3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes’ chromatin

How chromosomes are folded, spatially organized and regulated in three dimensions inside the cell nucleus are among the longest standing questions in cell biology. Genome-wide chromosome conformation capture (Hi-C) technique allowed identifying and characterizing spatial chromatin compartments in several mammalian species. Here, we present the first genome-wide analysis of chromatin interactions in chicken embryonic fibroblasts (CEF) and adult erythrocytes. We showed that genome of CEF is partitioned into topologically associated domains (TADs), distributed in accordance with gene density, transcriptional activity and CTCF-binding sites. In contrast to mammals, where all examined somatic cell types display relatively similar spatial organization of genome, chicken erythrocytes strongly differ from fibroblasts, showing pronounced A- and B- compartments, absence of typical TADs and formation of long-range chromatin interactions previously observed on mitotic chromosomes. Comparing mammalian and chicken genome architectures, we provide evidence highlighting evolutionary role of chicken TADs and their significance in genome activity and regulation.

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title: A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping
abstract: We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs "facing" one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.

title: Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions
abstract: The spatial organization of the genome is intimately linked to its biological function, yet our understanding of higher order genomic structure is coarse, fragmented and incomplete. In the nucleus of eukaryotic cells, interphase chromosomes occupy distinct chromosome territories, and numerous models have been proposed for how chromosomes fold within chromosome territories. These models, however, provide only few mechanistic details about the relationship between higher order chromatin structure and genome function. Recent advances in genomic technologies have led to rapid advances in the study of three-dimensional genome organization. In particular, Hi-C has been introduced as a method for identifying higher order chromatin interactions genome wide. Here we investigate the three-dimensional organization of the human and mouse genomes in embryonic stem cells and terminally differentiated cell types at unprecedented resolution. We identify large, megabase-sized local chromatin interaction domains, which we term ‘topological domains’, as a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. Finally, we find that the boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome.

title: Comparison of the 3D organization of sperm and fibroblast genomes using the Hi-C approach
abstract: The 3D organization of the genome is tightly connected to its biological function. The Hi-C approach was recently introduced as a method that can be used to identify higher-order chromatin interactions genome-wide. The aim of this study was to determine genome-wide chromatin interaction frequencies using the Hi-C approach in mouse sperm cells and embryonic fibroblasts. The obtained results demonstrated that the 3D genome organizations of sperm and fibroblast cells show a high degree of similarity both with each other and with the previously described mouse embryonic stem (ES) cells. Both A- and B-compartments and topologically associated domains (TADs) are present in spermatozoa and fibroblasts. Nevertheless, sperm cells and fibroblasts exhibited statistically significant differences between each other in the contact probabilities of defined loci. Tight packaging of the sperm genome resulted in an enrichment of long-range contacts compared with the fibroblasts. However, only 30% of the differences in the number of contacts are based on differences in the densities of their genome packages; the main source of the differences is the gain or loss of contacts that are specific for defined genome regions. An analysis of interchromosomal contacts in both cell types demonstrated that the large chromosomes showed a tendency to interact with each other more than with the small chromosomes and vice versa. We found that the dependence of the contact probability P(s) on genomic distance for sperm is in a good agreement with the fractal globular folding of chromatin. The similarity of the spatial DNA organization in sperm and somatic cell genomes suggests the stability of the 3D structure of genomes through generations.

title: Erez Lieberman-Aiden Principles of the Human Genome Comprehensive Mapping of Long-Range Interactions Reveals Folding
abstract: clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ):  April 4, 2012  www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/326/5950/289.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services,

title: Structural and functional diversity of Topologically Associating Domains
abstract: Recent studies have shown that chromosomes in a range of organisms are compartmentalized in different types of chromatin domains. In mammals, chromosomes form compartments that are composed of smaller Topologically Associating Domains (TADs). TADs are thought to represent functional domains of gene regulation but much is still unknown about the mechanisms of their formation and how they exert their regulatory effect on embedded genes. Further, similar domains have been detected in other organisms, including flies, worms, fungi and bacteria. Although in all these cases these domains appear similar as detected by 3C‐based methods, their biology appears to be quite distinct with differences in the protein complexes involved in their formation and differences in their internal organization. Here we outline our current understanding of such domains in different organisms and their roles in gene regulation.

title: Spatial partitioning of the regulatory landscape of the X-inactivation centre
abstract: In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment. A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the non-protein-coding Xist transcript. The extent of Xic sequences required for the proper regulation of Xist remains unknown. Here we use chromosome conformation capture carbon-copy (5C) and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region including Xist. We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. The Xist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters of Xist and Tsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle of cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre.

title: Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions
abstract: Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

title: Stable Chromosome Condensation Revealed by Chromosome Conformation Capture
abstract: Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.

title: Formation of new chromatin domains determines pathogenicity of genomic duplications
abstract: Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

title: Polymer physics predicts the effects of structural variants on chromatin architecture
abstract: Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer–promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.The authors present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer–promoter contacts. PRISMR correctly predicts ectopic contacts induced by pathogenic SVs at the mouse Epha4 locus.

title: CTCF: an architectural protein bridging genome topology and function
abstract: The eukaryotic genome is organized in the three-dimensional nuclear space in a specific manner that is both a cause and a consequence of its function. This organization is partly established by a special class of architectural proteins, of which CCCTC-binding factor (CTCF) is the best characterized. Although CTCF has been assigned various roles that are often contradictory, new results now help to draw a unifying model to explain the many functions of this protein. CTCF creates boundaries between topologically associating domains in chromosomes and, within these domains, facilitates interactions between transcription regulatory sequences. Thus, CTCF links the architecture of the genome to its function.

title: Comparative Hi-C Reveals that CTCF Underlies Evolution of Chromosomal Domain Architecture
abstract: Topological domains are key architectural building blocks of chromosomes, but their functional importance and evolutionary dynamics are not well defined. We performed comparative high-throughput chromosome conformation capture (Hi-C) in four mammals and characterized the conservation and divergence of chromosomal contact insulation and the resulting domain architectures within distantly related genomes. We show that the modular organization of chromosomes is robustly conserved in syntenic regions and that this is compatible with conservation of the binding landscape of the insulator protein CTCF. Specifically, conserved CTCF sites are co-localized with cohesin, are enriched at strong topological domain borders, and bind to DNA motifs with orientations that define the directionality of CTCF’s long-range interactions. Conversely, divergent CTCF binding between species is correlated with divergence of internal domain structure, likely driven by local CTCF binding sequence changes, demonstrating how genome evolution can be linked to a continuous flux of local conformation changes. We also show that large-scale domains are reorganized during genome evolution as intact modules.

title: The 3D organization of chromatin explains evolutionary fragile genomic regions.
abstract: Genomic rearrangements are a major source of evolutionary divergence in eukaryotic genomes, a cause of genetic diseases and a hallmark of tumor cell progression, yet the mechanisms underlying their occurrence and evolutionary fixation are poorly understood. Statistical associations between breakpoints and specific genomic features suggest that genomes may contain elusive “fragile regions” with a higher propensity for breakage. Here, we use ancestral genome reconstructions to demonstrate a near-perfect correlation between gene density and evolutionary rearrangement breakpoints. Simulations based on functional features in the human genome show that this pattern is best explained as the outcome of DNA breaks that occur in open chromatin regions coming into 3D contact in the nucleus. Our model explains how rearrangements reorganize the order of genes in an evolutionary neutral fashion and provides a basis for understanding the susceptibility of “fragile regions” to breakage.

