Hi-C analysis of genomic contacts revealed karyotype abnormalities in chicken HD3 cell line.

BACKGROUND: Karyotype abnormalities are frequent in immortalized continuous cell lines either transformed or derived from primary tumors. Chromosomal rearrangements can cause dramatic changes in gene expression and affect cellular phenotype and behavior during in vitro culture. Structural variations of chromosomes in many continuous mammalian cell lines are well documented, but chromosome aberrations in cell lines from other vertebrate models often remain understudied. The chicken LSCC-HD3 cell line (HD3), generated from erythroid precursors, was used as an avian model for erythroid differentiation and lineage-specific gene expression. However, karyotype abnormalities in the HD3 cell line were not assessed. In the present study, we applied high-throughput chromosome conformation capture to analyze 3D genome organization and to detect chromosome rearrangements in the HD3 cell line. RESULTS: We obtained Hi-C maps of genomic interactions for the HD3 cell line and compared A/B compartments and topologically associating domains between HD3 and several other cell types. By analysis of contact patterns in the Hi-C maps of HD3 cells, we identified more than 25 interchromosomal translocations of regions ≥ 200 kb on both micro- and macrochromosomes. We classified most of the observed translocations as unbalanced, leading to the formation of heteromorphic chromosomes. In many cases of microchromosome rearrangements, an entire microchromosome together with other macro- and microchromosomes participated in the emergence of a derivative chromosome, resembling "chromosomal fusions'' between acrocentric microchromosomes. Intrachromosomal inversions, deletions and duplications were also detected in HD3 cells. Several of the identified simple and complex chromosomal rearrangements, such as between GGA2 and GGA1qter; GGA5, GGA4p and GGA7p; GGA4q, GGA6 and GGA19; and duplication of the sex chromosome GGAW, were confirmed by FISH. CONCLUSIONS: In the erythroid progenitor HD3 cell line, in contrast to mature and immature erythrocytes, the genome is organized into distinct topologically associating domains. The HD3 cell line has a severely rearranged karyotype with most of the chromosomes engaged in translocations and can be used in studies of genome structure-function relationships. Hi-C proved to be a reliable tool for simultaneous assessment of the spatial genome organization and chromosomal aberrations in karyotypes of birds with a large number of microchromosomes.

Comparison and critical assessment of single-cell Hi-C protocols.

Advances in single-cell sequencing technologies make it possible to study the genome architecture in single cells. The rapid growth of the field has been fueled by the development of innovative single-cell Hi-C protocols. However, the protocols vary considerably in their efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. Here, we compare the two most commonly used single-cell Hi-C protocols. We use long-read sequencing to analyze molecular products of the Hi-C assay and show that whole-genome amplification step results in increased number of artifacts, larger coverage biases, and increased amount of noise compared to PCR-based amplification. Our comparison provides guidance for researchers studying chromatin architecture in individual cells.

Author Correction: Micronuclei in germ cells of hybrid frogs from Pelophylax esculentus complex contain gradually eliminated chromosomes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Micronuclei in germ cells of hybrid frogs from Pelophylax esculentus complex contain gradually eliminated chromosomes.

In most organisms, cells typically maintain genome integrity, as radical genome reorganization leads to dramatic consequences. However, certain organisms, ranging from unicellular ciliates to vertebrates, are able to selectively eliminate specific parts of their genome during certain stages of development. Moreover, partial or complete elimination of one of the parental genomes occurs in interspecies hybrids reproducing asexually. Although several examples of this phenomenon are known, the molecular and cellular processes involved in selective elimination of genetic material remain largely undescribed for the majority of such organisms. Here, we elucidate the process of selective genome elimination in water frog hybrids from the Pelophylax esculentus complex reproducing through hybridogenesis. Specifically, in the gonads of diploid and triploid hybrids, but not those of the parental species, we revealed micronuclei in the cytoplasm of germ cells. In each micronucleus, only one centromere was detected with antibodies against kinetochore proteins, suggesting that each micronucleus comprises a single chromosome. Using 3D-FISH with species-specific centromeric probe, we determined the role of micronuclei in selective genome elimination. We found that in triploid LLR hybrids, micronuclei preferentially contain P. ridibundus chromosomes, while in diploid hybrids, micronuclei preferentially contain P. lessonae chromosomes. The number of centromere signals in the nuclei suggested that germ cells were aneuploid until they eliminate the whole chromosomal set of one of the parental species. Furthermore, in diploid hybrids, misaligned P. lessonae chromosomes were observed during the metaphase stage of germ cells division, suggesting their possible elimination due to the inability to attach to the spindle and segregate properly. Additionally, we described gonocytes with an increased number of P. ridibundus centromeres, indicating duplication of the genetic material. We conclude that selective genome elimination from germ cells of diploid and triploid hybrids occurs via the gradual elimination of individual chromosomes of one of the parental genomes, which are enclosed within micronuclei.

