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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 is non-random and reproducible between individuals. A. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively (hatched marks indicate the paternal homolog). Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. This was the abnormal paternal homolog for RGS7 (sample ID: GM10958), abnormal maternal for CACNA1B (GM01921), abnormal paternal for ADORA2B:IVS1, and PMP22:IVS3 (GM06326), and normal maternal homolog for ACR (GM10273). B. Non-random DA was confirmed using cells from individuals in which the parental origin of the specific chromosomal rearrangement was unknown. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively. Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. RGS7 probe had greater probe target accessibility on the normal chromosome 1 (sample ID: L12-1980). CACNA1B had greater accessibility on chromosome 9 with heteromorphic variant (L13-72). ADORA2B:IVS1 and PMP22:IVS3 probes were brighter on the abnormal and normal chromosome 17s, respectively (L12-1980) while ACR showed greater accessibility to the normal chromosome 22 (L11-729). C. Quantification of probe signal fluorescence between homologs are shown by box plots of normalized integrated fluorescence intensity ratios. Single copy probes detecting DA (RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, ACR) exhibited large differences in hybridization intensities between homologs. This is indicated by the broad inter-quartile range of normalized intensity ratios from 0.55-1 (median intensity ratio, 0.87). By contrast, normalized intensity ratios for single copy FISH probes (CCNB1, Corf66, PMP22:IVS4-Ex 5, ADORA2B:Promoter-Ex1 and 1p36.3 intregenic region) with equal accessibility ranged from 0.07-0.31 (median intensity ratio, 0.14). Intensity differences between homologs were quantified by GVF from 125 metaphase cells for each probe category.
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Fig3: Differential accessibility is non-random and reproducible between individuals. A. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively (hatched marks indicate the paternal homolog). Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. This was the abnormal paternal homolog for RGS7 (sample ID: GM10958), abnormal maternal for CACNA1B (GM01921), abnormal paternal for ADORA2B:IVS1, and PMP22:IVS3 (GM06326), and normal maternal homolog for ACR (GM10273). B. Non-random DA was confirmed using cells from individuals in which the parental origin of the specific chromosomal rearrangement was unknown. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively. Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. RGS7 probe had greater probe target accessibility on the normal chromosome 1 (sample ID: L12-1980). CACNA1B had greater accessibility on chromosome 9 with heteromorphic variant (L13-72). ADORA2B:IVS1 and PMP22:IVS3 probes were brighter on the abnormal and normal chromosome 17s, respectively (L12-1980) while ACR showed greater accessibility to the normal chromosome 22 (L11-729). C. Quantification of probe signal fluorescence between homologs are shown by box plots of normalized integrated fluorescence intensity ratios. Single copy probes detecting DA (RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, ACR) exhibited large differences in hybridization intensities between homologs. This is indicated by the broad inter-quartile range of normalized intensity ratios from 0.55-1 (median intensity ratio, 0.87). By contrast, normalized intensity ratios for single copy FISH probes (CCNB1, Corf66, PMP22:IVS4-Ex 5, ADORA2B:Promoter-Ex1 and 1p36.3 intregenic region) with equal accessibility ranged from 0.07-0.31 (median intensity ratio, 0.14). Intensity differences between homologs were quantified by GVF from 125 metaphase cells for each probe category.

Mentions: Single copy probes from within genomic regions overlapping RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, and ACR showed preferential hybridization (based on probe fluorescence intensity) to the same homologous chromosome in different cells (non-random, p <5.0E-02, two proportion z-test; average of 80% metaphase cells [range 68-86%], n = 30–50 cells, Figures 2 and 3A). Interestingly, non-random DA was noted within PMP22:IVS3 and ADORA2B:IVS1, while adjacent single copy probes targeting different portions of these same genes (ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex5) showed similar hybridization intensities (e.g. equivalent accessibility) between homologs. Control single copy probes from within CCNB1 (Figure 1B, left panel), C9orf66 (Figure 1B, middle panel), and an intergenic region within 1p36.3 also exhibited equivalent accessibility between homologs. DA is not exclusive to chromosomes originating from one parent-of-origin. For example, single copy probes from within CACNA1B and ACR exhibited greater accessibility (i.e. brighter fluorescent intensities) to the maternally-derived chromosomal target, whereas RGS7, ADORA2B:IVS1, and PMP22:IVS3 exhibited increased accessibility to the paternally-derived homolog (Figures 2 and 3A).Figure 2