title: Chromatin architecture reorganization during stem cell differentiation
abstract: Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.

title: Gene density, transcription, and insulators contribute to the partition of the Drosophila genome into physical domains.
abstract: The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.

title: Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains.
abstract: Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

title: Stable Chromosome Condensation Revealed by Chromosome Conformation Capture
abstract: Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.

title: Investigation of the spatial genome organization of mouse sperm and fibroblasts by the Hi-C method
abstract: The spatial organization of eukaryotic genome plays an important role in the control of gene expression. The new Hi-C method allows investigation of chromosome contact in the three-dimensional architecture of the whole genome; however, it has not yet been applied to study the spatial configuration of the germ cells genome. Here we describe a protocol for production and quality control of Hi-C DNA libraries from mouse sperm cells and fibroblasts. Our results demonstrate that the Hi-C method can be used for studying the spatial organization of the densely packed sperm genome.

title: The chicken as a model for large-scale analysis of vertebrate gene function
abstract: The chicken has been an important experimental system for developmental biology, immunology and microbiology, having led to many fundamental discoveries. The increase in genomic resources, easy access to the embryo and the application of RNA interference mean that it will be easy and quick to use chick embryos to screen the function of many genes during embryonic development. So, it seems likely that the chicken will increasingly be the system of choice for many vertebrate biologists who are interested in gene function.

title: Emergence of the chicken as a model organism: implications for agriculture and biology.
abstract: Many of the features of the chicken make it an ideal model organism for phylogenetics and embryology, along with applications in agriculture and medicine. The availability of new tools such as whole genome gene expression arrays and single nucleotide polymorphism panels, coupled with the genome sequence, will enhance this position. These advances are reviewed and their implications are discussed.

title: Continental breakup and the ordinal diversification of birds and mammals
abstract: THE classical hypothesis for the diversification of birds and mammals proposes that most of the orders diverged rapidly in adaptive radiations after the Cretaceous/Tertiary (K/T) extinction event 65 million years ago1–3. Evidence is provided by the near-absence of fossils representing modern orders before the K/T boundary4,5. However, fossil-based estimates of divergence time are known to be conservative because of sampling biases6, and some molecular/time estimates point to earlier divergences among orders7–10. In an attempt to resolve this controversy, we have estimated times of divergence among avian and mammalian orders with a comprehensive set of genes that exhibit a constant rate of substitution. Here we report molecular estimates of divergence times that average about 50–90% earlier than those predicted by the classical hypothesis, and show that the timing of these divergences coincides with the Mesozoic fragmentation of emergent land areas. This suggests that continental breakup may have been an important mechanism in the ordinal diversification of birds and mammals.

title: The dynamics of chromosome evolution in birds and mammals
abstract: Comparative mapping, which compares the location of homologous genes in different species, is a powerful tool for studying genome evolution. Comparative maps suggest that rates of chromosomal change in mammals can vary from one to ten rearrangements per million years. On the basis of these rates we would expect 84 to 600 conserved segments in a chicken comparison with human or mouse. Here we build comparative maps between these species and estimate that numbers of conserved segments are in the lower part of this range. We conclude that the organization of the human genome is closer to that of the chicken than the mouse and by adding comparative mapping results from a range of vertebrates, we identify three possible phases of chromosome evolution. The relative stability of genomes such as those of the chicken and human will enable the reconstruction of maps of ancestral vertebrates.

title: Third Report on Chicken Genes and Chromosomes 2015
abstract: Opening insights into new technologies in avian genomics 
 
The chicken has long been a model organism for genetic and developmental studies. It is now beginning to take its place as a model genome, opening up the fields of phylogenetics and comparative genomics like never before. This report comes at a time of huge technological advances (particularly in sequencing methodologies) and summarizes the current efforts to complete the gaps in the genome. It describes the progress that has been made in genomic annotation, particularly with respect to noncoding RNAs and genetic variants. Reviews of comparative genomics, avian evolution and sex determination are included as well as transcriptomic case studies and developments in epigenetic studies. The Third Report on Chicken Genes and Chromosomes also features the National Avian Research Facility and how it has developed into a resource for the study of avian biology, genetics, infection and disease. In this volume researchers interested in genetics, genomics and evolution will find detailed information that has not been available until now.

title: Comparative genomics reveals insights into avian genome evolution and adaptation
abstract: Birds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.

title: Evolutionary stasis: the stable chromosomes of birds.
abstract: Evolution at the molecular level is manifested in a variety of types of change in DNA sequences, including changes in the structure and organisation of chromosomes. However, in birds chromosomal evolution occurs at an unusually slow rate and recent whole-genome comparisons have shown that many chromosomes have remained more or less intact during avian evolution. Here I discuss progress in the development of genetic maps of natural bird populations, which has revealed that the evolutionary stasis of chromosomes often extends to conservation of gene order. The evolutionary stability of bird chromosomes, which might relate to a low frequency of transposable elements, will facilitate the transfer of genomic information from model to non-model organisms and might have a connection to the rarity of postzygotic incompatibilities observed in birds.

title: Second report on chicken genes and chromosomes 2005
abstract: Schmid, M, Nanda, I, Hoehn, H, Schartl, M, Haaf, T, Buerstedde, J-M, Arakawa, H, Caldwell, RB, Weigend, S, Burt, DW, Smith, J, Griffin, DK, Masabanda, JS, Groenen, MAM, Crooijmans, RPMA, Vignal, A, Fillon, V, Morisson, M, Pitel, F, Vignoles, M, Garrigues, A, Gellin, J, Rodionov, AV, Galkina, SA, Lukina, NA, Ben-Ari, G, Blum, S, Hillel, J, Twito, T, Lavi, U, David, L, Feldman, MW, Delany, ME, Conley, CA, Fowler, VM, Hedges, SB, Godbout, R, Katyal, S, Smith, C, Hudson, Q, Sinclair, A & Mizuno, S 2005, 'Second report on chicken genes and chromosomes 2005', Cytogenetic and Genome Research, vol. 109, no. 4, pp. 415-79.

title: Arrangements of macro- and microchromosomes in chicken cells
abstract: Arrangements of chromosome territories in nuclei of chicken fibroblasts and neurons were analysed employing multicolour chromosome painting, laser confocal scanning microscopy and three-dimensional (3D) reconstruction. The chicken karyotype consists of 9 pairs of macrochromosomes and 30 pairs of microchromosomes. Although the latter represent only 23% of the chicken genome they contain almost 50% of its genes. We show that territories of microchromosomes in fibroblasts and neurons were clustered within the centre of the nucleus, while territories of the macrochromosomes were preferentially located towards the nuclear periphery. In contrast to these highly consistent radial arrangements, the relative arrangements of macrochromosome territories with respect to each other (side-by-side arrangements) were variable. A stringent radial arrangement of macro- and microchromosomes was found in mitotic cells. Replication labelling studies revealed a pattern of DNA replication similar to mammalian cell nuclei: gene dense, early replicating chromatin mostly represented by microchromosomes, was located within the nuclear interior, surrounded by a rim of late replicating chromatin. These results support the evolutionary conservation of several features of higher-order chromatin organization between mammals and birds despite the differences in their karyotypes.