Giant poly(A)-rich RNP aggregates form at terminal regions of avian lampbrush chromosomes.

The cell nucleus comprises a number of chromatin-associated domains. Certain chromatin-associated domains are nucleated by nascent RNA and accumulate non-nascent transcripts in the form of ribonucleoprotein (RNP) aggregates. In the transcriptionally active nucleus of the growing avian oocyte, RNP-rich structures, here termed giant terminal RNP aggregates (GITERA), form at the termini of lampbrush chromosomes. Using GITERA as an example, we aimed to explore mechanisms of RNP aggregate formation at certain chromosomal loci to establish whether they accumulate non-nascent RNA and to analyze protein composition in RNP aggregates. We found that GITERA on chicken and pigeon lampbrush chromosomes do not contain nascent transcripts. At the same time, RNA fluorescent in situ hybridization (FISH) and in situ reverse transcription demonstrated that GITERA accumulate poly(A)-rich RNA. Moreover, subtelomere chromosome regions adjacent to GITERA are transcriptionally active as shown by detection of incorporated BrUTP and the elongating form of RNA polymerase II. GITERA on both chicken and pigeon lampbrush chromosomes are enriched in splicing factors but not in heterogeneous nuclear RNP (hnRNP) L and K. A subtype of GITERA concentrates hnRNP I/PTB and p54nrb/NonO. Interestingly, hnRNP I/PTB and p54nrb/NonO in such subtype of GITERA were revealed in long threads. The resemblance of these threads to amyloid-like fibers is discussed. Our data suggest that transcription of subtelomeric sequences serves as a seeding event for accumulation of non-nascent RNA and associated RNP proteins. Such accumulation leads to GITERA formation in terminal chromosomal regions in avian oocyte nucleus. 3'-processed transcripts derived from other chromosomal loci may be attracted to GITERA by binding to the same RNP proteins or to their interaction partners.

LAMPBRUSH CHROMOSOMES AS A MODEL FOR EXPLORATION INTO LOCI OF NUCLEAR DOMAINS FORMATION.

Nuclear domains can be divided into two major groups: those arising freely in nucleoplasm and those forming at specific chromosomal loci as a result of their activity. The advantages of giant transcriptionally active lampbrush chromosomes for the investigation of nuclear bodies formed in particular chromosomal regions have been demonstrated in a series of studies. We propose to use two strategies to analyze the loci of nuclear domains formation on lampbrush chromosomes typical for avian and amphibian oocytes. The first approach implies consecutive mapping of BAC-clones, containing the fragments of DNA assigned to genomic coordinates, in close proximity to the nuclear domains. The second approach is based on mechanical microdissection of chromosomal regions adjacent to a particular nuclear structure. DNA of dissected material can be amplified by PCR with degenerate primers and mapped by fluorescent in situ hybridization (FISH) on chromosomal spreads. Utilization of high-throughput sequencing (next generation sequencing, NGS) technologies also proves to be prospective for subsequent deciphering of regions underlying nuclear structures formation. Deciphered fragments can be aligned against reference genome assembly to define precisely the loci responsible for nuclear domains assembly. In this review, the possibilities of using two complementary strategies for investigation of nuclear domains associated with lampbrush chromosomes are demonstrated.

[Cajal bodies and histone locus bodies: molecular structure and function].

