Lampbrush chromosomes LBCs are transcriptionally active chromosomes found in the germinal vesicle GV of large oocytes of many vertebrate and invertebrate animals and also in the giant single-celled alga Acetabularia. These cells are all in prophase of the first meiotic division. LBCs probably represent the most active transcriptional state that can be attained by cells that must give rise to diploid progeny. Polyploidy permits cells to reach higher rates of transcription per nucleus but precludes a return to diploidy. In this sense LBCs represent a relatively inefficient transcriptional compromise employed by large meiotic cells.
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Giant lampbrush chromosomes LBCs typical for growing oocytes of various animal species are characterized by a specific chromomere-loop appearance and massive transcription. Chromomeres represent universal units of chromatin packaging at LBC stage. While quite good progress has been made in investigation of LBCs structure and function, chromomere organization still remains poorly understood. To extend our knowledge on chromomere organization, we applied microdissection to chicken LBCs.
In particular, 31 and 5 individual chromomeres were dissected one by one along the macrochromosome 4 and one microchromosome, respectively. The data on genomic context of individual chromomeres was obtained by high-throughput sequencing of the corresponding chromomere DNA. Alignment of adjacent chromomeres to chicken genome assembly provided information on chromomeres size and genomic boarders, indicating that prominent marker chromomeres are about 4—5 Mb in size, while common chromomeres of 1.
Analysis of genomic features showed that the majority of chromomere-loop complexes combine gene-dense and gene-poor regions, while massive loopless DAPI-positive chromomeres lack genes and are remarkably enriched with different repetitive elements. Generally, the results obtained suggest that chromomeres of LBCs do not correspond unambiguously to any type of well-established spatial domains of interphase nucleus in chicken somatic cells.
In highly extended chromosomes, such as polytene chromosomes, lampbrush chromosomes LBCs , and pachytene chromosomes, a chromomere is defined as a universal unit of chromatin packaging Vlad and Macgregor, While our understanding of structure and function of chromomeres in polytene chromosomes has considerably grown in recent years, chromomere organization in LBCs still remains poorly understood.
Chromomeres of lampbrush chromosomes, being typical for animal growing oocytes, are regarded as condensed and apparently transcriptionally inactive chromatin domains Vlad and Macgregor, ; Macgregor, LBC chromomeres can be seen in both fixed and living chromosome preparations. An array of chromomeres constitutes an axis of each LBC, with neighboring chromomeres being connected by thin decondensed chromatin threads interchromomeric fibers. Besides, apart from chromomeres with numerous pairs of extended loops, there are some chromomeres lacking recognizable loops.
From structural point of view LBC chromomeres are thought to represent a rosette of microloops, which are connected by protein clips at their bases. Nevertheless, while there is some data on overall structure, protein composition, and epigenetic status of LBC chromomeres, their genomic context has not been a focus of previous studies. Morphologically discrete chromomeres can be mechanically dissected from a single copy of LBC by glass needles.
To extend our knowledge on chromomere organization and genomic context, we performed microdissection of all prominent chromomeres from lampbrush macrochromosome 4 and one of the microchromosomes in a chicken lampbrush chromosome set. The data on cytogenetic and genomic features of individual chromomeres were obtained by high-resolution fluorescence in situ hybridization FISH and high-throughput sequencing procedure.
Finally, LBC chromomeres were compared with chromatin domains earlier identified by Hi-C technique in interphase nucleus of chicken embryonic fibroblasts.
Generally, the results obtained in the present study suggest that chromomeres of LBCs do not correspond unambiguously to any type of well-established chromatin domains of interphase nucleus of somatic cells. Mitotic metaphase chromosomes were obtained from chicken embryonic fibroblasts according to standard protocols. All institutional and national guidelines for the care and use of laboratory and farm animals were followed.
The animal studies received approval — of the Ethical committee of Saint-Petersburg State University. In brief, individual chromomeres were dissected one after another along the length of macrochromosome 4 and one of the microchromosomes under phase contrast microscopy. Metaphase chromosomes were pre-treated with 0.
Post-hybridization washings included two changes of 0. Avidin-Alexa Molecular Probes Inc. The DNA-library preparation was performed according to the manufacturer's recommendations with some modifications Ion Torrent, Life Technologies.
Quality and quantity of the fragments were evaluated by high-resolution capillary electrophoresis using Shimadzu MultiNA Japan.
To get rid of dimers of primers, the samples were purified using magnetic beads Agencourt AMPure XP Beckman Coulter followed by a capillary electrophoresis analysis. Final concentrations of the DNA-libraries were assessed using Qubit 2. To get rid of terminal adapter sequences and remove poor quality base calls from the end, the sequence reads were trimmed and filtered by length.
