Chromosomal G-dark Bands Determine the Spatial Organization of Centromeric Heterochromatin in the Nucleus

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric α-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or throughtrans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric α-satellite DNA in human lymphoid cells synchronized at G0/G1is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric α-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that “chromosomal environment” plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.

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Architectural proteins CTCF and cohesin have distinct roles in modulating the higher order structure and expression of the CFTR locus

Nucleic Acids Research ◽

10.1093/nar/gku648 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9612-9622 ◽

Cited By ~ 22

Author(s):

Nehal Gosalia ◽

Daniel Neems ◽

Jenny L. Kerschner ◽

Steven T. Kosak ◽

Ann Harris

Keyword(s):

Nuclear Periphery ◽

Three Dimensional ◽

Higher Order ◽

Ctcf Binding ◽

Cftr Gene ◽

Dimensional Structure ◽

Order Structure ◽

Cis Acting ◽

Architectural Proteins ◽

Enhancer Blocking

Abstract Higher order chromatin structures across the genome are maintained in part by the architectural proteins CCCTC binding factor (CTCF) and the cohesin complex, which co-localize at many sites across the genome. Here, we examine the role of these proteins in mediating chromatin structure at the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encompasses nearly 200 kb flanked by CTCF-binding enhancer-blocking insulator elements and is regulated by cell-type-specific intronic enhancers, which loop to the promoter in the active locus. SiRNA-mediated depletion of CTCF or the cohesin component, RAD21, showed that these two factors have distinct roles in regulating the higher order organization of CFTR. CTCF mediates the interactions between CTCF/cohesin binding sites, some of which have enhancer-blocking insulator activity. Cohesin shares this tethering role, but in addition stabilizes interactions between the promoter and cis-acting intronic elements including enhancers, which are also dependent on the forkhead box A1/A2 (FOXA1/A2) transcription factors (TFs). Disruption of the three-dimensional structure of the CFTR gene by depletion of CTCF or RAD21 increases gene expression, which is accompanied by alterations in histone modifications and TF occupancy across the locus, and causes internalization of the gene from the nuclear periphery.

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Spatial Organization of Chromatin: Transcriptional Control of Adaptive Immune Cell Development

Frontiers in Immunology ◽

10.3389/fimmu.2021.633825 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jagan M. R. Pongubala ◽

Cornelis Murre

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Chromatin Structure ◽

Transcriptional Control ◽

Spatial Organization ◽

Structural Integrity ◽

Immune Cell ◽

Three Dimensional ◽

Spatial Proximity ◽

Gene Promoters

Higher-order spatial organization of the genome into chromatin compartments (permissive and repressive), self-associating domains (TADs), and regulatory loops provides structural integrity and offers diverse gene regulatory controls. In particular, chromatin regulatory loops, which bring enhancer and associated transcription factors in close spatial proximity to target gene promoters, play essential roles in regulating gene expression. The establishment and maintenance of such chromatin loops are predominantly mediated involving CTCF and the cohesin machinery. In recent years, significant progress has been made in revealing how loops are assembled and how they modulate patterns of gene expression. Here we will discuss the mechanistic principles that underpin the establishment of three-dimensional (3D) chromatin structure and how changes in chromatin structure relate to alterations in gene programs that establish immune cell fate.

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The role of nuclear organization in trans-splicing based expression of heat shock protein 90 in Giardia lamblia

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009810 ◽

2021 ◽

Vol 15 (9) ◽

pp. e0009810

Author(s):

Vinithra Iyer ◽

Sheetal Tushir ◽

Shreekant Verma ◽

Sudeshna Majumdar ◽

Srimonta Gayen ◽

...

Keyword(s):

Three Dimensional ◽

Heat Shock Protein 90 ◽

Spatial Proximity ◽

Chromosome 5 ◽

Chromosome Conformation ◽

Cis Acting ◽

Trans Splicing ◽

In Trans

Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that remains partially understood. The canonical cis-acting nucleotide elements such as 5’SS-GU, 3’SS-AG, polypyrimidine tract and branch point adenine are present in the independent pre-mRNAs and therefore trans-splicing of Hsp90 must be assisted by spliceosomes in vivo. Using an approach of RNA-protein pull down, we showed that an RNA helicase selectively interacts with HspN pre-mRNA. Our experiments involving high resolution chromosome conformation capture technology as well as DNA FISH show that the trans-spliced genes of Giardia are in three-dimensional spatial proximity in the nucleus. Altogether our study provides a glimpse into the in vivo mechanisms involving protein factors as well as chromatin structure to facilitate the unique inter-molecular post-transcriptional stitching of split genes in G. lamblia.

