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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

Correspondence of metaphase chromosome accessibility with epigenetic marks associated with open chromatin in interphase. Genome browser tracks show integrated ChIP-seq signal intensities of open chromatin features (y-axis) determined by ENCODE. Genomic locations for a set of representative single copy probe intervals is provided (GRCh37) along x-axis, probe size in kilobase pairs is represented by black bar, and genes are shown in blue. A. Genomic regions with equivalent accessibility show a higher density of open chromatin epigenetic features than regions with DA (panel B). C. The distributions of integrated intensities for each open chromatin feature were plotted around the 95% confidence interval for all probe intervals provided in Table 1, and grouped according to whether the probes showed DA (black bars) or equivalent accessibility (red bars). Group means of the integrated intensity values are shown on the y-axis (y = log 10) and individual features of open chromatin are indicated on the x-axis. The mean integrated ChIP-seq intensities of open chromatin features were significantly different by ANOVA (p =1.0E-04), in particular for all histone marks and DNase I HS, between DA and sequences with equivalent accessibility.
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Fig7: Correspondence of metaphase chromosome accessibility with epigenetic marks associated with open chromatin in interphase. Genome browser tracks show integrated ChIP-seq signal intensities of open chromatin features (y-axis) determined by ENCODE. Genomic locations for a set of representative single copy probe intervals is provided (GRCh37) along x-axis, probe size in kilobase pairs is represented by black bar, and genes are shown in blue. A. Genomic regions with equivalent accessibility show a higher density of open chromatin epigenetic features than regions with DA (panel B). C. The distributions of integrated intensities for each open chromatin feature were plotted around the 95% confidence interval for all probe intervals provided in Table 1, and grouped according to whether the probes showed DA (black bars) or equivalent accessibility (red bars). Group means of the integrated intensity values are shown on the y-axis (y = log 10) and individual features of open chromatin are indicated on the x-axis. The mean integrated ChIP-seq intensities of open chromatin features were significantly different by ANOVA (p =1.0E-04), in particular for all histone marks and DNase I HS, between DA and sequences with equivalent accessibility.

Mentions: The source of the differences in single copy FISH probe accessibility between metaphase homologs is not known, however other markers of localized, sequence specific chromosome accessibility during interphase are well established [27]. We compared common epigenetic chromosomal modifications diagnostic for open chromatin during interphase to the same genomic intervals that show DA or equivalent accessibility in metaphase (n = 93 genomic regions, Table 1). Interphase epigenetic patterns for single copy intervals detecting equivalent probe accessibility to both homologs showed higher integrated signal intensities. In particular, Deoxyribonuclease I hypersensitivity (DNase I HS), and open chromatin features marked by modifications such as Histone 3 lysine 4 mono-methylation (H3K4me1) and Histone 3 lysine 27 acetylation (H3K27ac) (Figure 7A). These targets exhibited higher integrated signal intensities for DNase HS and histone marks of open chromatin than other marks associated with transcriptionally active chromatin (i.e. H3K36me3, H4K20me1). By contrast, homologous chromosomal intervals exhibiting DA generally had lower integrated signal intensities for the same open chromatin features (Figure 7B), which would be consistent with diminished levels of open chromatin marks at less accessible metaphase loci. Collectively, the average integrated signal intensities of all open chromatin marks (DNase I HS, FAIRE, H3K4me1, H3K9ac, H3K27ac, H3K4me2) in the DA genomic intervals was significantly lower (μ = 2830, σ = 1900) relative to intervals with equivalent accessibility (μ = 4330, σ = 3650) (F = 62.28, p = 1.0E-04; Figure 7C and Table 1).Figure 7


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

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

Correspondence of metaphase chromosome accessibility with epigenetic marks associated with open chromatin in interphase. Genome browser tracks show integrated ChIP-seq signal intensities of open chromatin features (y-axis) determined by ENCODE. Genomic locations for a set of representative single copy probe intervals is provided (GRCh37) along x-axis, probe size in kilobase pairs is represented by black bar, and genes are shown in blue. A. Genomic regions with equivalent accessibility show a higher density of open chromatin epigenetic features than regions with DA (panel B). C. The distributions of integrated intensities for each open chromatin feature were plotted around the 95% confidence interval for all probe intervals provided in Table 1, and grouped according to whether the probes showed DA (black bars) or equivalent accessibility (red bars). Group means of the integrated intensity values are shown on the y-axis (y = log 10) and individual features of open chromatin are indicated on the x-axis. The mean integrated ChIP-seq intensities of open chromatin features were significantly different by ANOVA (p =1.0E-04), in particular for all histone marks and DNase I HS, between DA and sequences with equivalent accessibility.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4269072&req=5

Fig7: Correspondence of metaphase chromosome accessibility with epigenetic marks associated with open chromatin in interphase. Genome browser tracks show integrated ChIP-seq signal intensities of open chromatin features (y-axis) determined by ENCODE. Genomic locations for a set of representative single copy probe intervals is provided (GRCh37) along x-axis, probe size in kilobase pairs is represented by black bar, and genes are shown in blue. A. Genomic regions with equivalent accessibility show a higher density of open chromatin epigenetic features than regions with DA (panel B). C. The distributions of integrated intensities for each open chromatin feature were plotted around the 95% confidence interval for all probe intervals provided in Table 1, and grouped according to whether the probes showed DA (black bars) or equivalent accessibility (red bars). Group means of the integrated intensity values are shown on the y-axis (y = log 10) and individual features of open chromatin are indicated on the x-axis. The mean integrated ChIP-seq intensities of open chromatin features were significantly different by ANOVA (p =1.0E-04), in particular for all histone marks and DNase I HS, between DA and sequences with equivalent accessibility.
Mentions: The source of the differences in single copy FISH probe accessibility between metaphase homologs is not known, however other markers of localized, sequence specific chromosome accessibility during interphase are well established [27]. We compared common epigenetic chromosomal modifications diagnostic for open chromatin during interphase to the same genomic intervals that show DA or equivalent accessibility in metaphase (n = 93 genomic regions, Table 1). Interphase epigenetic patterns for single copy intervals detecting equivalent probe accessibility to both homologs showed higher integrated signal intensities. In particular, Deoxyribonuclease I hypersensitivity (DNase I HS), and open chromatin features marked by modifications such as Histone 3 lysine 4 mono-methylation (H3K4me1) and Histone 3 lysine 27 acetylation (H3K27ac) (Figure 7A). These targets exhibited higher integrated signal intensities for DNase HS and histone marks of open chromatin than other marks associated with transcriptionally active chromatin (i.e. H3K36me3, H4K20me1). By contrast, homologous chromosomal intervals exhibiting DA generally had lower integrated signal intensities for the same open chromatin features (Figure 7B), which would be consistent with diminished levels of open chromatin marks at less accessible metaphase loci. Collectively, the average integrated signal intensities of all open chromatin marks (DNase I HS, FAIRE, H3K4me1, H3K9ac, H3K27ac, H3K4me2) in the DA genomic intervals was significantly lower (μ = 2830, σ = 1900) relative to intervals with equivalent accessibility (μ = 4330, σ = 3650) (F = 62.28, p = 1.0E-04; Figure 7C and Table 1).Figure 7

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