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

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

Differential accessibility is non-random and reproducible between individuals. A. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively (hatched marks indicate the paternal homolog). Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. This was the abnormal paternal homolog for RGS7 (sample ID: GM10958), abnormal maternal for CACNA1B (GM01921), abnormal paternal for ADORA2B:IVS1, and PMP22:IVS3 (GM06326), and normal maternal homolog for ACR (GM10273). B. Non-random DA was confirmed using cells from individuals in which the parental origin of the specific chromosomal rearrangement was unknown. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively. Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. RGS7 probe had greater probe target accessibility on the normal chromosome 1 (sample ID: L12-1980). CACNA1B had greater accessibility on chromosome 9 with heteromorphic variant (L13-72). ADORA2B:IVS1 and PMP22:IVS3 probes were brighter on the abnormal and normal chromosome 17s, respectively (L12-1980) while ACR showed greater accessibility to the normal chromosome 22 (L11-729). C. Quantification of probe signal fluorescence between homologs are shown by box plots of normalized integrated fluorescence intensity ratios. Single copy probes detecting DA (RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, ACR) exhibited large differences in hybridization intensities between homologs. This is indicated by the broad inter-quartile range of normalized intensity ratios from 0.55-1 (median intensity ratio, 0.87). By contrast, normalized intensity ratios for single copy FISH probes (CCNB1, Corf66, PMP22:IVS4-Ex 5, ADORA2B:Promoter-Ex1 and 1p36.3 intregenic region) with equal accessibility ranged from 0.07-0.31 (median intensity ratio, 0.14). Intensity differences between homologs were quantified by GVF from 125 metaphase cells for each probe category.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Differential accessibility is non-random and reproducible between individuals. A. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively (hatched marks indicate the paternal homolog). Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. This was the abnormal paternal homolog for RGS7 (sample ID: GM10958), abnormal maternal for CACNA1B (GM01921), abnormal paternal for ADORA2B:IVS1, and PMP22:IVS3 (GM06326), and normal maternal homolog for ACR (GM10273). B. Non-random DA was confirmed using cells from individuals in which the parental origin of the specific chromosomal rearrangement was unknown. The light gray and black shading represents the brighter hybridization to either the normal or abnormal homolog, respectively. Bars depicting higher percentages correspond to the more accessible, brighter homolog in a given cell. RGS7 probe had greater probe target accessibility on the normal chromosome 1 (sample ID: L12-1980). CACNA1B had greater accessibility on chromosome 9 with heteromorphic variant (L13-72). ADORA2B:IVS1 and PMP22:IVS3 probes were brighter on the abnormal and normal chromosome 17s, respectively (L12-1980) while ACR showed greater accessibility to the normal chromosome 22 (L11-729). C. Quantification of probe signal fluorescence between homologs are shown by box plots of normalized integrated fluorescence intensity ratios. Single copy probes detecting DA (RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, ACR) exhibited large differences in hybridization intensities between homologs. This is indicated by the broad inter-quartile range of normalized intensity ratios from 0.55-1 (median intensity ratio, 0.87). By contrast, normalized intensity ratios for single copy FISH probes (CCNB1, Corf66, PMP22:IVS4-Ex 5, ADORA2B:Promoter-Ex1 and 1p36.3 intregenic region) with equal accessibility ranged from 0.07-0.31 (median intensity ratio, 0.14). Intensity differences between homologs were quantified by GVF from 125 metaphase cells for each probe category.
Mentions: Single copy probes from within genomic regions overlapping RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, and ACR showed preferential hybridization (based on probe fluorescence intensity) to the same homologous chromosome in different cells (non-random, p <5.0E-02, two proportion z-test; average of 80% metaphase cells [range 68-86%], n = 30–50 cells, Figures 2 and 3A). Interestingly, non-random DA was noted within PMP22:IVS3 and ADORA2B:IVS1, while adjacent single copy probes targeting different portions of these same genes (ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex5) showed similar hybridization intensities (e.g. equivalent accessibility) between homologs. Control single copy probes from within CCNB1 (Figure 1B, left panel), C9orf66 (Figure 1B, middle panel), and an intergenic region within 1p36.3 also exhibited equivalent accessibility between homologs. DA is not exclusive to chromosomes originating from one parent-of-origin. For example, single copy probes from within CACNA1B and ACR exhibited greater accessibility (i.e. brighter fluorescent intensities) to the maternally-derived chromosomal target, whereas RGS7, ADORA2B:IVS1, and PMP22:IVS3 exhibited increased accessibility to the paternally-derived homolog (Figures 2 and 3A).Figure 2

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