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History of chromosome rearrangements reflects the spatial organization of yeast chromosomes

Journal of Bioinformatics and Computational Biology ◽

10.1142/s021972001641002x ◽

2016 ◽

Vol 14 (02) ◽

pp. 1641002 ◽

Cited By ~ 4

Author(s):

Ekaterina E. Khrameeva ◽

Geoffrey Fudenberg ◽

Mikhail S. Gelfand ◽

Leonid A. Mirny

Keyword(s):

Dna Repair ◽

Spatial Organization ◽

Nuclear Periphery ◽

Three Dimensional ◽

Genomic Rearrangements ◽

Evolutionary Time ◽

Chromosomal Organization ◽

Cellular Processes ◽

History Of ◽

Organizational Principles

Three-dimensional (3D) organization of genomes affects critical cellular processes such as transcription, replication, and deoxyribo nucleic acid (DNA) repair. While previous studies have investigated the natural role, the 3D organization plays in limiting a possible set of genomic rearrangements following DNA repair, the influence of specific organizational principles on this process, particularly over longer evolutionary time scales, remains relatively unexplored. In budding yeast S.cerevisiae, chromosomes are organized into a Rabl-like configuration, with clustered centromeres and telomeres tethered to the nuclear periphery. Hi-C data for S.cerevisiae show that a consequence of this Rabl-like organization is that regions equally distant from centromeres are more frequently in contact with each other, between arms of both the same and different chromosomes. Here, we detect rearrangement events in Saccharomyces species using an automatic approach, and observe increased rearrangement frequency between regions with higher contact frequencies. Together, our results underscore how specific principles of 3D chromosomal organization can influence evolutionary events.

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EPCO-31. EPIGENOMIC INTRATUMORAL HETEROGENEITY OF GLIOBLASTOMA IN THREE-DIMENSIONAL SPACE

Neuro-Oncology ◽

10.1093/neuonc/noaa215.310 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii76-ii76

Author(s):

Radhika Mathur ◽

Sriranga Iyyanki ◽

Stephanie Hilz ◽

Chibo Hong ◽

Joanna Phillips ◽

...

Keyword(s):

Spatial Organization ◽

Dimensional Space ◽

Three Dimensional ◽

Spatial Location ◽

Therapy Resistance ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Intratumoral Heterogeneity ◽

Tumor Evolution ◽

Open Chromatin

Abstract Treatment failure in glioblastoma is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and generation of therapy-resistant clones. While ITH in glioblastoma has been well-characterized at the genomic and transcriptomic levels, the extent of ITH at the epigenomic level and its biological and clinical significance are not well understood. In collaboration with neurosurgeons, neuropathologists, and biomedical imaging experts, we have established a novel topographical approach towards characterizing epigenomic ITH in three-dimensional (3-D) space. We utilize pre-operative MRI scans to define tumor volume and then utilize 3-D surgical neuro-navigation to intra-operatively acquire 10+ samples representing maximal anatomical diversity. The precise spatial location of each sample is mapped by 3-D coordinates, enabling tumors to be visualized in 360-degrees and providing unprecedented insight into their spatial organization and patterning. For each sample, we conduct assay for transposase-accessible chromatin using sequencing (ATAC-Seq), which provides information on the genomic locations of open chromatin, DNA-binding proteins, and individual nucleosomes at nucleotide resolution. We additionally conduct whole-exome sequencing and RNA sequencing for each spatially mapped sample. Integrative analysis of these datasets reveals distinct patterns of chromatin accessibility within glioblastoma tumors, as well as their associations with genetically defined clonal expansions. Our analysis further reveals how differences in chromatin accessibility within tumors reflect underlying transcription factor activity at gene regulatory elements, including both promoters and enhancers, and drive expression of particular gene expression sets, including neuronal and immune programs. Collectively, this work provides the most comprehensive characterization of epigenomic ITH to date, establishing its importance for driving tumor evolution and therapy resistance in glioblastoma. As a resource for further investigation, we have provided our datasets on an interactive data sharing platform – The 3D Glioma Atlas – that enables 360-degree visualization of both genomic and epigenomic ITH.

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New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis

Cells ◽

10.3390/cells10071819 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1819

Author(s):

Tatyana Karamysheva ◽

Svetlana Romanenko ◽

Alexey Makunin ◽

Marija Rajičić ◽

Alexey Bogdanov ◽

...

Keyword(s):

In Situ Hybridization ◽

Sex Chromosomes ◽

Interphase Nucleus ◽

Spatial Organization ◽

Three Dimensional ◽

Dna Probes ◽

B Chromosomes ◽

Apodemus Flavicollis ◽

Yellow Necked Mouse

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

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Heterogeneous chromatin mobility derived from chromatin states is a determinant of genome organisation in S. cerevisiae

10.1101/106344 ◽

2017 ◽

Cited By ~ 4

Author(s):

Sven A. Sewitz ◽

Zahra Fahmi ◽

Latifa Aljebali ◽

Jeremy Bancroft ◽

Otávio J. B. Brustolini ◽

...