The review provides modern classification of evolutionarily conserved coilin-containing nuclear bodies of somatic and germ cells that is based on the characteristic features of their molecular composition and the nature of their functions. The main differences between Cajal bodies and histone locus bodies, which are involved in the biogenesis of small nuclear spliceosomal and nucleolar RNAs and in the 3'-end processing of histone precursor messenger RNA, respectively, are considered. It is shown that a significant contribution to the investigation of the diversity of coilin-containing bodies was made by the studies on the architecture of the RNA processing machinery in oocyte nuclei in a number of model organisms. The characteristics features of the molecular composition of coilin-containing bodies in the nuclei of growing oocytes (the so-called germinal vesicles) of vertebrates, including amphibians and birds, are described.

[Spatial arrangement of macro-, midi- and microchromosomes in transcriptionally active nuclei of growing oocytes in birds of the order galliformes].

Three-dimensional genome organization in the cell nucleus reflects its functional state and is one of the regulatory levels of gene expression. Thus, a detailed exploration of the interrelations between the genome spatial organization and functioning is essential. In this work, three-dimensional genome organization in growing oocytes of Galliform birds, with giant transcriptionally active nucleus characterized by nearly absolute lack of structural constraints on chromosome decondensation, is analyzed in detail. Radial distribution of three groups of chromosomes with different size and gene density in the nuclei of chicken and Japanese quail oocytes was analyzed using confocal laser scanning microscopy followed by the 3D-reconstruction. The chromosome position relative to the nuclear center was estimated by analyzing its localization in the certain radial zones of the nucleus and direct distance measurements from the centre of the nucleus to the terminal regions and the center of gravity of the chromosome. It has been shown that, in the transcriptionally active nuclei of avian oocytes, chromosomes are located at a significant distance from the nuclear envelope and the gene-rich microchromosomes have no preferential location close to the center of the nucleus and are localized mainly at the periphery of the region occupied by the whole chromosome set. Therefore the radial distribution of lampbrush chromosomes in the oocyte nucleus does not obey the regularity of the spatial arrangement of chromosomes in the interphase nucleus according to which the gene-rich chromosome territories are located at the nuclear center and the gene-poor ones are at the nuclear periphery. With the help of visualization of 3D-preserved lampbrush chromosomes in the intact nucleus, we have confirmed the presence of the repulsion forces between the lateral loops of lampbrush half-bivalents and the lack of interactions between the heterochromatic segments of different bivalents at the lampbrush stage of oogenesis.

[Organization of centromere regions of chromosomes in the lampbrush phase].

Centromeres have a pivotal role in the complex of structural elements that are required for precise segregation of eukaryotic chromosomes during two types of cellular divisions--mitosis and meiosis. Data of ultrastructural and cytomolecular analysis indicate significant changes in molecular composition and functional morphology of centromeres during preparation for the first meiotic division. The review is devoted to modern concepts of morpho-functional organization of chromosomal centromere regions in growing oocytes in birds and amphibians. Structure, molecular composition as well as domain organization of centromeres in the lampbrush phase are characterized; data of cytogenetic analysis are presented. Special attention is given to the significance and regulation of satellite DNA transcription in the nuclei of developing oocytes. Possible functions of centromere "protein bodies" formed at the primary constriction of meiotic bivalents are discussed.

Precise centromere positioning on chicken chromosome 3.

Despite the progress of the chicken (Gallus gallus) genome sequencing project, the centromeric sequences of most macrochromosomes remain unknown. This makes it difficult to determine centromere positions in the genome sequence assembly. Using giant lampbrush chromosomes from growing oocytes, we analyzed in detail the pericentromeric region of chicken chromosome 3. Without knowing the DNA sequence, the centromeres at the lampbrush stage are detectable by immunostaining with antibodies against cohesin subunits. Immunostaining for cohesin followed by FISH with 23 BAC clones, covering the region from 0 to 23 Mb on chicken chromosome 3 (GGA3), allowed us to map the GGA3 centromere between BAC clones WAG38P15 and WAG54M22 located at position 2.3 and 2.5 Mb, respectively. This corresponds to the gap between 2 supercontigs at the 2.4-Mb position in the current GGA3 sequence assembly (build 2.1). Furthermore, we have determined that the current putative centromeric gap at position 11.6-13.1 Mb corresponds in fact to a long cluster of tandem chicken erythrocyte nuclear membrane repeats (CNM).

