Open Access

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

Molecular Cytogenetics20147:70

https://doi.org/10.1186/s13039-014-0070-y

Received: 31 July 2014

Accepted: 6 October 2014

Published: 19 November 2014

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.

Keywords

Metaphase single copy FISHHomologous chromosome structure3-D super resolution microscopyDifferential chromatin accessibilityAllelic differencesHuman mitotic chromosomesMolecular cytogenetics

Background

Homologous metaphase chromosome structures are heterogeneous at optical, sub-optical and atomic resolution [1]–[5]. This heterogeneity is manifest as distinctive chromosomal banding patterns superimposed on a highly conserved banding framework [6],[7]. Within the same cell, each chromosome of a homologous pair may be laterally and longitudinally asymmetric [8],[9] or display differences in DNA methylation [10], and replication timing [11]–[14]. Differences in chromosome band resolution and histone modifications are distributed along the length of the mitotic metaphase chromosomes [15]. In fact, phosphorylation of core histones-H3 and H4 at specific residues is retained in metaphase chromosomes, as an intermediate step in chromosome condensation [16]. By contrast, lysine methylation and acetylation of histones are transient chromosome marks, with the loss of acetylation observed on all core histones in G2/M-arrested cells [17],[18]. High fidelity mitotic metaphase chromosome condensation is essential for accurate transmission and differentiation of the genome into daughter cells, however this process tolerates some degree of structural heterogeneity between chromosome homologs [1]. Despite advances in modeling higher order chromosome condensation, the locus-specific accessibility of chromatin within highly condensed metaphase chromosomes is not well understood. Some progress, however, has been made through investigations of histone and nonhistone proteins that reorganize chromatin into its condensed state [19].

We have noted reproducible differences in chromatin accessibility between homologous metaphase chromosomes in specific genomic regions using locus-specific short (1.5-5 kb), fluorescence in situ hybridization (FISH) probes [20],[21]. These differences manifest as variation in hybridization intensities between homologs at single cell resolution. This phenomenon has been observed for ~10% of the 305 genomic probes that we have reported [20]–[25], however the reasons for such variation were not understood. The remaining genomic regions show no significant differences in hybridization intensities between allelic loci on metaphase chromosomes.

In this study, we investigated locus-specific targets in metaphase chromosome regions that show consistent differences in DNA probe fluorescence intensity between homologs. Evidence is presented that these differences in hybridization of DNA probes result from their differential accessibility (DA) to their respective genomic targets. Using optical, and super-resolution microscopy with short target, unique sequence single copy FISH probes; these allelic chromosome regions exhibit consistent, non-random differences between their respective chromosome structures. Further, sequence analyses of interphase epigenetic marks at these loci suggest the possibility that such differences may be related to the presence of specific chromatin modifications.

Results

Differential hybridization patterns detected on normal metaphase chromosome

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

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.

Table 1

Comparison of open chromatin features to single copy genomic regions with and without DA

  

Open chromatin features

SC probe probe location [GRCh37]