For comparative analysis the chromomere borders were defined according to the alignment of sequenced reads to the reference genome and by excluding single reads distant from the main cluster of reads. The chromomeres with ambiguous borders were excluded from the analysis. The number of TADs per chromomere was counted using JuiceBox heatmaps and the defined chromomere borders. To analyze the genomic organization of LBC chromomeres, we applied mechanical microdissection to chicken lampbrush chromosomes followed by preparation of DNA-libraries of isolated chromomeres.
In particular, all prominent chromomeres were dissected one by one along the chicken lampbrush macrochromosome 4 starting from the q-ter chromosome region: chromomeres 1—31 Supplementary Figure S1. Additionally, we isolated chromomeres constituting one of chicken microchromosomes in the lampbrush form. To verify the quality and specificity of the dissected samples, the DNA fragments were mapped on chicken metaphase chromosomes. Among 31 DNA probes marking chicken LBC4, 27 probes demonstrated bright and specific hybridization signal on a corresponding pair of homologous chromosomes in metaphase plates Figures 1A—C.
The remainder four probes gave a major hybridization signal on GGA4 as well as additional minor signals dispersed across the karyotype, which might be due to excess of interspersed DNA-repeats in microdissected material.
Figure 1 FISH-mapping of microdissected lampbrush chromosome chromomeres on chicken metaphase chromosomes. Chromomere ID numbers are indicated.
Chromosomes are counterstained with DAPI. In most cases, a hybridization signal was observed in a single chromomere similar in size and morphology to the dissected chromomere indicating a tendency of chromomeres to maintain their integrity as individual chromatin domains.
At the same time, in some cases we observed a FISH signal in several neighboring chromomeres, which can be explained by different degrees of LBC's condensation during the oocyte growth. That is, chromatin of an individual chromomere dissected from a more compact lampbrush chromosome may be included into several smaller chromomeres in less compact chromosomes.
The majority of DNA probes hybridized to small and medium-sized loose chromomeres, with the hybridization signal being also revealed in RNP-matrix of extended lateral loops Figures 2B, C. To investigate the genomic context of LBC chromomeres, we applied high-throughput sequencing of individual chromomeres microdissected from chicken lampbrush chromosomes. Such marker chromomeres are typical for certain regions of the largest chicken lampbrush marcrochromosomes.
In the present study, we sequenced DNA-material of 24 neighboring chromomeres covering the LBC 4 along its length samples macro1—6, 11—23, 27—31 , and five chromomeres constituting one of the chicken microchromosomes samples micro1—5.
In case of LBC4, all 24 samples of individual chromomere-loop complexes were successfully assigned to GGA4 reference genome assembly with neighboring dissected chromomeres being mapped to adjacent genomic regions Figure 3. Besides, the results of genome mapping allowed identifying the dissected lampbrush microchromosome as chromosome Figure 3 Genomic context of individual lampbrush chromosome chromomeres.
The sequencing reads corresponding to individual chromomeres are shown in different colors. In particular, according to our assessments the amount of DNA in the majority of small and medium-sized chromomeres is about 1.
The microdissected chromomere samples were also analyzed with regard to gene density and repeat content Figure 3. Besides, the sequencing data was compared with the results of FISH-mapping on lampbrush chromosomes. Some chromomeres demonstrated relatively higher gene density and lower content of repetitive sequences chromomeres of LBC11, chromomeres 22 and 13 of LBC4.
Thus the genomic coordinates of the double loop bridge region were determined precisely, which provides prospects to determine the DNA sequences underlying the formation of such structures. Besides, high-throughput sequencing data demonstrated that DNA of massive loopless chromomeres was significantly enriched by repetitive DNA-elements of different nature and comprised smaller amount of genes as compared to neighboring regions.
We conclude that the described complex approach combining cytological, cytogenetic, and genomic analysis allows to correlate morphologically distinct chromatin domains—lampbrush chromosome chromomeres in complex with arising lateral loops—with particular deciphered genomic regions. In particular, similar to other vertebrates chicken genome proved to be folded into large-scale epigenetically distinct domains: A-compartments containing open and transcriptionally active chromatin and B-compartments with silent chromatin.
Within compartments the chromatin is packaged into submegabase-sized topologically associated domains TADs , which represent local contact-enriched self-interacting chromatin domains.
In other words, a single chromomere may contain chromatin belonging to both A and B compartments of interphase nucleus. The majority of dissected chromomeres contained different proportions of A and B compartments with only single chromomeres being fully overlapped by a compartment of one type.
Individual chromomeres are shown in different colors and numbered according to chromomere ID. A- and B-compartments are shown in red and blue, correspondingly. The genomic regions corresponding to LBC chromomeres are shown in blue. It should be taken into account that genomic borders of microdissected chromomeres were mapped with some precision, which was determined by the accuracy of chromomere identification and isolation during microdissection and by the sequencing depth.