Keyword(s):

Heat Shock ◽

Spatial Clustering ◽

Genome Structure ◽

Nuclear Periphery ◽

Three Dimensional ◽

Normal Growth ◽

Heterogeneous Distribution ◽

Chromatin States ◽

Protein Occupancy ◽

Self Organisation

AbstractSpatial organisation of the genome is essential for regulating gene activity, yet the mechanisms that shape this three-dimensional organisation in eukaryotes are far from understood. Here, we combine bioinformatic determination of chromatin states during normal growth and heat shock, and computational polymer modelling of genome structure, with quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to spatial self-organisation of the genome. We observe that more than forty percent of chromatin-associated proteins display a poised and heterogeneous distribution along the chromosome, creating a heteropolymer. This distribution changes upon heat shock in a concerted, state-specific manner. Simulating yeast chromosomes as heteropolymers, in which the mobility of each segment depends on its cumulative protein occupancy, results in functionally relevant structures, which match our experimental data. This thermodynamically driven self-organisation achieves spatial clustering of poised genes and mechanistically contributes to the directed relocalisation of active genes to the nuclear periphery upon heat shock.One Sentence SummaryUnequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.

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H3K9me2 orchestrates inheritance of spatial positioning of peripheral heterochromatin through mitosis

eLife ◽

10.7554/elife.49278 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 10

Author(s):

Andrey Poleshko ◽

Cheryl L Smith ◽

Son C Nguyen ◽

Priya Sivaramakrishnan ◽

Karen G Wong ◽

...

Keyword(s):

Cell Division ◽

Spatial Organization ◽

Nuclear Periphery ◽

Nuclear Lamina ◽

Positional Information ◽

Specific Gene ◽

Cell Type ◽

Histone 3 ◽

Spatial Positioning ◽

Peripheral Heterochromatin

Cell-type-specific 3D organization of the genome is unrecognizable during mitosis. It remains unclear how essential positional information is transmitted through cell division such that a daughter cell recapitulates the spatial genome organization of the parent. Lamina-associated domains (LADs) are regions of repressive heterochromatin positioned at the nuclear periphery that vary by cell type and contribute to cell-specific gene expression and identity. Here we show that histone 3 lysine 9 dimethylation (H3K9me2) is an evolutionarily conserved, specific mark of nuclear peripheral heterochromatin and that it is retained through mitosis. During mitosis, phosphorylation of histone 3 serine 10 temporarily shields the H3K9me2 mark allowing for dissociation of chromatin from the nuclear lamina. Using high-resolution 3D immuno-oligoFISH, we demonstrate that H3K9me2-enriched genomic regions, which are positioned at the nuclear lamina in interphase cells prior to mitosis, re-associate with the forming nuclear lamina before mitotic exit. The H3K9me2 modification of peripheral heterochromatin ensures that positional information is safeguarded through cell division such that individual LADs are re-established at the nuclear periphery in daughter nuclei. Thus, H3K9me2 acts as a 3D architectural mitotic guidepost. Our data establish a mechanism for epigenetic memory and inheritance of spatial organization of the genome.

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Spatial organization of transcript elongation and splicing kinetics

10.1101/2021.01.28.428713 ◽

2021 ◽

Author(s):

Alyssa D. Casill ◽

Adam J. Haimowitz ◽

Brian Kosmyna ◽

Charles C. Query ◽

Kenny Ye ◽

...

Keyword(s):

Alternative Splicing ◽

Spatial Organization ◽

Dimensional Space ◽

Three Dimensional ◽

Regulatory Elements ◽

Multiple Control ◽

Pol Ii ◽

Topologically Associated Domains ◽

Transcript Elongation ◽

Kinetics Of

SummaryThe organization of the genome in three-dimensional space has been shown to play an important role in gene expression. Specifically, facets of genomic interaction such as topologically associated domains (TADs) have been shown to regulate transcription by bringing regulatory elements into close proximity1. mRNA production is an intricate process with multiple control points including regulation of Pol II elongation and the removal of non-coding sequences via pre-mRNA splicing2. The connection between genomic compartments and the kinetics of RNA biogenesis and processing has been largely unexplored. Here, we measure Pol II elongation and splicing kinetics genome-wide using a novel technique that couples nascent RNA-seq with a mathematical model of transcription and co-transcriptional RNA processing. We uncovered multiple layers of spatial organization of these rates: the rate of splicing is coordinated across introns within individual genes, and both elongation and splicing rates are coordinated within TADs, as are alternative splicing outcomes. Overall, our work establishes that the kinetics of transcription and splicing are coordinated by the spatial organization of the genome and suggests that TADs are a major platform for coordination of alternative splicing.

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Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.11.1918926 ◽

1991 ◽

Vol 39 (11) ◽

pp. 1495-1506 ◽

Cited By ~ 22

Author(s):

P M Motte ◽

R Loppes ◽

M Menager ◽

R Deltour

Keyword(s):

Electron Microscopy ◽

In Situ Hybridization ◽

Spatial Organization ◽

Higher Plants ◽

Three Dimensional ◽

Spatial Arrangement ◽

Thin Sections ◽

Cytoplasmic Dna ◽

Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

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