Gene interval or cytoband

DNase-OS

FAIR-OS

H3K4me

H3K9Ac

H3K27Ac

H3K4me2

chr1:1171789-1175143*

1p36.3, intergenic

11592

8710

544.0

168.4

115.6

120.6

chr1:1181574-1185503

FAM132A:Ex1-IVS1

7394

9695

596.5

143.0

102.8

160.9

chr1:1628792-1633615

CDK11B:IVS6

10378

14274

326.3

222.1

121.8

177.6

chr1:1632683-1637407

CDK11B:IVS6

12139

12829

290.7

125.8

51.8

51.8

chr1:240965538-240967390

RGS7:IVS13-IVS14

1420

9976

200.6

74.0

12.1

29.6

chr1:240988582-240990678*

RGS7:IVS4-IVS5

2840

8999

125.3

55.3

7.4

59.2

chr4:3242502-3246008

HTT:Ex67

9225

15222

248.0

142.8

81.4

51.8

chr5:1421588-1425427

SLC6A3:IVS4-IVS5

4702

8574

172.0

80.3

66.6

14.8

chr5:9355970-9358454

SEMA5A:IVS3

2827

16953

235.4

103.6

36.5

29.6

chr5:9361501-9365307

SEMA5A:IVS3

12398

40009

3017.6

597.4

1993.0

1530.2

chr5:9371425-9374496

SEMA5A:IVS3

1397

18058

531.5

48.5

81.4

59.2

chr5:11042187-11044508

CTNND2:IVS16

2221

15462

253.7

80.3

32.6

44.4

chr5:11071700-11076039

CTNND2:IVS16

4422

19382

344.8

158.4

118.5

77.8

chr5:11084988-11089067

CTNND2:IVS15

2942

16403

297.3

81.4

81.4

22.2

chr5:68462247-68464721*

CCNB1:Ex1-IVS3

31707

29162

1400.2

2378.9

1953.4

2076.3

chr7:73506616-73509661

L1MK1:IVS14

32349

30750

3473.0

4213.1

5870.8

4186.7

chr7:73534615-73536880

L1MK1:Ex1-IVS3

3639

9068

237.2

47.3

0

22.2

chr8:116658428-116661455

TRPS1:IVS1

3738

15369

650.0

224.2

78.6

396.1

chr8:116661938-116665132

TRPS1:IVS1

2031

15754

316.0

112.2

59.2

57.9

chr9:213762-215844*

C9orf66:Ex1

10945

15191

868.4

1550.5

503.2

1667.6

chr9:133587757-133589963

ABL1:Ex1b-IVS1b

32515

25514

616.8

3043.7

2278.6

1563.2

chr9:133616347-133618188

ABL1:IVS1b

1917

10733

341.9

83.6

42.0

71.1

chr9:133733132-133735051

ABL1:IVS3

2859

8103

188.8

50.9

74.2

37.0

chr9:133735369-133737639

ABL1:IVS3

2211

11425

259.7

62.8

56.1

95.6

chr9:133745513-133749828

ABL1:IVS4-IVS6

18884

37627

4959.4

477.6

982.4

998.7

chr9:133759487-133764440

ABL1:Ex11

7053

15356

322.9

174.4

142.9

74.0

chr9:140952206-140954439*

CACNA1B:Ex29-IVS31

4956

8277

302.2

88.8

33.8

51.8

chr9:140969092-140971796

CACNA1B:IVS33-IVS34

4127

7686

151.2

69.2

44.4

37.0

chr11:133180187-133182699*

OPCML:IVS1

2306

11280

202.1

77.4

37.0

133.8

chr12:11958559-11960434

ETV6:IVS2

2403

19900

2947.6

327.2

751.8

757.8

chr12:11992883-11994726

ETV6:IVS2

2257

16709

786.4

134.8

384.3

62.9

chr12:11992883-11995741

ETV6:IVS3

2988

23954

887.0

206.0

453.6

232.7

chr13:100626271-100630715

13q32.3, intergenic

11678

18628

216.6

76.2

65.8

23.2

chr13:100643221-100648153

13q32.3, intergenic

7452

25389

687.8

115.4

83.2

221.4

chr15:22690247-22693115

15q11.2, intergenic

1705

4996

347.0

85.2

47.4

29.6

chr15:22853681-22855541

TUBGCP5:IVS11-IVS13

1049

21106

132.0

75.4

96.9

22.2

chr15:23864038-23868139

15q11.2, intergenic

4260

17789

272.4

74.0

38.6

79.6

chr15:23883747-23886037

15q11.2, intergenic

1969

6908

84.0

76.3

10.5

29.6

chr15:23886989-23890525

MAGEL2:Promoter- 3′UTR

7602