title: Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones
abstract: We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues.

title: An Appraisal of Nuclear Organisation in Interphase Embryonic Fibroblasts of Chicken, Turkey and Duck
abstract: Determining the nuclear ‘addresses’ of chromosome territories is a well-documented means of assaying for nuclear organisation in many cell types and species. Data in avian species are however limited at best, despite the pivotal role played by birds (particularly chickens) in agriculture, and as model organisms in developmental biology. That is, studies have hitherto focussed mostly on mammals (especially humans) and have demonstrated the importance of chromosome territory positioning in embryology, disease and evolution. Thus a detailed study of nuclear organisation in many species, many cell types and many developmental stages in birds is warranted, however, before this is achieved, ‘baseline’ needs to be established to determine precisely the relative locations of chromosome territories in at least 1 cell type of at least 1 bird. With this in mind we hybridised FISH probes from chicken chromosomes 1–28 to embryonic fibroblast nuclei, determining nuclear addresses using a newly developed plug-in to the image analysis package ImageJ. In our experience, evenly spaced representative BAC clones yielded more consistent results than hybridisation of chromosome paints. Results suggested that chromosome territory distribution best fitted a chromosome size-based (rather than gene density-based) pattern. Identical BAC clones were then hybridised to turkey and duck in a comparative genomic strategy. Observations were consistent with those seen in chicken (although, less well-defined in duck), providing preliminary evidence of conservation throughout evolution.

title: Three-dimensional architecture of tandem repeats in chicken interphase nucleus
abstract: Tandem repeats belong to a class of genomic repetitive elements that form arrays of head-to-tail monomers. Due to technical difficulties in sequencing and assembly of large tandem repeat arrays, it remains largely unknown by which mechanisms tandem-repeat-containing regions aid in maintenance of ordered radial genome organization during interphase. Here we analyzed spatial distribution of several types of tandem repeats in interphase nuclei of chicken MDCC-MSB1 cells and somatic tissues relative to heterochromatin compartments and nuclear center. We showed that telomere and subtelomere repeats generally localize at the nuclear or chromocenters periphery. A tandem repeat known as CNM, typical for centromere regions of gene-dense microchromosomes, forms interchromosome clusters and occupies DAPI-positive chromocenters that appear predominantly within the nuclear interior. In contrast, centromere-specific tandem repeats of the majority of gene-poor macrochromosomes are embedded into the peripheral layer of heterochromatin. Chicken chromocenters rarely comprise centromere sequences of both macro- and microchromosomes, whose territories localize in different radial nuclear zones. Possible mechanisms of observed tandem repeats positioning and its implication in highly ordered arrangement of chromosome territories in chicken interphase nucleus are discussed.

title: RNA synthesis in chicken erythrocytes.
abstract: CHICKEN erythrocyte nuclei were reported not to synthesize RNA1, and consequently have been used to study reactivation of nuclear synthesis2–4. In some ducks, however, mature erythrocyte nuclei continue to synthesize some RNA5–7, and we have found that the erythrocytes of adult chickens synthesize substantial amounts of RNA. We have found none in which the erythrocytes have no synthetic activity.

title: A chicken red cell inhibitor of transcription associated with the terminally differentiated state
abstract: When a red cell nuclear extract (RCE) from adult chickens was injected into Xenopus oocytes along with the chicken beta globin gene, transcript levels were dramatically reduced compared to injection of DNA alone. The inhibitory action of the RCE was not specific to the beta globin gene since the Herpes thymidine kinase and Xenopus 5S RNA gene transcript levels were similarly reduced. Transcriptional repression was observed even after passage of the RCE through oocyte cytoplasm to the nucleus. The inhibitory activity binds to DNA cellulose, which suggests that the inhibitor either binds to DNA or associates with DNA-binding proteins. Nuclease digestion of the chromatin assembled on injected beta globin DNA revealed that inhibition was not associated with local changes in chromatin structure. Extracts from 9-d chicken embryonic erythroid cells, in which the endogenous beta globin gene is actively expressed, did not inhibit transcription. The inhibitory activity is, therefore, restricted to transcriptionally quiescent, adult erythrocytes. Since the inhibitory effects were seen with both polymerase II and III directed genes, we speculate that the activity may be part of the extreme transcriptional repression which occurs in the terminally differentiated erythrocyte.

title: Electron-microscope observations on the organization of the nucleus in chicken erythrocytes and a superunit thread hypothesis for chromosome structure.
abstract: Previously it was shown that when condensed chromatin from several different types of cell is stained with uranyl-lead and examined in thin sections in the electron microscope, the stain is distributed into a dot-dash pattern arising from threads, with lesser-staining intermediate areas. We now show that when a section through chicken erythrocyte chromatin is stained with ethanolic phosphotungstic acid (PTA) the stain distribution is homogeneous. This shows that the lesser-staining regions after uranyl-lead, cannot be an overlap artifact. We conclude that the stains and hence the molecules in chromatin are distributed between 2 phases, an o - and an e -phase, so called because the structural units in chromatin are arranged in an orderly way at the surface of the nucleus and give rise to oddly ( o ) and evenly ( e ) numbered bands. Measurements of electron density per unit thickness, proportional to the number of stain molecules per unit volume, are made in thin sections through erythrocytes and reticulocytes from adult hen, 4-day-old chicks and 17-day embryos. The results indicate differences in the packing of the molecules in chromatin and further show that the e -phase is quite likely to have a higher DNA to protein ratio than the o -phase. After uranyl-lead stain the visibility of the dot-dash pattern in cells from adult hen is relatively low due, we propose, to closer packing. In micrographs through condensed chromatin treated with uranyl-lead the eye selects out only the densely stained dots and dashes, width 17 nm. When erythrocyte chromatin is partially or completely disrupted in various ways, threads 25-30 nm then become visible. We propose that condensed chromatin in intact cells contains structural units which consist of a central element, width 17 nm previously referred to as the unit thread, forming the e -phase, surrounded by a cylindrical shell forming the o -phase. This socalled superunit thread is similar in width, about 25-30 nm, to that reported by other workers in preparations of chromosomes spread on water surfaces. The hypothesis therefore helps explain what appeared to be discrepancies in thread dimensions. Certain other ultrastructural features of erythrocyte nuclei are also reported which are either pertinent to the general aim of this study, namely the way in which nucleoproteins fold up in chromosomes, or to biochemical studies, to be reported shortly, in which attempts are made to locate the proteins removed from isolated erythrocyte nuclei during subsequent washing in salt solutions.

title: STRUCTURE IN NUCLEATED ERYTHROCYTES
abstract: The structure of the nucleated erythrocyte of frog and chicken has been investigated by electron microscopy and correlated with the distribution of haemoglobin and DNA-containing material determined by haem absorption and Feulgen staining in the light microscope. The nuclei of both species are found to contain haemoglobin which is continuous with the haemoglobin in the cytoplasm through holes or pores in the nuclear envelope. In addition the nucleus of the frog erythrocyte sometimes contains a single invagination which is lined by the nuclear envelope. The structure of the nuclear envelope and the pores and the organisation of the nucleus are similar to those already described for other somatic cells. Erythrocytes differ from the cells previously studied in that a continuity, via the nuclear pores, of chemical substance in the interior of the nucleus and in the cytoplasm can be directly demonstrated. This is due to the fact that the cytoplasm of erythrocytes is simple, consisting predominantly of haemoglobin, and that haemoglobin is easily recognised by its specific absorption. The static pictures obtained by electron microscopy have been supplemented by observations in phase-contrast of the changes in refraction of the cell contents due to the diffusion of the haemoglobin from the nucleus into the cytoplasm during haemolysis.

