Differentially accessible, single copy sequences form contiguous domains along metaphase chromosomes that are conserved among multiple tissues

Background During mitosis, chromatin engages in a dynamic cycle of condensation and decondensation. Condensation into distinct units to ensure high fidelity segregation is followed by rapid and reproducible decondensation to produce functional daughter cells. Factors contributing to the reproducibility of chromatin structure between cell generations are not well understood. We investigated local metaphase chromosome condensation along mitotic chromosomes within genomic intervals showing differential accessibility (DA) between homologs. DA was originally identified using short sequence-defined single copy (sc) DNA probes of < 5 kb in length by fluorescence in situ hybridization (scFISH) in peripheral lymphocytes. These structural differences between metaphase homologs are non-random, stable, and heritable epigenetic marks which have led to the proposed function of DA as a marker of chromatin memory. Here, we characterize the organization of DA intervals into chromosomal domains by identifying multiple DA loci in close proximity to each other and examine the conservation of DA between tissues. Results We evaluated multiple adjacent scFISH probes at 6 different DA loci from chromosomal regions 2p23, 3p24, 12p12, 15q22, 15q24 and 20q13 within peripheral blood T-lymphocytes. DA was organized within domains that extend beyond the defined boundaries of individual scFISH probes. Based on hybridizations of 2 to 4 scFISH probes per domain, domains ranged in length from 16.0 kb to 129.6 kb. Transcriptionally inert chromosomal DA regions in T-lymphocytes also demonstrated conservation of DA in bone marrow and fibroblast cells. Conclusions We identified novel chromosomal regions with allelic differences in metaphase chromosome accessibility and demonstrated that these accessibility differences appear to be aggregated into contiguous domains extending beyond individual scFISH probes. These domains are encompassed by previously established topologically associated domain (TAD) boundaries. DA appears to be a conserved feature of human metaphase chromosomes across different stages of lymphocyte differentiation and germ cell origin, consistent with its proposed role in maintenance of intergenerational cellular chromosome memory.

Tools for Drawing Informative Idiograms

Each species has a typical karyotype, which represents the phenotypic appearance of the somatic chromosomes including number, size, and morphology. Idiogram is a diagrammatic representation of the chromosomes showing their relative size, homologous groups and different cytogenetic landmarks. Chromosomal analysis of cytological preparations is an essential component of many investigations which involves the calculation of karyotypic parameters and generation of idiogram. Although various tools are available for karyotype analysis, here we demonstrate karyotype analysis using our recently developed tool named KaryoMeasure. KaryoMeasure is a semi-automated free and user-friendly karyotype analysis software that facilitates data collection from different digital images of the metaphase chromosome spreads and calculates a wide variety of chromosomal and karyotypic parameters along with the related standard errors. KaryoMeasure draws idiograms of both diploid and allopolyploid species into a vector-based SVG or PDF image file.

Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

Analysis of the Metaphase Chromosome Karyotypes in Imaginal Discs of Aedes communis, Ae. punctor, Ae. intrudens, and Ae. rossicus (Diptera: Culicidae) Mosquitoes

In this study, cytogenetic analysis of the metaphase chromosomes from imaginal discs of Aedes (Diptera: Culicidae) mosquitoes—Aedes communis, Ae. punctor, Ae. intrudens, and Ae. rossicus—was performed. The patterns of C-banding and DAPI staining of the heteroсhromatin and the length of the chromosomes demonstrate species specificity. In particular, the Ae. punctor chromosomes are the shortest compared with Ae. communis, Ae. intrudens, and Ae. rossicus, and they also carry additional C and DAPI bands in intercalary regions. The Ae. intrudens chromosomes are the longest, they have pericentromeric C bands, and they almost lack any DAPI bands near the centromere of chromosome 3 versus Ae. communis, which has the largest pericentromeric DAPI blocks in all three chromosome pairs. Ae. rossicus also possesses DAPI bands in the centromeric regions of all chromosomes, but their staining is weaker compared with those of Ae. communis. Therefore, the analysis of karyotypes is a tool for species-level identification of these mosquitoes.

Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to disassemble bridging fibers, and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochores fibers.