Abstract
BackgroundCondensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. This study reports molecular cytogenetic data showing quantifiable localized differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy DNA probes used in fluorescence in situ hybridization.ResultsFourteen probes (from chromosomes 1, 5, 9, 11, 15, 17, 22) targeting genic and intergenic regions were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization between homologs were non-random for 8 genomic regions (RGS7, CACNA1B, GABRA5, SNRPN, HERC2, PMP22:IVS3, ADORA2B:IVS1, ACR) and were not unique to known imprinted domains or specific chromosomes. DNA probes within CCNB1, C9orf66, ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex 5, and intergenic region 1p36.3 showed no DA (equivalent accessibility), while OPCML showed unbiased DA. To pinpoint probe locations, we performed 3D-structured illumination microscopy (3D-SIM). This showed that genomic regions with DA had 3.3-fold greater volumetric, integrated probe intensities and broad distributions of probe depths along axial and lateral axes of the 2 homologs, compared to a low copy probe target (NOMO1) with equivalent accessibility. Genomic regions with equivalent accessibility were also enriched for epigenetic marks of open interphase chromatin (DNase I HS, H3K27Ac, H3K4me1) to a greater extent than regions with DA.ConclusionsThis study provides evidence that DA is non-random and reproducible; it is locus specific, but not unique to known imprinted regions or specific chromosomes. Non-random DA was also shown to be heritable within a 2 generation family. DNA probe volume and depth measurements of hybridized metaphase chromosomes further show locus-specific chromatin accessibility differences by super-resolution 3D-SIM. Based on these data and the analysis of interphase epigenetic marks of genomic intervals with DA, we conclude that there are localized differences in compaction of homologs during mitotic metaphase and that these differences may arise during or preceding metaphase chromosome compaction. Our results suggest new directions for locus-specific structural analysis of metaphase chromosomes, motivated by the potential relationship of these findings to underlying epigenetic changes established during interphase.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-014-0070-y) contains supplementary material, which is available to authorized users.
Highlights
Condensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known
To illustrate different hybridization behaviours between homologs with short-target, single copy fluorescence in situ hybridization (FISH) probes, we compare examples of normal metaphase chromosomes hybridized with probes that show differences in accessibility to probes with equivalent accessibility
Single copy probes with differences in fluorescence intensities between homologs (CACNA1B, HERC2, and PMP22:IVS3 genes) are shown in Figure 1A, Table 1 and are contrasted with hybridized probes that show similar fluorescence intensities to each homolog (CCNB1, C9orf66, BCR, Figure 1B and Table 1)
Summary
Condensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. Homologous metaphase chromosome structures are heterogeneous at optical, sub-optical and atomic resolution [1,2,3,4,5]. This heterogeneity is manifest as distinctive chromosomal banding patterns superimposed on a highly conserved banding framework [6,7]. Differences in chromosome band resolution and histone modifications are distributed along the length of the mitotic metaphase chromosomes [15]. High fidelity mitotic metaphase chromosome condensation is essential for accurate transmission and differentiation of the genome into daughter cells, this process tolerates some degree of structural heterogeneity between chromosome homologs [1]. Some progress has been made through investigations of histone and nonhistone proteins that reorganize chromatin into its condensed state [19]
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