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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 among related individuals. A. Schematic of a two probe two color single copy FISH strategy to distinguish chromosome 15 homologs is shown. The hemizygous deletion on proximal chromosome 15q is identified by the loss of probe UBE3A (green) on one homolog and the presence of HERC2, GABRA5, SNPRN (red, pink). The deletion occurs on the paternal homolog in individual II-1 (mother) and on the maternal homolog in the children (III-1 and III-2). DA for probes outside of the deletion is represented by a bright hybridization on one homolog (red circle) and weak fluorescence hybridization on the other one (pink circle). The deleted chromosome is gray and the normal chromosome is white. B. DA detected by HERC2, GABRA5, SNPRN showed that the paternal chromosome in the three individuals (deletion in II-1; normal in III-1 and III-2) contained the brighter fluorescence intensities (HERC2 II-1, 73.3% of metaphase cells III-1, 84.6%; GABRA5 II-1, 68% III-2, 77.8%; SNRPN II-1, 82.6% III-2, 75.0%) and was more accessible.
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Fig4: Differential accessibility is non-random among related individuals. A. Schematic of a two probe two color single copy FISH strategy to distinguish chromosome 15 homologs is shown. The hemizygous deletion on proximal chromosome 15q is identified by the loss of probe UBE3A (green) on one homolog and the presence of HERC2, GABRA5, SNPRN (red, pink). The deletion occurs on the paternal homolog in individual II-1 (mother) and on the maternal homolog in the children (III-1 and III-2). DA for probes outside of the deletion is represented by a bright hybridization on one homolog (red circle) and weak fluorescence hybridization on the other one (pink circle). The deleted chromosome is gray and the normal chromosome is white. B. DA detected by HERC2, GABRA5, SNPRN showed that the paternal chromosome in the three individuals (deletion in II-1; normal in III-1 and III-2) contained the brighter fluorescence intensities (HERC2 II-1, 73.3% of metaphase cells III-1, 84.6%; GABRA5 II-1, 68% III-2, 77.8%; SNRPN II-1, 82.6% III-2, 75.0%) and was more accessible.

Mentions: We also examined if DA was heritable in 3 members of an Angelman Syndrome (AS) family with a chromosome 15q12 microdeletion (Table 2) at loci adjacent to the rearrangement [13,26]. In this family, the unaffected mother (II-1, Figure 4) inherited the microdeletion from her father (not available for study); and passed on the deleted chromosome to her AS children (III-1, III-2, Figure 4). A dual probe-dual labeling and color detection FISH strategy (Figure 4A) was utilized to distinguish the chromosome 15 homologs based on the presence or absence of the microdeletion. A 4.9 kb single copy FISH probe within the deletion interval (UBE3A:IVS7-IVS8, Table 2) served as a control (green circle in Figure 4A) to track the abnormal chromosome 15. Single copy probes detecting DA (dark and light red circles in Figure 4A) targeted intact sequences outside the deletion interval that occurred both within the AS imprinted domain (GABRA5 [2.77 kb], SNRPN [2.09 kb]) and adjacent to the imprinted domain (HERC2 [1.81 kb]). Irrespective of their imprinted status, probes within GABRA5, SNRPN, and HERC2 all showed a bias in non-random hybridization. The paternally inherited chromosome 15, which was deleted in II-1 and intact in III-1 and III-2, consistently exhibited greater probe accessibility (Figure 4B). Previously, we have reported biased early-replication during S phase at the same loci on the paternally-derived chromosome [13]. The variance in the fraction of cells reported to have DA among different samples (Table 2) for all single copy probes described above (RGS7, CACNA1B, OPCML, GABRA5, SNRPN, HERC2, ADORA2B:IVS1, PMP22:IVS3, and ACR) was not significant (σ2 = 9.72, p = 8.65E-01, μ = 35 cells analyzed per sample, Bartlett’s test for homogeneity of variance).Figure 4


