusters of CNEs coincide with topologically associating domains (TADs), indicating ancient origins and stability of TAD locations. This has suggested further hypotheses about the still elusive origin of CNEs, and has provided a comparative genomics-based method of estimating the position of TADs around developmentally regulated genes in genomes where chromatin conformation capture data is missing. To enable researchers in gene regulation and chromatin biology to start deciphering this phenomenon, we developedCNEr, a R/Bioconductor toolkit for large-scale identification of CNEs and for studying their genomic properties. We applyCNErto two novel genome comparisons - fruit fly vs tsetse fly, and two sea urchin genomes - and report novel insights gained from their analysis. We also show how to reveal interesting characteristics of CNEs by coupling CNEr with existing Bioconductor packages.CNEris available at Bioconductor (https://bioconductor.org/packages/CNEr/) and maintained at github (https://github.com/ge11232002/CNEr).

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