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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

Visualization of metaphase chromosome equivalent accessibility in 2- and 3-dimensions. A. Epifluorescence image of metaphase cell hybridized with a low copy probe (3.4 kb) within NOMO1. 3D structured illumination microscopy of hybridized probe volume (panel B) and probe depth (panel C) for the homologs in panel A are presented. B. Both homologs show equivalent hybridization accessibility, where the fluorescence is embedded within the chromosome and protrudes above the surface. Reconstructed volume view in the left homolog was generated by up-righting it (arrow 1) and turning it clockwise about the z-axis (arrow 2) (see orientation schematic). Volume view in the right homolog was generated by up-righting it (arrow 1) and turning it counter-clockwise (arrow 2) (see schematic). C. Crosshairs are centered over the maximal fluorescent intensity projection along the XY, XZ and YZ axes for each chromosome 16 homolog. The axial projection (depth) of the probe fluorescence spans 15 of 18 0.1 μm reconstructed optical sections for both homologs, depicting equivalent chromatin accessibility (white rectangles delineate boundaries along the Z axis).
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Fig6: Visualization of metaphase chromosome equivalent accessibility in 2- and 3-dimensions. A. Epifluorescence image of metaphase cell hybridized with a low copy probe (3.4 kb) within NOMO1. 3D structured illumination microscopy of hybridized probe volume (panel B) and probe depth (panel C) for the homologs in panel A are presented. B. Both homologs show equivalent hybridization accessibility, where the fluorescence is embedded within the chromosome and protrudes above the surface. Reconstructed volume view in the left homolog was generated by up-righting it (arrow 1) and turning it clockwise about the z-axis (arrow 2) (see orientation schematic). Volume view in the right homolog was generated by up-righting it (arrow 1) and turning it counter-clockwise (arrow 2) (see schematic). C. Crosshairs are centered over the maximal fluorescent intensity projection along the XY, XZ and YZ axes for each chromosome 16 homolog. The axial projection (depth) of the probe fluorescence spans 15 of 18 0.1 μm reconstructed optical sections for both homologs, depicting equivalent chromatin accessibility (white rectangles delineate boundaries along the Z axis).

Mentions: Using super-resolution, 3-dimensional structured illumination microscopy (3D-SIM), we demonstrated reproducible and significant differences in probe volume (p = 3.72E-07, n = 22 metaphase cells) and depth (p = 1.41E-07, n = 22) between homologous regions of three DA probes (PMP22:IVS3, HERC2, ACR). The distribution of probe volume and depth was broad in regions with DA (Additional file 2: Figure S2A) relative to those with equivalent accessibility (Additional file 2: Figure S2B). For example, a 1.81 kb single copy probe detecting DA within HERC2 (Figure 5A) exhibited a large difference between homologs (Figure 5B, 0.22 μm3 left panel and 0.001 μm3 right panel). Notably, the axial distributions (i.e. depth) of the probe fluorescence from the accessible (Figure 5C, left panel) and less accessible (Figure 5C, right panel) homologs were 1.70 μm and 0.80 μm, respectively. These differences in volume and depth projections can also be viewed by traversing through cross-sections of the hybridized chromosomes (Additional file 3: Movie S1, probe PMP22:IVS3). The hybridization signals of accessible and DA probes were contained within different focal planes of metaphase chromatin, and there was large variation in the number of reconstructed optical sections hybridized to the same target on different homologs (Figure 5C). By contrast, a probe detecting 5 distinct targets on chromosome 16 (NOMO1, Figure 6A) with equivalent accessibility to both homologs showed similar probe volumes (Figure 6B, 0.60 μm3, left panel and 0.89 μm3, right panel) and depths (Figure 6C, 1.4 μm both panels) (also see Additional file 4: Movie S2). Hybridization to each of these low copy targets were assessed for volume and depth differences as a single fluorescent target due to their close genomic proximity (~1 Mb apart). Among all cells, differences in NOMO1 probe volume (p = 1.30E-01, n = 20 metaphase cells analyzed) and depth (p = 8.90E-01, n = 20 metaphase cells) between homologs were not significant (Additional file 2: Figure S2B). These findings provide direct evidence that DA is due to the genomic target sequence being less accessible on one of the chromosome homologs.Figure 5