That is, we analyzed 27 deciphered chromomeres and estimated that one chromomere may comprise from 0. On the average, chromomeres contained 2. In general, our data suggest that lampbrush chromosome chromomeres do not correspond unambiguously to any type of spatial genomic domains previously identified in the interphase nucleus of somatic cells.
The described approach allowed us to correlate particular chromomere-loop complexes with the deciphered genomic regions.
Until this study, the DNA composition of lampbrush chromosome chromomeres has remained unknown with few exceptions. In particular, some data were obtained for a small number of chromomeres consisting of massive arrays of tandemly repeated sequences.
However, as early as in H. Macgregor suggested that while some chromomeres bear highly uniform DNA such as clusters of repetitive sequences , the others have a less uniform content Macgregor, In the present study, we for the first time established genomic properties of an array of regular chromomeres from chicken LBCs including all morphologically distinct chromomeres from macrochromosome 4 and microchromosome Apparently, chromomeres of meiotic lampbrush chromosomes have little in common with chromomeres of polytene chromosomes.
On the contrary, the data obtained in our study imply that chromomeres of chicken LBCs generally do not correspond to TADs identified in chicken embryonic fibroblasts. In particular, LBC chromomeres are larger structural units of chromatin organization, and genomic regions corresponding to several somatic TADs are involved in their formation. A pattern of transcription during the lampbrush stage of meiosis dramatically differs from one in somatic cells due to a supposed role of LBCs in accumulation of maternal RNAs in growing oocytes.
Such a peculiar pattern of transcription leads to a distinctive pattern of untranscribed regions gathering in chromomeres. This can underlay the discrepancy in organization between LBC chromomeres and compact chromatin domains of somatic cells.
It was previously suggested that lampbrush chromosome chromomeres appear as a result of massive transcription taking place on the lateral loops Callan, That is, lateral loops with RNP-matrix consisting of nascent transcripts and associated RNA-binding factors push apart dense transcriptionally inactive chromatin domains leading to their insularity.
Further single-cell Hi-C studies of oocyte nucleus with a lampbrush chromosome set together with high-resolution FISH-mapping are required to determine chromatin domains with higher frequency of self-interactions and their correspondence to lampbrush chromomeres.
The animal study was reviewed and approved by the Ethical committee of Saint-Petersburg State University approval — AK conceived the study and supervised the project. AZ planned and carried out most of the principal cytogenetic experiments. AZ and OP performed high-throughput sequencing. AZ and AM performed bioinformatic analysis. All authors read and approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Dmitrii Polev St. Petersburg State University.
Lampbrush chromosome immature eggs of most animals, except mammals. They were first described by Walther Flemming in They are highly extended meiotic half-bivalents, each consisting of 2 sister chromatids. Lampbrush chromosomes are clearly visible even in the light microscope , where they are seen to be organized into a series of chromomeres with large chromatin loops extended laterally. Amphibian and avian lampbrush chromosomes can be microsurgically isolated from oocyte nucleus germinal vesicle with either forceps or needles. Each lateral loop contains one or several transcription units with polarized RNP-matrix coating the DNA axis of the loop.
Induction of human lampbrush chromosomes
Giant lampbrush chromosomes LBCs typical for growing oocytes of various animal species are characterized by a specific chromomere-loop appearance and massive transcription. Chromomeres represent universal units of chromatin packaging at LBC stage. While quite good progress has been made in investigation of LBCs structure and function, chromomere organization still remains poorly understood. To extend our knowledge on chromomere organization, we applied microdissection to chicken LBCs. In particular, 31 and 5 individual chromomeres were dissected one by one along the macrochromosome 4 and one microchromosome, respectively.
Are lampbrush chromosomes unique to meiotic cells?
Metrics details. Over the past two decades, chromosome microdissection has been widely used in diagnostics and research enabling analysis of chromosomes and their regions through probe generation and establishing of chromosome- and chromosome region-specific DNA libraries. However, relatively small physical size of mitotic chromosomes limited the use of the conventional chromosome microdissection for investigation of tiny chromosomal regions. In the present study, we developed a workflow for mechanical microdissection of giant transcriptionally active lampbrush chromosomes followed by the preparation of whole-chromosome and locus-specific fluorescent in situ hybridization FISH -probes and high-throughput sequencing. In particular, chicken Gallus g.
We'd like to understand how you use our websites in order to improve them. Register your interest. The lampbrush chromosomes and assorted nuclear bodies of amphibian and avian oocytes provide uniquely advantageous and amenable experimental material for cell biologists to study the structure and function of the eukaryotic nucleus, and in particular to address the processes of nuclear gene expression. Recent findings discussed here include the molecular analysis of the actively elongating RNA polymerase complexes associated with lampbrush chromosome loops and of the association between loop nascent transcripts and RNA processing components. In addition, several types of chromosome structure that do not outwardly resemble simple extended loops and that may house novel nuclear functions have recently been studied in detail.