12049

198.2

88.8

51.8

50.9

chr15:25016909-25018586

15q11.2, intergenic

1670

5764

216.6

38.2

48.1

111.5

chr15:25052358-25054037

15q11.2, intergenic

671

6051

83.8

51.9

7.4

0

chr15:25068481-25070727*

SNRPN: Promoter:IVS1

1524

7291

149.1

74.4

19.3

22.2

chr15:25199392-25201602

SNRPN:IVS4

6799

10253

258.1

1391.0

937.3

486.3

chr15:25613407-25617676

UBE3A:IVS7-IVS8

2728

26796

3025.0

81.4

182.8

96.0

chr15:27117096-27119866*

GABRA5:IVS3

5815

8082

140.0

40.8

37.0

29.6

chr15:28509526-28511337*

HERC2:IVS12-IVS13

4580

10908

854.2

313.8

728.2

465.3

chr15:102388168-102389774

OR4F13P:IVS3-Ex5

950

8872

64.0

47.6

37.0

29.6

chr16:15013674-15017156

16p13.11, intergenic

814

984

248.3

51.8

62.2

53.0

chr16:16412325-16415807

PKD1P1:IVS2-IVS7

473

268

168.0

76.8

71.4

51.8

chr16:16452359-16455837

16p13.11, intergenic

418

670

98.7

54.1

65.2

44.4

chr16:16234893-16236784

ABCC1:IVS30-Ex31

4867

11513

451.2

103.6

89.3

45.6

chr16:18440574-18444056

16p12.3, intergenic

110

0

181.5

74.0

73.4

31.2

chr16:18484058-18487536

16p12.3, intergenic

616

907

167.7

89.9

51.8

19.1

chr17:905599-910582

ABR:IVS21-3′UTR

10824

19481

304.9

174.9

132.8

2.9

chr17:941273-943865

ABR:IVS16

3508

6315

170.9

89.1

74.0

71.8

chr17:2591614-2594572

CLUH:IVS25-3′UTR

10756

11515

576.5

96.2

384.9

163.2

chr17:2596810-2599164

CLUH:IVS13-IVS19

5323

6551

301.9

112.5

63.3

37.0

chr17:2603297-2606091

CLUH:IVS3-IVS9

7600

7093

222.1

57.0

51.8

123.2

chr17:18128679-18133300

LLGL1:Promoter-Ex2

36213

27618

1016.0

647.8

219.2

743.9

chr17:18143933-18146387

LLGL1:Ex17-IVS22

7467

8690

136.8

114.2

37.0

16.8

chr17:15133018-15136902*

PMP22:IVS4-Ex5

5482

14180

207.3

113.3

60.8

34.6

chr17:15150757-15153084*

PMP22:IVS3

4694

12616

321.0

77.7

28.7

44.4

chr17:15174803-15176657

17p12, intergenic

2574

11

163.0

89.3

37.0

31.4

chr17:15847751-15849832*

ADORA2B:Promoter-Ex1

10751

9889

763.1

241.5

79.0

980.8

chr17:15868752-15870532*

ADORA2B:IVS1

2859

17457

711.4

95.0

78.8

84.7

chr17:18150509-18152632

FLII:IVS15-Ex21

23767

14999

2289.9

74.7

439.4

75.6

chr17:18153505-18154823

FLII:IVS12-IVS14

3547

3937

47.1

36.7

36.8

0

chr17:19286892-19288934

MFAP4:IVS3-Ex6

3415

5897

286.5

139.3

95.8

54.8

chr17:37861465-37863632

ERBB2:IVS5-IVS6

5114

6170

296.6

90.6

78.3

84.4

chr17:37882684-37886219

ERBB2:IVS27-Ex31

9666

16440

1561.2

617.8

331.8

836.4

chr17:38500482-38504359

RARA:IVS2

17458

19211

3584.2

597.6

650.9

813.2

chr17:38512106-38514271

RARA: IVS8-Ex9

5468

6830

177.2

130.6

125.8

88.3

chr17:38608442-38610468

IGFBP4:IVS1-IVS3

4526

10512

230.8

39.5

29.6

62.7

chr17:38613433-38617530

IGFBP4:Ex4

8748

18085

557.1

155.4

88.8

74.0

chr17:80290070-80293112

SECTM1:Ex1-IVS1

13714

13008

2005.4

294.1

202.4

1002.5

chr20:10642756-10644909

JAG1:IVS2-IVS3

2943

15006

1104.1

118.6

49.2

225.7

chr21:36259933-36264124

RUNX1:IVS2

26119

30777

1920.4

2050.4

1478.7

2915.8

chr21:39454065-39456057

DSCR4:IVS2

2440

7032

155.4

65.3

24.6

