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Localized, non-random differences in chromatin accessibility between homologous metaphase chromosomes.

Khan WA, Rogan PK, Knoll JH - Mol Cytogenet (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Western Ontario, London, ON N6A 5C1 Canada ; Cytognomix, Inc, London, ON N6G 4X8 Canada.

ABSTRACT

Background: Condensation 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.

Results: Fourteen 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.

Conclusions: This 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.

No MeSH data available.


Related in: MedlinePlus

Differential accessibility and equivalent accessibility patterns between metaphase chromosome homologs detected by single copy probes. A. Human chromosomes hybridized with single copy FISH probes developed from CACNA1B (2.23 kb), HERC2 (1.81 kb), and PMP22:IVS3 (2.32 kb) (left to right) show differential hybridization between homologs. Arrows indicate the homolog with less fluorescence (or less accessibility). B. Examples of human cells with single copy FISH probes developed from within CCNB1 (2.47 kb), C9orf66 (2.08 kb), and BCR (3.4 kb) (left to right) that show similar fluorescence intensities (or equivalent accessibility) between homologous regions. Chromosomes were counterstained with DAPI (converted to gray scale in image) and probes were labelled with digoxigenin d-UTP and detected with Cy3 digoxin antibody.
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Fig1: Differential accessibility and equivalent accessibility patterns between metaphase chromosome homologs detected by single copy probes. A. Human chromosomes hybridized with single copy FISH probes developed from CACNA1B (2.23 kb), HERC2 (1.81 kb), and PMP22:IVS3 (2.32 kb) (left to right) show differential hybridization between homologs. Arrows indicate the homolog with less fluorescence (or less accessibility). B. Examples of human cells with single copy FISH probes developed from within CCNB1 (2.47 kb), C9orf66 (2.08 kb), and BCR (3.4 kb) (left to right) that show similar fluorescence intensities (or equivalent accessibility) between homologous regions. Chromosomes were counterstained with DAPI (converted to gray scale in image) and probes were labelled with digoxigenin d-UTP and detected with Cy3 digoxin antibody.

Mentions: Our previous studies demonstrated consistent differences in hybridization intensities for single copy probes in at least two-thirds of the metaphase cells. DA was probe and genomic interval specific and not related to either probe labeling or the individual samples hybridized. To illustrate different hybridization behaviours between homologs with short-target, single copy 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 (i.e. differential accessibility or DA) 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 (i.e. equivalent accessibility) to each homolog (CCNB1, C9orf66, BCR, Figure 1B and Table 1).Figure 1


Localized, non-random differences in chromatin accessibility between homologous metaphase chromosomes.

Khan WA, Rogan PK, Knoll JH - Mol Cytogenet (2014)

Differential accessibility and equivalent accessibility patterns between metaphase chromosome homologs detected by single copy probes. A. Human chromosomes hybridized with single copy FISH probes developed from CACNA1B (2.23 kb), HERC2 (1.81 kb), and PMP22:IVS3 (2.32 kb) (left to right) show differential hybridization between homologs. Arrows indicate the homolog with less fluorescence (or less accessibility). B. Examples of human cells with single copy FISH probes developed from within CCNB1 (2.47 kb), C9orf66 (2.08 kb), and BCR (3.4 kb) (left to right) that show similar fluorescence intensities (or equivalent accessibility) between homologous regions. Chromosomes were counterstained with DAPI (converted to gray scale in image) and probes were labelled with digoxigenin d-UTP and detected with Cy3 digoxin antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269072&req=5

Fig1: Differential accessibility and equivalent accessibility patterns between metaphase chromosome homologs detected by single copy probes. A. Human chromosomes hybridized with single copy FISH probes developed from CACNA1B (2.23 kb), HERC2 (1.81 kb), and PMP22:IVS3 (2.32 kb) (left to right) show differential hybridization between homologs. Arrows indicate the homolog with less fluorescence (or less accessibility). B. Examples of human cells with single copy FISH probes developed from within CCNB1 (2.47 kb), C9orf66 (2.08 kb), and BCR (3.4 kb) (left to right) that show similar fluorescence intensities (or equivalent accessibility) between homologous regions. Chromosomes were counterstained with DAPI (converted to gray scale in image) and probes were labelled with digoxigenin d-UTP and detected with Cy3 digoxin antibody.
Mentions: Our previous studies demonstrated consistent differences in hybridization intensities for single copy probes in at least two-thirds of the metaphase cells. DA was probe and genomic interval specific and not related to either probe labeling or the individual samples hybridized. To illustrate different hybridization behaviours between homologs with short-target, single copy 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 (i.e. differential accessibility or DA) 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 (i.e. equivalent accessibility) to each homolog (CCNB1, C9orf66, BCR, Figure 1B and Table 1).Figure 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Western Ontario, London, ON N6A 5C1 Canada ; Cytognomix, Inc, London, ON N6G 4X8 Canada.

ABSTRACT

Background: Condensation 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.

Results: Fourteen 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.

Conclusions: This 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.

No MeSH data available.


Related in: MedlinePlus