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

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

Visualization of metaphase chromosome equivalent accessibility in 2- and 3-dimensions. A. Epifluorescence image of metaphase cell hybridized with a low copy probe (3.4 kb) within NOMO1. 3D structured illumination microscopy of hybridized probe volume (panel B) and probe depth (panel C) for the homologs in panel A are presented. B. Both homologs show equivalent hybridization accessibility, where the fluorescence is embedded within the chromosome and protrudes above the surface. Reconstructed volume view in the left homolog was generated by up-righting it (arrow 1) and turning it clockwise about the z-axis (arrow 2) (see orientation schematic). Volume view in the right homolog was generated by up-righting it (arrow 1) and turning it counter-clockwise (arrow 2) (see schematic). C. Crosshairs are centered over the maximal fluorescent intensity projection along the XY, XZ and YZ axes for each chromosome 16 homolog. The axial projection (depth) of the probe fluorescence spans 15 of 18 0.1 μm reconstructed optical sections for both homologs, depicting equivalent chromatin accessibility (white rectangles delineate boundaries along the Z axis).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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Fig6: Visualization of metaphase chromosome equivalent accessibility in 2- and 3-dimensions. A. Epifluorescence image of metaphase cell hybridized with a low copy probe (3.4 kb) within NOMO1. 3D structured illumination microscopy of hybridized probe volume (panel B) and probe depth (panel C) for the homologs in panel A are presented. B. Both homologs show equivalent hybridization accessibility, where the fluorescence is embedded within the chromosome and protrudes above the surface. Reconstructed volume view in the left homolog was generated by up-righting it (arrow 1) and turning it clockwise about the z-axis (arrow 2) (see orientation schematic). Volume view in the right homolog was generated by up-righting it (arrow 1) and turning it counter-clockwise (arrow 2) (see schematic). C. Crosshairs are centered over the maximal fluorescent intensity projection along the XY, XZ and YZ axes for each chromosome 16 homolog. The axial projection (depth) of the probe fluorescence spans 15 of 18 0.1 μm reconstructed optical sections for both homologs, depicting equivalent chromatin accessibility (white rectangles delineate boundaries along the Z axis).
Mentions: Using super-resolution, 3-dimensional structured illumination microscopy (3D-SIM), we demonstrated reproducible and significant differences in probe volume (p = 3.72E-07, n = 22 metaphase cells) and depth (p = 1.41E-07, n = 22) between homologous regions of three DA probes (PMP22:IVS3, HERC2, ACR). The distribution of probe volume and depth was broad in regions with DA (Additional file 2: Figure S2A) relative to those with equivalent accessibility (Additional file 2: Figure S2B). For example, a 1.81 kb single copy probe detecting DA within HERC2 (Figure 5A) exhibited a large difference between homologs (Figure 5B, 0.22 μm3 left panel and 0.001 μm3 right panel). Notably, the axial distributions (i.e. depth) of the probe fluorescence from the accessible (Figure 5C, left panel) and less accessible (Figure 5C, right panel) homologs were 1.70 μm and 0.80 μm, respectively. These differences in volume and depth projections can also be viewed by traversing through cross-sections of the hybridized chromosomes (Additional file 3: Movie S1, probe PMP22:IVS3). The hybridization signals of accessible and DA probes were contained within different focal planes of metaphase chromatin, and there was large variation in the number of reconstructed optical sections hybridized to the same target on different homologs (Figure 5C). By contrast, a probe detecting 5 distinct targets on chromosome 16 (NOMO1, Figure 6A) with equivalent accessibility to both homologs showed similar probe volumes (Figure 6B, 0.60 μm3, left panel and 0.89 μm3, right panel) and depths (Figure 6C, 1.4 μm both panels) (also see Additional file 4: Movie S2). Hybridization to each of these low copy targets were assessed for volume and depth differences as a single fluorescent target due to their close genomic proximity (~1 Mb apart). Among all cells, differences in NOMO1 probe volume (p = 1.30E-01, n = 20 metaphase cells analyzed) and depth (p = 8.90E-01, n = 20 metaphase cells) between homologs were not significant (Additional file 2: Figure S2B). These findings provide direct evidence that DA is due to the genomic target sequence being less accessible on one of the chromosome homologs.Figure 5

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