65.9

chr21:39463783-39466136

DSCR4:IVS2

2017

10359

126.5

63.2

51.8

25.6

chr21:39473031-39475467

DSCR4:IVS2

2256

10103

137.4

86.9

37.0

0.2

chr22:19338598-19342289

HIRA:IVS21-IVS24

3429

12123

325.1

150.0

59.2

37.0

chr22:23578368-23581572

BCR:IVS1

6792

19899

2489.1

283.0

240.2

284.8

chr22:23604414-23607814

BCR:IVS4

11921

14381

1989.6

425.4

621.7

362.3

chr22:23623055-23625566

BCR:IVS8

21132

16241

2543.9

311.6

501.8

451.4

chr22:51175125-51178674*

ACR:Ex1-IVS3

11986

12916

175.2

85.1

37.0

58.9

chrX:592626-595515

SHOX:IVS2-Ex3

7316

7254

125.9

55.3

29.6

37.0

chrX:597816-600430

SHOX:IVS3

6234

7821

64.0

74.0

41.5

44.4

chrX:602538-605057

SHOX:IVS5

4319

4191

147.9

29.6

7.0

14.8

chrX:7891853-7895877

PNPLA4:Ex1-IVS2

19932

42372

1854.3

2340.4

2553.7

1715.7

chrX:8440844-8443508

Xp22.31, intergenic

1639

12112

151.2

59.2

30.0

45.5

chrX:8505855-8509075

KAL1:IVS9-IVS10

4319

14875

147.9

106.6

14.8

44.4

chrX:9613498-9617784

TBL1X:IVS4

4522

20294

429.2

164.6

115.6

133.7

chrX:9685383-9689409

TBL1X:Ex18

3938

43044

336.8

101.4

65.5

90.3

Probes from 93 genomic regions exhibiting DA (bold) or equivalent accessibility by metaphase FISH listed by chromosome number and GRCh37 genomic coordinates. Single copy intervals marked with * were characterized by FISH in this study; the other intervals were previously reported.20–25 Single copy probes that overlapped genes are specified relative to their exonic (Ex), intronic (IVS) or untranslated (UTR) positions. Single copy probes from intergenic regions were specified by cytogenetic location. Integrated signal intensities of the open chromatin features from ENCODE 27 are shown. As appropriate, values are shown with one significant digit after the decimal.

A potential alternative explanation is that differences in probe fluorescence might be related to polymorphic copy number differences in the genome. The genomic intervals covering each of the probes were examined for common copy number variants (CCNV) in the normal population. Two probes within the same genomic interval (CDK11B:IVS6; Table 1) overlapped a ~55 kb CCNV (chr1:1,616,989-1,672,591[GRCh37]), but neither exhibited DA. The remaining single copy probes (Table 1) either did not overlap any CCNVs or were known to overlap pathogenic CNV intervals. Population CCNVs cannot account for hybridization intensity differences between homologous chromosomes.

Chromatin accessibility to homologous metaphase chromosomes is non-random for most differentially accessible targets

FISH probes from chromosomes 1, 5, 9, 11, 15, 17 and 22 showing DA were hybridized to patient samples, in which specific homologs could be distinguished by the presence of a chromosome rearrangement (e.g. a translocation, inversion or heteromorphism) (Table 2). We investigated whether the same homolog in a sample was more likely to have a brighter probe hybridization signal than its counterpart (e.g. non-random), or whether hybridization intensity differences were random (e.g. the brighter signal occurred with equal frequency between homologs).
Table 2