title: Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei
abstract: Chromatin folding in eukaryotes fits the genome into the limited volume of the cell nucleus. Formation of higher-order chromatin structures attenuates DNA accessibility, thus contributing to the control of essential genome functions such as transcription, DNA replication, and repair. The 30-nm fiber is thought to be the first hierarchical level of chromatin folding, but the nucleosome arrangement in the compact 30-nm fiber was previously unknown. We used cryoelectron tomography of vitreous sections to determine the structure of the compact, native 30-nm fiber of avian erythrocyte nuclei. The predominant geometry of the 30-nm fiber revealed by subtomogram averaging is a left-handed two-start helix with approximately 6.5 nucleosomes per 11 nm, in which the nucleosomes are juxtaposed face-to-face but are shifted off their superhelical axes with an axial translation of approximately 3.4 nm and an azimuthal rotation of approximately 54°. The nucleosomes produce a checkerboard pattern when observed in the direction perpendicular to the fiber axis but are not interdigitated. The nucleosome packing within the fibers shows larger center-to-center internucleosomal distances than previously anticipated, thus excluding the possibility of core-to-core interactions, explaining how transcription and regulation factors can access nucleosomes.

title: Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM)
abstract: The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native environment. Reproducible high-resolution AFM images of chromatin compaction at several levels can be demonstrated. An extended beads-on-a-string (width of ∼ 15-20nm, height of ∼ 2-3nm for each individual nucleosome) can be consistently observed. Furthermore, superbeads (width of ∼ 40nm, height of ∼ 7nm) are demonstrated. Visualization of the solenoid conformation at the level of 30nm chromatin fiber is attained either by using AFM or by using electron microscopy. In addition, tightly coiled chromatin fibers (∼ 50-60nm and ∼ 90-110nm) can be revealed. Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.

title: Structure of the chromosomal material in inactive nuclei of chicken red blood cells
abstract: Electron microscope sections have been cut of chicken red blood cell nuclei by a novel procedure that produces zones of material below 100 Å in thickness. After fixing and staining, electron dense structures are observed that have dimensions closely correlating with those determined for nucleosomes. These structures appear as wedge-shaped or as circular objects with a diameter of approximately 90–100 Å. Five to eight of these objects are arranged in a spiral around a central core giving rise to a 250–300 Å structure which may represent a superbead or a cross section of a solenoidal fibre.

title: Ultrastructural and biochemical observations on interphase nuclei isolated from chicken erythrocytes.
abstract: Adult hen erythrocyte nuclei are isolated from cells or haemolysed in situ by acting on the plasma membrane with rotating knives or with non-ionic detergents. When the isolation medium contains magnesium ions (1 mM), sucrose (0-4 M) and Tris buffer (0.01 M, pH 7-5) called SMTOG (see text), the ultrastructure in thin sections through the condensed chromatin bodies, after staining with either uranyl-lead or phosphotungstic acid (PTA), is similar to that found in the intact cell. Hence it can be concluded that the 2 phases which comprise chromatin, the o- and e-phase, survive nuclear isolation. These are so called because the structural units in chromatin are arranged at the surface of the nucleus into one or more layers and give rise to oddly (o) and evenly (e) numbered bands. The 0-phase is also largely retained after extensive washing in 0-07 M NaC1 as shown by electron microscopy and biochemical measurements; only 6% of the total nuclear protein is removed, a value small compared with the fractional amount of the chromatin protein calculated to lie in the o-phase, about 70%. After extensive washing in saline-EDTA there are structural changes in chromatin, but biochemical data show that the molecules in the o-phase are also largely retained; loss of protein amounts to between 5 and 11%. These data suggest that the o-phase is a structural component of the chromatin bodies. They support the hypothesis that condensed chromatin is formed by folding superunit threads. These units consist of a central thread-like element about 17 nm diameter which stains preferentially with uranyl-lead and forms the e-phase, with an outer cylindrical shell forming the o-phase of total diameter about 28nm. The 5-10% proteins removed by salt washes are located exclusively in a particulate component, quite likely the chromatin. They have been examined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. There are about 10 or more protein species, ranging in molecular weight from 21000 upwards. The groups of large granules previously found in the nuclear sap of intact erythrocytes are shown to be associated with an amorphous or finely fibrillar body.

title: MENT, a Heterochromatin Protein That Mediates Higher Order Chromatin Folding, Is a New Serpin Family Member*
abstract: Terminal cell differentiation is correlated with the extensive sequestering of previously active genes into compact transcriptionally inert heterochromatin. In vertebrate blood cells, these changes can be traced to the accumulation of a developmentally regulated heterochromatin protein, MENT. Cryoelectron microscopy of chicken granulocyte chromatin, which is highly enriched with MENT, reveals exceptionally compact polynucleosomes, which maintain a level of higher order folding above that imposed by linker histones. The amino acid sequence of MENT reveals a close structural relationship with serpins, a large family of proteins known for their ability to undergo dramatic conformational transitions. Conservation of the “hinge region” consensus in MENT indicates that this ability is retained by the protein. MENT is distinguished from the other serpins by being a basic protein, containing several positively charged surface clusters, which are likely to be involved in ionic interactions with DNA. One of the positively charged domains bears a significant similarity to the chromatin binding region of nuclear lamina proteins and with the A·T-rich DNA-binding motif, which may account for the targeting of MENT to peripheral heterochromatin. MENT ectopically expressed in a mammalian cell line is transported into nuclei and is associated with intranuclear foci of condensed chromatin.

title: Stage-specific expression and localization of MENT, a nuclear protein associated with chromatin condensation in terminally differentiating avian erythroid cells.
abstract: Polyclonal antibodies have been raised against a non-histone protein (MENT) which has been previously shown to be associated with the repressed chromatin of mature chicken erythrocytes and to promote the in vitro condensation of chromatin of immature erythrocyte nuclei. Here we report that the expression pattern of MENT closely follows chromatin condensation in maturing avian erythrocytes of definitive and primary lineages. Accumulation of MENT correlates more strongly with chromatin condensation than does accumulation of histone H5. In addition to being present in erythrocytes, the protein was also found in neutrophil nuclei and an immunofluorescence reaction was observed with embryonic (nucleated) thrombocytes. MENT was not detected in other chicken tissues (brain, liver, testis). In intact erythrocytes, MENT immunofluorescence was found in foci close to the nuclear periphery, while in isolated, decondensed nuclei, the fluorescence signal was uniformly distributed. In neutrophil nuclei, containing approximately 10 times more MENT than adult erythrocytes, intense staining associated with the peripheral heterochromatin was observed. These findings are discussed in regard to a possible mechanism for chromatin condensation by MENT.