[Chicken lampbrush chromosomes: transcription of tandemly repetitive DNA sequences].

The transcribed part of the genome includes both protein-coding sequences and a variety of sequences with unknown functions. Amphibian and avian lampbrush chromosomes represent a convenient experimental system for studying cell functions and the regulation of transcription of protein-noncoding DNA. Taking lumpy loops formed on chicken (Gallusgallus domesticus) chromosome 2 at the lampbrush stage as an example, we applied an approach allowing RNA sources to be identified in the lateral loops of lampbrush chromosomes. This approach involves a bioinformatic analysis of data from the chicken genome sequencing project and a high-resolution mapping of transcripts on microsurgically isolated bivalents. As a result, a novel tandemly repetitive DNA sequence, LL2R (lumpy loop 2 repeat), of approximately 440 bp in size was identified in the chicken genome, its transcripts taking part in the formation of lumpy loops with a massive RNP matrix on chromosome 2 in growing oocytes.

[Lampbrush chromosomes of the Japanese quail (Coturnix coturnix japonica): a new version of cytogenetic maps].

Avian oocyte chromosomes are transfomed into giant transcriptionally active lampbrush chromosomes (LBCs) at meiosis 1 diplotene. These chromosomes are a convenient tool for high-resulution cytogenetic analysis. Using differential staining with fluorochromes DAPI and CMA3, we have constructed detailed cytological maps for lampbrush macrochromosomes 1-5 and ZW of the Japanese quail Coturnix coturnix japonica. We also performed a comparative analysis ofmitotic chromosomes and LBCs corresponding to them. We estimated the decondensation coefficient during LBC formation and determined the centromere indices for mitotic and diplotene chromosomes and thus found that different chromosomes and chromosomal regions demonstrate unequal degrees of decondensation.

Polymorphic heterochromatic segments in Japanese quail microchromosomes.

Using highly extended lampbrush chromosomes from diplotene oocytes, we have examined the distribution of heterochromatin protein 1 beta (HP1 beta) and histone H3 modifications on chicken (Gallus gallus) and Japanese quail (Coturnix japonica) (2n = 78) microchromosomes. Acrocentric microchromosomes of chicken and submetacentric microchromosomes of quail differ in several morphological features. In addition to pericentromeric and subtelomeric blocks of constitutive heterochromatin, which are enriched in HP1 beta protein and repressive histone modifications, not completely condensed but heterochromatic segments were found to be an attribute of the short arms of submetacentric microchromosomes in Japanese quail. These heterochromatic regions are variable in length and do not form chiasmata in female germ cells. Dissimilarity in the centromere positions in chicken and Japanese quail microchromosomes is proposed to be due to the accumulation of repetitive sequences on the short arms of quail microchromosomes. Transcriptional activation of polymorphic heterochromatic segments of quail microchromosomes during the lampbrush stage is demonstrated.

Avian lampbrush chromosomes: a powerful tool for exploration of genome expression.

Lampbrush chromosomes (LBCs) are highly extended bivalents that function in the growing oocytes of many animals. Due to their distinctive chromomere-loop organization and intense transcriptional activity of lateral loops the LBCs, mainly amphibian ones, have served as a powerful system for exploring the general principles of chromosome organization and function. The exploitation of avian LBCs has considerably broadened the opportunities for comparative genome research and for cytogenetic analysis of domestic species. In this review we highlight the advantages of avian LBCs for research in different areas including integration of genome organization studies with studies on gene activity in vivo, analysis of co-transcriptional events occurring on nascent transcripts and investigation of chromosome-associated intranuclear domains. Recent findings concerning the organization of transcriptionally active and silent chromatin together with involvement of cohesin and condensin complexes into maintenance of structural integrity of LBCs are presented. The biological significance of the LBC phenomenon is discussed. The intensive transcription on LBCs shows some specific features: very long transcription units, deregulated termination, and transcription of non-coding satellite repeats. Here, based on the modern view on a role of RNA interference machinery in regulation of genome expression, we suggest a mechanism of initiation of satellite DNA transcription and offer a novel interpretation of the 'classical' hypothesis that sought to explain the significance of widespread transcription during oocyte growth.