Comparison of the somatic TADs and lampbrush chromomere-loop complexes in transcriptionally active prophase I oocytes

In diplotene oocyte nuclei of all vertebrate species, except mammals, chromosomes lack interchromosomal contacts and chromatin is linearly compartmentalized into distinct chromomere-loop complexes forming lampbrush chromosomes. However, the mechanisms underlying the formation of chromomere-loop complexes remain unexplored. Here we aimed to juxtapose somatic topologically associating domains (TADs), recently identified in chicken embryonic fibroblasts, with chromomere-loop complexes in lampbrush meiotic chromosomes. By measuring 3D-distances and colocalization between linear equidistantly located genomic loci, positioned within one TAD or separated by a TAD border, we confirmed the presence of predicted TADs in chicken embryonic fibroblast nuclei. Using three-colored FISH with BAC probes we mapped equidistant genomic regions included in several sequential somatic TADs on isolated chicken lampbrush chromosomes. Eight genomic regions, each comprising two or three somatic TADs, were mapped to non-overlapping neighboring lampbrush chromatin domains - lateral loops, chromomeres or chromomere-loop complexes. Genomic loci from the neighboring somatic TADs could localize in one lampbrush chromomere-loop complex, while genomic loci belonging to the same somatic TAD could be localized in neighboring lampbrush chromomere-loop domains. In addition, FISH-mapping of BAC probes to the nascent transcripts on the lateral loops indicates transcription of at least 17 protein-coding genes and 2 non-coding RNA genes during the lampbrush stage of chicken oogenesis, including genes involved in oocyte maturation and early embryo development.

Identification of Genomic Loci Responsible for the Formation of Nuclear Domains Using Lampbrush Chromosomes

In the cell nuclei, various types of nuclear domains assemble as a result of transcriptional activity at specific chromosomal loci. Giant transcriptionally active lampbrush chromosomes, which form in oocyte nuclei of amphibians and birds enable the mapping of genomic sequences with high resolution and the visualization of individual transcription units. This makes avian and amphibian oocyte nuclei an advantageous model for studying locus-specific nuclear domains. We developed two strategies for identification and comprehensive analysis of the genomic loci involved in nuclear domain formation on lampbrush chromosomes. The first approach was based on the sequential FISH-mapping of BAC clones containing genomic DNA fragments with a known chromosomal position close to the locus of a nuclear domain. The second approach involved mechanical microdissection of the chromosomal region adjacent to the nuclear domain followed by the generation of FISH-probes and DNA sequencing. Furthermore, deciphering the DNA sequences from the dissected material by high throughput sequencing technologies and their mapping to the reference genome helps to identify the genomic region responsible for the formation of the nuclear domain. For those nuclear domains structured by nascent transcripts, identification of genomic loci of their formation is a crucial step in the identification of scaffold RNAs.

Chicken Microchromosomes in the Lampbrush Phase: A Cytogenetic Description

Lampbrush chromosomes are giant, transcriptionally active, meiotic chromosomes found in oocytes of all vertebrates with the exception of mammals. Lampbrush chromosomes offer a convenient tool for cytogenetic mapping and, in particular, have been instrumental in mapping genes and linkage groups on chicken (GGA) chromosomes. Whereas cytogenetic maps of macrochromosome GGA1-10 and microchromosome GGA11-16 lampbrush bivalents have been established, identification and description of smaller microchromosome bivalents are still missing. In this work, we used specific FISH probes for the identification of 12 chicken lampbrush chromosomes formed by GGA17-28. Our observations on chromomere and lateral loop arrangement and chiasma position allowed us to construct the respective cytogenetic maps for these microchromosomes. For the 10 smallest chicken microchromosomes, GGA29-38, no individual molecular tags are available, yet they can be collectively marked using the PO41 repeat. The reported results contribute to building of working cytogenetic maps of the chicken karyotype.