title: Chromatin compaction in terminally differentiated avian blood cells: the role of linker histone H5 and non-histone protein MENT
abstract: Chromatin has a tendency to shift from a relatively decondensed (active) to condensed (inactive) state during cell differentiation due to interactions of specific architectural and/or regulatory proteins with DNA. A promotion of chromatin folding in terminally differentiated avian blood cells requires the presence of either histone H5 in erythrocytes or non-histone protein, myeloid and erythroid nuclear termination stage-specific protein (MENT), in white blood cells (lymphocytes and granulocytes). These highly abundant proteins assist in folding of nucleosome arrays and self-association of chromatin fibers into compacted chromatin structures. Here, we briefly review structural aspects and molecular mode of action by which these unrelated proteins can spread condensed chromatin to form inactivated regions in the genome.

title: Changes in histone acetyl content and in nuclear non-histone protein composition of avian erythroid cells at different stages of maturation.
abstract: Duck erythroid cells were fractionated by centrifugation in bovine serum albumin density gradients. Differences in cell density were correlated with the stage of maturation as determined by biochemical and morphological parameters. Avian erythroid cells increase in density as they mature. Cell populations corresponding to known stages in differentiation were compared with regard to histone composition and degree of acetylation, nature and amount of the non-histone nuclear proteins, and DNA and RNA synthetic capacities. Although the total histone to DNA ratio of the chromatin does not vary appreciably during maturation, the relative proportion of the erythroid cell-specific histone F2c increases at least 2-fold with the increasing age of the cell, whereas the other histones, except F2a2, decrease. The degree of acetylation of histones F2a1 and F3 decreases with the increasing age of the cell. The non-histone protein complement of the erythroid cell nucleus decreases 6-fold during maturation. Analysis of the non-histone proteins in polyacrylamide gels containing sodium dodecyl sulfate shows a limited heterogeneity and a remarkable similarity in their electrophoretic patterns at different stages in maturation. Quantitative rather than qualitative changes predominate as maturation proceeds. The decline in non-histone protein content with age is striking particularly in the lower molecular weight components. However, a protein band of molecular weight 55,000 is conserved selectively during maturation and it represents the predominant component of the non-histone nuclear proteins of the mature erythrocyte. The nucleoprotein changes occurring during erythrocyte maturation parallel a decrease in the amount of chromatin-associated RNA and the rate of RNA synthesis. The average RNA synthetic capacity of the isolated erythroblasts and early polychromatic erythrocytes is about 8 times higher than that of the mature erythrocyte fraction.

title: Chromosome-Specific Intron Size Differences in the Avian CHD Gene Provide an Efficient Method for Sex Identification in Birds
abstract: to this problem for some species (e.g. Griffiths and Holland 1990, Quinn et al. 1990, Rabenold et al. 1991, Dvorak et al. 1992, Longmire et al. 1993, Ogawa et al. 1997), but their use requires considerable xpertise, and often their taxonomic range is limited. More recently, PCR-based approaches that are technically simpler and that have broad taxonomic utility have been developed (Ogawa et al. 1997). The discovery of a highly conserved gene (CHD) that is linked to the W and a non-W chromosome in

title: The chicken erythrocyte epigenome
abstract: BackgroundTranscriptional regulation is impacted by multiple layers of genome organization. A general feature of transcriptionally active chromatin is sensitivity to DNase I and association with acetylated histones. However, very few of these active DNase I-sensitive domains, such as the chicken erythrocyte β-globin domain, have been identified and characterized. In chicken polychromatic erythrocytes, dynamically acetylated histones associated with DNase I-sensitive, transcriptionally active chromatin prevent histone H1/H5-induced insolubility at physiological ionic strength.ResultsHere, we identified and mapped out all the transcriptionally active chromosomal domains in the chicken polychromatic erythrocyte genome by combining a powerful chromatin fractionation method with next-generation DNA and RNA sequencing. Two classes of transcribed chromatin organizations were identified on the basis of the extent of solubility at physiological ionic strength. Highly transcribed genes were present in multigenic salt-soluble chromatin domains ranging in length from 30 to over 150 kb. We identified over 100 highly expressed genes that were organized in broad dynamically highly acetylated, salt-soluble chromatin domains. Highly expressed genes were associated with H3K4me3 and H3K27ac and produced discernible antisense transcripts. The moderately- and low-expressing genes had highly acetylated, salt-soluble chromatin regions confined to the 5′ end of the gene.ConclusionsOur data provide a genome-wide profile of chromatin signatures in relation to expression levels in chicken polychromatic erythrocytes.

title: NEMO: a tool for analyzing gene and chromosome territory distributions from 3D-FISH experiments
abstract: Three-dimensional fluorescence in situ hybridization (3D-FISH) is used to study the organization and the positioning of chromosomes or specific sequences such as genes or RNA in cell nuclei. Many different programs (commercial or free) allow image analysis for 3D-FISH experiments. One of the more efficient open-source programs for automatically processing 3D-FISH microscopy images is Smart 3D-FISH, an ImageJ plug-in designed to automatically analyze distances between genes. One of the drawbacks of Smart 3D-FISH is that it has a rather basic user interface and produces its results in various text and image files thus making the data post-processing step time consuming. We developed a new Smart 3D-FISH graphical user interface, NEMO, which provides all information in the same place so that results can be checked and validated efficiently. NEMO gives users the ability to drive their experiments analysis in either automatic, semi-automatic or manual detection mode. We also tuned Smart 3D-FISH to better analyze chromosome territories.


AVAILABILITY
NEMO is a stand-alone Java application available for Windows and Linux platforms. The program is distributed under the creative commons licence and can be freely downloaded from https://www-lgc.toulouse.inra.fr/nemo

title: A simple and effective method for ultrastructural analysis of mitosis in Drosophila S2 cells

title: NIH Image to ImageJ: 25 years of image analysis
abstract: For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.

title: Juicebox Provides a Visualization System for Hi-C Contact Maps with Unlimited Zoom.
abstract: Hi-C experiments study how genomes fold in 3D, generating contact maps containing features as small as 20 bp and as large as 200 Mb. Here we introduce Juicebox, a tool for exploring Hi-C and other contact map data. Juicebox allows users to zoom in and out of Hi-C maps interactively, just as a user of Google Earth might zoom in and out of a geographic map. Maps can be compared to one another, or to 1D tracks or 2D feature sets.

title: Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition
abstract: Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin ‘ground state’ could potentially provide insights into reprogramming cells to a state of totipotency.

title: Identification of alternative topological domains in chromatin
abstract: Chromosome conformation capture experiments have led to the discovery of dense, contiguous, megabase-sized topological domains that are similar across cell types and conserved across species. These domains are strongly correlated with a number of chromatin markers and have since been included in a number of analyses. However, functionally-relevant domains may exist at multiple length scales. We introduce a new and efficient algorithm that is able to capture persistent domains across various resolutions by adjusting a single scale parameter. The ensemble of domains we identify allows us to quantify the degree to which the domain structure is hierarchical as opposed to overlapping, and our analysis reveals a pronounced hierarchical structure in which larger stable domains tend to completely contain smaller domains. The identified novel domains are substantially different from domains reported previously and are highly enriched for insulating factor CTCF binding and histone marks at the boundaries.