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 among related individuals. A. Schematic of a two probe two color single copy FISH strategy to distinguish chromosome 15 homologs is shown. The hemizygous deletion on proximal chromosome 15q is identified by the loss of probe UBE3A (green) on one homolog and the presence of HERC2, GABRA5, SNPRN (red, pink). The deletion occurs on the paternal homolog in individual II-1 (mother) and on the maternal homolog in the children (III-1 and III-2). DA for probes outside of the deletion is represented by a bright hybridization on one homolog (red circle) and weak fluorescence hybridization on the other one (pink circle). The deleted chromosome is gray and the normal chromosome is white. B. DA detected by HERC2, GABRA5, SNPRN showed that the paternal chromosome in the three individuals (deletion in II-1; normal in III-1 and III-2) contained the brighter fluorescence intensities (HERC2 II-1, 73.3% of metaphase cells III-1, 84.6%; GABRA5 II-1, 68% III-2, 77.8%; SNRPN II-1, 82.6% III-2, 75.0%) and was more accessible.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Differential accessibility is non-random among related individuals. A. Schematic of a two probe two color single copy FISH strategy to distinguish chromosome 15 homologs is shown. The hemizygous deletion on proximal chromosome 15q is identified by the loss of probe UBE3A (green) on one homolog and the presence of HERC2, GABRA5, SNPRN (red, pink). The deletion occurs on the paternal homolog in individual II-1 (mother) and on the maternal homolog in the children (III-1 and III-2). DA for probes outside of the deletion is represented by a bright hybridization on one homolog (red circle) and weak fluorescence hybridization on the other one (pink circle). The deleted chromosome is gray and the normal chromosome is white. B. DA detected by HERC2, GABRA5, SNPRN showed that the paternal chromosome in the three individuals (deletion in II-1; normal in III-1 and III-2) contained the brighter fluorescence intensities (HERC2 II-1, 73.3% of metaphase cells III-1, 84.6%; GABRA5 II-1, 68% III-2, 77.8%; SNRPN II-1, 82.6% III-2, 75.0%) and was more accessible.
Mentions: We also examined if DA was heritable in 3 members of an Angelman Syndrome (AS) family with a chromosome 15q12 microdeletion (Table 2) at loci adjacent to the rearrangement [13,26]. In this family, the unaffected mother (II-1, Figure 4) inherited the microdeletion from her father (not available for study); and passed on the deleted chromosome to her AS children (III-1, III-2, Figure 4). A dual probe-dual labeling and color detection FISH strategy (Figure 4A) was utilized to distinguish the chromosome 15 homologs based on the presence or absence of the microdeletion. A 4.9 kb single copy FISH probe within the deletion interval (UBE3A:IVS7-IVS8, Table 2) served as a control (green circle in Figure 4A) to track the abnormal chromosome 15. Single copy probes detecting DA (dark and light red circles in Figure 4A) targeted intact sequences outside the deletion interval that occurred both within the AS imprinted domain (GABRA5 [2.77 kb], SNRPN [2.09 kb]) and adjacent to the imprinted domain (HERC2 [1.81 kb]). Irrespective of their imprinted status, probes within GABRA5, SNRPN, and HERC2 all showed a bias in non-random hybridization. The paternally inherited chromosome 15, which was deleted in II-1 and intact in III-1 and III-2, consistently exhibited greater probe accessibility (Figure 4B). Previously, we have reported biased early-replication during S phase at the same loci on the paternally-derived chromosome [13]. The variance in the fraction of cells reported to have DA among different samples (Table 2) for all single copy probes described above (RGS7, CACNA1B, OPCML, GABRA5, SNRPN, HERC2, ADORA2B:IVS1, PMP22:IVS3, and ACR) was not significant (σ2 = 9.72, p = 8.65E-01, μ = 35 cells analyzed per sample, Bartlett’s test for homogeneity of variance).Figure 4

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