title: Comparing clusterings---an information based distance
abstract: This paper proposes an information theoretic criterion for comparing two partitions, or clusterings, of the same data set. The criterion, called variation of information (VI), measures the amount of information lost and gained in changing from clustering C to clustering C^'. The basic properties of VI are presented and discussed. We focus on two kinds of properties: (1) those that help one build intuition about the new criterion (in particular, it is shown the VI is a true metric on the space of clusterings), and (2) those that pertain to the comparability of VI values over different experimental conditions. As the latter properties have rarely been discussed explicitly before, other existing comparison criteria are also examined in their light. Finally we present the VI from an axiomatic point of view, showing that it is the only ''sensible'' criterion for comparing partitions that is both aligned to the lattice and convexely additive. As a consequence, we prove an impossibility result for comparing partitions: there is no criterion for comparing partitions that simultaneously satisfies the above two desirable properties and is bounded.

title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α)
abstract: Viruses that infect birds pose major threats—to the global supply of chicken, the major, universally-acceptable meat, and as zoonotic agents (e.g. avian influenza viruses H5N1 and H7N9). Controlling these viruses in birds as well as understanding their emergence into, and transmission amongst, humans will require considerable ingenuity and understanding of how different species defend themselves. The type I interferon-coordinated response constitutes the major antiviral innate defence. Although interferon was discovered in chicken cells, details of the response, particularly the identity of hundreds of stimulated genes, are far better described in mammals. Viruses induce interferon-stimulated genes but they also regulate the expression of many hundreds of cellular metabolic and structural genes to facilitate their replication. This study focusses on the potentially anti-viral genes by identifying those induced just by interferon in primary chick embryo fibroblasts. Three transcriptomic technologies were exploited: RNA-seq, a classical 3′-biased chicken microarray and a high density, “sense target”, whole transcriptome chicken microarray, with each recognising 120–150 regulated genes (curated for duplication and incorrect assignment of some microarray probesets). Overall, the results are considered robust because 128 of the compiled, curated list of 193 regulated genes were detected by two, or more, of the technologies.

title: Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes
abstract: Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor–encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1.

title: MEME-ChIP: motif analysis of large DNA datasets
abstract: Advances in high-throughput sequencing have resulted in rapid growth in large, high-quality datasets including those arising from transcription factor (TF) ChIP-seq experiments. While there are many existing tools for discovering TF binding site motifs in such datasets, most web-based tools cannot directly process such large datasets.  The MEME-ChIP web service is designed to analyze ChIP-seq ‘peak regions’—short genomic regions surrounding declared ChIP-seq ‘peaks’. Given a set of genomic regions, it performs (i) ab initio motif discovery, (ii) motif enrichment analysis, (iii) motif visualization, (iv) binding affinity analysis and (v) motif identification. It runs two complementary motif discovery algorithms on the input data—MEME and DREME—and uses the motifs they discover in subsequent visualization, binding affinity and identification steps. MEME-ChIP also performs motif enrichment analysis using the AME algorithm, which can detect very low levels of enrichment of binding sites for TFs with known DNA-binding motifs. Importantly, unlike with the MEME web service, there is no restriction on the size or number of uploaded sequences, allowing very large ChIP-seq datasets to be analyzed. The analyses performed by MEME-ChIP provide the user with a varied view of the binding and regulatory activity of the ChIP-ed TF, as well as the possible involvement of other DNA-binding TFs. Availability: MEME-ChIP is available as part of the MEME Suite at http://meme.nbcr.net.  t.bailey@uq.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.

title: Novel Insights into Chromosome Evolution in Birds, Archosaurs, and Reptiles
abstract: Homologous synteny blocks (HSBs) and evolutionary breakpoint regions (EBRs) in mammalian chromosomes are enriched for distinct DNA features, contributing to distinct phenotypes. To reveal HSB and EBR roles in avian evolution, we performed a sequence-based comparison of 21 avian and 5 outgroup species using recently sequenced genomes across the avian family tree and a newly-developed algorithm. We identified EBRs and HSBs in ancestral bird, archosaurian (bird, crocodile, and dinosaur), and reptile chromosomes. Genes involved in the regulation of gene expression and biosynthetic processes were preferably located in HSBs, including for example, avian-specific HSBs enriched for genes involved in limb development. Within birds, some lineage-specific EBRs rearranged genes were related to distinct phenotypes, such as forebrain development in parrots. Our findings provide novel evolutionary insights into genome evolution in birds, particularly on how chromosome rearrangements likely contributed to the formation of novel phenotypes.

title: CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription
abstract: Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

title: Formation of Chromosomal Domains by Loop Extrusion.
abstract: Topologically associating domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes, yet the mechanisms of TAD formation remain unclear. Here, we propose that loop extrusion underlies TAD formation. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. Using polymer simulations, we show that this model produces TADs and finer-scale features of Hi-C data. Each TAD emerges from multiple loops dynamically formed through extrusion, contrary to typical illustrations of single static loops. Loop extrusion both explains diverse experimental observations-including the preferential orientation of CTCF motifs, enrichments of architectural proteins at TAD boundaries, and boundary deletion experiments-and makes specific predictions for the depletion of CTCF versus cohesin. Finally, loop extrusion has potentially far-ranging consequences for processes such as enhancer-promoter interactions, orientation-specific chromosomal looping, and compaction of mitotic chromosomes.

title: Unusual compartmentalization of CTCF and other transcription factors in the course of terminal erythroid differentiation.
abstract: It is demonstrated that in chicken embryonic and mature erythrocyte nuclei the distribution of a versatile transcription factor CTCF differs drastically from its distribution in nuclei of proliferating erythroid and non-erythroid cells. In the latter case CTCF was distributed throughout the whole nucleus volume, being concentrated in many small compartments (punctuate nuclear staining). In contrast, in embryonic and mature erythrocytes CTCF was concentrated in a limited number of large compartments. These large CTCF-containing compartments were not observed in other cells. Occasionally, but not in all cells, some of these compartments were localized close to nucleoli but did not colocalize with them. In mature erythrocytes a clear exclusion of CTCF-containing compartments from the chromatin domain was observed. This exclusion correlated with a tight association of CTCF with the nuclear matrix. Concentration in relatively large compartments and exclusion from the chromatin domain in nuclei of mature erythrocytes were also observed for RNA polymerase II and several transcription factors. The data are discussed in the context of a hypothesis postulating that relocalization of different components of the transcriptional machinery from the chromatin domain into the interchromatin compartment is an important step of the terminal inactivation of chicken erythrocyte nuclei.

title: The fractal globule as a model of chromatin architecture in the cell
abstract: The fractal globule is a compact polymer state that emerges during polymer condensation as a result of topological constraints which prevent one region of the chain from passing across another one. This long-lived intermediate state was introduced in 1988 (Grosberg et al. 1988) and has not been observed in experiments or simulations until recently (Lieberman-Aiden et al. 2009). Recent characterization of human chromatin using a novel chromosome conformational capture technique brought the fractal globule into the spotlight as a structural model of human chromosome on the scale of up to 10 Mb (Lieberman-Aiden et al. 2009). Here, we present the concept of the fractal globule, comparing it to other states of a polymer and focusing on its properties relevant for the biophysics of chromatin. We then discuss properties of the fractal globule that make it an attractive model for chromatin organization inside a cell. Next, we connect the fractal globule to recent studies that emphasize topological constraints as a primary factor driving formation of chromosomal territories. We discuss how theoretical predictions, made on the basis of the fractal globule model, can be tested experimentally. Finally, we discuss whether fractal globule architecture can be relevant for chromatin packing in other organisms such as yeast and bacteria.

title: A pathway for mitotic chromosome formation
abstract: Tracking mitotic chromosome formation How cells pack DNA into fully compact, rod-shaped chromosomes during mitosis has fascinated cell biologists for more than a century. Gibcus et al. delineated the conformational transition trajectory from interphase chromatin to mitotic chromosomes minute by minute during the cell cycle. The mitotic chromosome is organized in a spiral staircase architecture in which chromatin loops emanate radially from a centrally located helical scaffold. The molecular machines condensin I and II play distinct roles in these processes: Condensin II is essential for helical winding, whereas condensin I modulates the organization within each helical turn. Science, this issue p. eaao6135 Mitotic chromosome folding involves formation of increasingly compacted helically arranged nested loop arrays.  During mitosis, cells compact their chromosomes into dense rod-shaped structures to ensure their reliable transmission to daughter cells. Our work explores how cells achieve this compaction. We integrate genetic, genomic, and computational approaches to characterize the key steps in mitotic chromosome formation from the G2 nucleus to metaphase, and we identify roles of specific molecular machines, condensin I and II, in these major conformational transitions.  We used chicken DT-40 cells expressing an analog-sensitive CDK1 to produce cell cultures that synchronously enter mitosis. We collected cells at key time points during mitotic entry; analyzed chromosome organization by microscopy, chromosome conformation capture, and polymer simulations; and delineated a pathway of mitotic chromosome formation. We used engineered cell lines to study the function of condensin complexes, which are critical for mitotic chromosome formation. We fused condensin I and II subunits to plant auxin-inducible degron domains, thus enabling their rapid depletion in late G2 just before mitotic entry. These cell lines allowed us to determine the roles of condensin I and II in specific steps of the mitotic chromosome morphogenesis pathway.  Our analysis of G2 chromosomes reveals hallmarks of interphase chromosomes, including topologically associating domains and compartments. Upon entry into prophase, this organization is lost within minutes, and by late prophase, chromosomes are folded as arrays of consecutive loops condensed around a central axis. These loops project with random but mutually correlated angles from the axis. During prometaphase, the loop array undergoes two major reorganizations. First, it acquires a helical arrangement of loops. Polymer simulations of Hi-C data show that the centrally located axis acquires a helical twist so that consecutive loops emanate as the steps of a spiral staircase. Second, the chromatin loops become nested with ~400-kb outer loops split up by ~80-kb inner loops. As prometaphase proceeds, chromosomes shorten through progressive helical winding, with the numbers of loops per turn increasing. As a result, the size of a helical turn grows from ~3 Mb (~40 loops) to ~12 Mb (~150 loops). Depletion of condensin I or II before mitotic entry revealed their differing roles in mitotic chromosome formation. Either condensin can mediate loop array formation. However, condensin II is required for the helical twisting of the scaffold from which loops emanate, whereas condensin I modulates the size and arrangement of nested inner loops.  We describe a pathway of mitotic chromosome folding that unifies many previous observations. In prophase, condensins mediate the loss of interphase organization and the formation of arrays of consecutive loops. In prometaphase, chromosomes adopt a spiral staircase–like structure with a helically arranged axial scaffold of condensin II at the bases of chromatin loops. The condensin II loops are further compacted by condensin I into clusters of smaller nested loops that are additionally collapsed by chromatin-to-chromatin attractions. The combination of nested loops distributed around a helically twisted axis plus dense chromatin packing achieves the 10,000-fold compaction of chromatin into linearly organized chromosomes that is required for accurate chromosome segregation when cells divide. A pathway for mitotic chromosome formation. In prophase, condensins mediate the loss of interphase chromosome conformation, and loop arrays are formed. In prometaphase, the combined action of condensin I (blue spheres in the bottom diagram) and II (red spheres) results in helically arranged nested loop arrays. Mitotic chromosomes fold as compact arrays of chromatin loops. To identify the pathway of mitotic chromosome formation, we combined imaging and Hi-C analysis of synchronous DT40 cell cultures with polymer simulations. Here we show that in prophase, the interphase organization is rapidly lost in a condensin-dependent manner, and arrays of consecutive 60-kilobase (kb) loops are formed. During prometaphase, ~80-kb inner loops are nested within ~400-kb outer loops. The loop array acquires a helical arrangement with consecutive loops emanating from a central “spiral staircase” condensin scaffold. The size of helical turns progressively increases to ~12 megabases during prometaphase. Acute depletion of condensin I or II shows that nested loops form by differential action of the two condensins, whereas condensin II is required for helical winding.

title: Upgrading short-read animal genome assemblies to chromosome level using comparative genomics and a universal probe set.
abstract: Most recent initiatives to sequence and assemble new species' genomes de novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of subchromosomal-sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification, and physical mapping to chromosomes. Multigenome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon (Columbia livia) and peregrine falcon (Falco peregrinus) to chromosome levels comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food, and/or environmental reasons. Pigeon has a typical avian karyotype (2n = 80), while falcon (2n = 50) is highly rearranged compared to the avian ancestor. By using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved noncoding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE "deserts." This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species, while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome.

title: Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program.
abstract: Mammalian genomes are partitioned into domains that replicate in a defined temporal order. These domains can replicate at similar times in all cell types (constitutive) or at cell type-specific times (developmental). Genome-wide chromatin conformation capture (Hi-C) has revealed sub-megabase topologically associating domains (TADs), which are the structural counterparts of replication domains. Hi-C also segregates inter-TAD contacts into defined 3D spatial compartments that align precisely to genome-wide replication timing profiles. Determinants of the replication-timing program are re-established during early G1 phase of each cell cycle and lost in G2 phase, but it is not known when TAD structure and inter-TAD contacts are re-established after their elimination during mitosis. Here, we use multiplexed 4C-seq to study dynamic changes in chromatin organization during early G1. We find that both establishment of TADs and their compartmentalization occur during early G1, within the same time frame as establishment of the replication-timing program. Once established, this 3D organization is preserved either after withdrawal into quiescence or for the remainder of interphase including G2 phase, implying 3D structure is not sufficient to maintain replication timing. Finally, we find that developmental domains are less well compartmentalized than constitutive domains and display chromatin properties that distinguish them from early and late constitutive domains. Overall, this study uncovers a strong connection between chromatin re-organization during G1, establishment of replication timing, and its developmental control.

title: CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation.
abstract: Genome function, replication, integrity, and propagation rely on the dynamic structural organization of chromosomes during the cell cycle. Genome folding in interphase provides regulatory segmentation for appropriate transcriptional control, facilitates ordered genome replication, and contributes to genome integrity by limiting illegitimate recombination. Here, we review recent high-resolution chromosome conformation capture and functional studies that have informed models of the spatial and regulatory compartmentalization of mammalian genomes, and discuss mechanistic models for how CTCF and cohesin control the functional architecture of mammalian chromosomes.

title: Emerging Evidence of Chromosome Folding by Loop Extrusion
abstract: Chromosome organization poses a remarkable physical problem with many biological consequences: how can molecular interactions between proteins at the nanometer scale organize micron-long chromatinized DNA molecules, insulating or facilitating interactions between specific genomic elements? The mechanism of active loop extrusion holds great promise for explaining interphase and mitotic chromosome folding, yet remains difficult to assay directly. We discuss predictions from our polymer models of loop extrusion with barrier elements, and review recent experimental studies that provide strong support for loop extrusion, focusing on perturbations to CTCF and cohesin assayed via Hi-C in interphase. Finally, we discuss a likely molecular mechanism of loop extrusion by SMC complexes.

title: Chromatin organization by an interplay of loop extrusion and compartmental segregation
abstract: Mammalian chromatin is organized on length scales ranging from individual nucleosomes to chromosomal territories. At intermediate scales two dominant features emerge in interphase: (i) alternating regions (<5Mb) of active and inactive chromatin that spatially segregate into different compartments, and (ii) domains (<1Mb), i.e. regions that preferentially interact internally, which are also termed topologically associating domains (TADs) and are central to gene regulation. There is growing evidence that TADs are formed by active extrusion of chromatin loops by cohesin, whereas compartments are established by a phase separation process according to local chromatin states. Here we use polymer simulations to examine how the two processes, loop extrusion and compartmental segregation, work collectively and potentially interfere in shaping global chromosome organization. Our integrated model faithfully reproduces Hi-C data from previously puzzling experimental observations, where targeting of the TAD-forming machinery led to changes in compartmentalization. Specifically, depletion of chromatin-associated cohesin reduced TADs and revealed hidden, finer compartments, while increased processivity of cohesin led to stronger TADs and reduced compartmentalization, and depletion of the TAD boundary protein, CTCF, weakened TADs while leaving compartments unaffected. We reveal that these experimental perturbations are special cases of a general polymer phenomenon of active mixing by loop extrusion. This also predicts that interference with chromatin epigenetic states or nuclear volume would affect compartments but not TADs. Our results suggest that chromatin organization on the megabase scale emerges from competition of non-equilibrium active loop extrusion and epigenetically defined compartment structure.

title: Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure
abstract: BackgroundIn homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation.ResultsWe found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation.ConclusionsOur findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.

title: Erythroblast cell lines transformed by a temperature‐sensitive mutant of avian erythroblastosis virus: A model system to study erythroid differentiation in vitro
abstract: A continuous chicken erythroblast cell line transformed by the temperature‐sensitive mutant ts34 of avian erythroblastosis virus was developed. This cell line, designated HD3, could be induced to terminally differentiate by shift to the nonpermissive temperature. The differentiated cells resembled erythrocytes as judged by morphology, expression of hemoglobin as determined by benzidine staining and radioimmunoassay, and by the expression of differentiation‐specific cell surface antigens. Terminal differentiation was dependent on an erythropoietin‐like activity present in anemic chicken serum. In contrast, induction of differentiation in the same cells by butyric acid was erythropoietin independent and did not lead to the formation of erythrocytes. In addition, we found that the responsiveness to temperature inducibility and to butyric acid could be dissociated in variant sublines of HD3 and that both types of differentiation inducers appear to act via different pathways.

title: Organization of the Mitotic Chromosome
abstract: Chromosome Conundrum The three-dimensional organization of chromosomal DNA within the cell nucleus plays an important role in gene regulation. Naumova et al. (p. 948, published online 7 November; see the Perspective by Kleckner et al.) used chromosome conformation capture-based methods in human tissue culture cells to analyze the higher order folding of human chromosomes across the cell cycle. During interphase the chromosomes showed locus-specific compart-mentalization. In mitotic cells, on the other hand, the chromosome organization was more linear, consistent with arrays of consecutive chromatin loops. Chromosome conformation changes dramatically during the cell cycle and is unlikely to carry epigenetic information.  Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type–specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.

title: Linkage of the major histocompatibility (B) complex and the nucleolar organizer in the chicken. Assignment to a microchromosome.
abstract: The linkage relationship and chromosomal locations of the major histocompatibility (B) complex and nucleolar organizers (18S + 28S ribosomal RNA genes) were studied in normal and aneuploid chickens. The Balloantigens were defined by hemagglutination, using monospecific alloantisera. A chicken having three B haplotypes was detected and used in test matings to normal disomic chickens. Additional cases of birds having three different haplotypes were generated in the progeny of such matings. Analysis of the segregation patterns of B haplotypes suggested that the chickens with an additional haplotype were trisomics. Chickens having three B haplotypes also displayed a maximum of three nucleoli in somatic cells instead of the normal two nucleoli of diploids. This indicated the presence of an additional nucleolus organizing region (NOR). Cytogenetic and cytochemical studies were performed on cells of normal and putative trisomic chickens. All chickens displayed a normal array of chromosomes for pairs 1 through 9. Silver staining differentiated Ag-NORs on the long arms of two and three microchromosomes in disomic and trisomic types, respectively. Viable tetrasomic chickens, produced from inter se matings of trisomics, displayed four nucleoli and four Ag-NORs in somatic cell preparations. These results indicate that the DNA sequences encoding the B histocompatibility antigens and the 18S + 28S ribosomal RNAs are linked on an acrocentric microchromosome in the domestic chicken.

title: Architecture and organization of chicken microchromosome 16: order of the NOR, MHC-Y, and MHC-B subregions.
abstract: Here we present a high-resolution cytogenomic analysis of chicken microchromosome 16. We established the location of the major histocompatibility complex (MHC)-B and -Y subregions relative to each other and to the nucleolus organizer region (NOR) encoding the 18S-5.8S-28S ribosomal DNA. To do so, we employed multicolor fluorescence in situ hybridization using large-insert bacterial artificial chromosome clones with fully sequenced inserts or repetitive sequence probes specific for the subregion of interest. We show that the MHC-Y and -B regions are located on the same side of the NOR, rather than opposite ends, as previously proposed. On the q arm, the MHC-Y is closely adjacent to the NOR, whereas the MHC-B is distal near the q-terminus. A relatively large GC-rich region separates the 2 MHC subregions and includes a specialized structure, a secondary constriction. We propose that the GC-rich large physical distance is the basis for the lack of genetic linkage between the NOR and MHC-B and between the MHC-Y and -B. An integrated model for GGA 16 is presented that incorporates gene complex order in the context of key architectural features including p and q arms, primary (centromere) and secondary constrictions, telomeres, as well as AT- and GC-rich regions.

title: Nucleolar changes in maturing avian erythroblasts and erythrocytes
abstract: SummaryMaturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Ring shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.

title: DNA synthesis in purified populations of avian erythroid cells.
abstract: By the use of equilibrium density-gradient centrifugation erythroblasts and early polychromatic erythrocyte8 have been isolated from avian anaemic bone marrow. Cells from both the unfractionated and purified preparations have been characterized in terms of their histological type, size, haemoglobin content and ability to synthesize DNA. Erythroblasts were the only cells to synthesize DNA and it appeared that their progeny, the polychromatic erythrocyte, failed to enter a new S phase. The experimental system described allows biochemical characterization of earlier stages of avian erythropoiesis than has previously been possible.