Cell lines and single copy FISH probes used to assess chromatin accessibility

Sample ID: cytogenetics

Probes for tracking homologs

Cytoband, gene: interval

Status

GM10958: 46,XX, t(1;11) (q31.2;q25) pat

1q43.3, RGS7:IVS4-IVS5 1p36.3, intergenic

DA Equivalent

GM10273: 46,XX, t(11;22)(p13;q12.2) pat

22q13.3, ACR:Ex1-IVS3

DA

GM01921: 47,XY, t(8;14)(q13;q13), inv(9)(p11q13) mat, +21

9q34.3, CACNA1B:Ex29-IVS31 9p24.3, C9orf66:Ex1

DA Equivalent

GM06326: 46, X, t(Y;17) (q11.21;q21) pat

17p12, PMP22:IVS3 & ADORA2B:IVS1 17p12, PMP22:IVS4-Ex5 & ADORA2B:Promoter-Ex1

DA Equivalent

GM10958: 46,XX, t(1;11) (q31.2;q25) pat

11q25, OPCML:IVS1

DA

GM10273: 46,XX, t(11;22) (p13;q12.2) pat

11q25, OPCML:IVS1

DA

II-2: 46,XX.ish del (15) (q11.2q13) (D15S10-,UBE3A-) pat

15q12, SNRPN: Promoter:IVS1 & GABRA5:IVS3 15q13.1, HERC2:IVS12-IVS13

DA DA

III-1: 46,XY.ish del(15) (q11.2q13) (D15S10-,UBE3A-) mat

Same as II-2

DA

III-2: 46,XX.ish del(15) (q11.2q13) (D15S10-,UBE3A-) mat

Same as II-2

DA

L12-1980: 46,XX, t(1;17) (p10;q10)

1q43.3, RGS7:IVS4-IVS5 17p12, PMP22:IVS3 & ADORA2B:IVS1

DA DA

L13-72: 46,XX,9qh+

9q34.3, CACNA1B:Ex29-IVS31

DA

L11-729: 46,XY, t(7;22) (q32;q13.33)

22q13.3, ACR:Ex1-IVS3

DA

Cytogenetic nomenclature for each of the samples is indicated. Parental origins of the rearrangements are indicated when known (mat = maternal, pat = paternal). Cells are from human lymphocytes (L12-1980, L13-72, L11-729) or lymphoblastoid cell lines [GM10958, GM10273, GM01921, GM06326, and family II-1 (mother), III-1 (child), III-2 (child)].

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

Detection of DA within cytogenetically-distinguishable homologous regions of known parental origin. Genomic coordinates of single copy probes detecting DA within 5 different chromosomal regions are indicated. Schematic of the normal and derivative (der) or inverted (inv) chromosome with homologous target are shown. Specific chromosomes are highlighted (white rectangles), ‘mat’ and ‘pat’ refer to the maternal or paternal origin of the altered homolog, respectively. Brighter probe intensity was recurrently observed on the same homolog for a probe for each cell line. RGS7 probe had greater target accessibility on the der chromosome 11 (paternal, GM10958). CACNA1B had greater target accessibility on the inv chromosome 9; (maternal, GM01921). ADORA2B:IVS1 and PMP22:IVS3 hybridizations were brighter on the derivative chromosome 17 (paternal, GM06326) and ACR:Ex1-IVS3 hybridizations were brighter on the normal chromosome 22 (maternal, GM10273).

Figure 3

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.

The non-random nature of DA was confirmed in a set of independent samples (L12-1980, L13-72, L11-729, Table 2) with distinguishable homologs (Additional file 1: Figure S1), of which parental origins were not known. Non-random DA was observed for probes from within RGS7, CACNA1B, PMP22: IVS3, ADORA2B:IVS1 and ACR, in which the accessible homolog exhibited significantly brighter probe hybridizations (p <5.0E-02; average of 74% metaphase cells [range 69-85%], n =25-50 metaphases per cell line, Figure 3B). Single copy probes from within PMP22:IVS3 (in cell line, GM06326) and RGS7 (GM10958) showed the brighter probe signal hybridized to the abnormal (i.e. derivative) chromosome homolog in the majority of cells analyzed (Figure 3A). By contrast, the same probes when mapped to an additional cell line with a structural alteration (L12-1980), showed that the normal chromosome homolog (Figure 3B) had a more intense hybridization signal. This indicates that DA is not influenced by the presence of particular chromosome rearrangements. Although chromatin accessibility for most DA targets exhibited a non-random preference for one homolog, one DA probe (OPCML; 2.53 kb) had a random pattern. This finding was confirmed on two different cell lines with cytogenetically distinguishable chromosome 11s (Table 2 and Additional file 1: Figure S1).

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

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.

Quantification of hybridizations confirm variation in fluorescence intensities between homologs for probes detecting DA versus equivalent accessibility

The extent of variation in DNA probe hybridization intensity between homologs was quantified by gradient vector flow (GVF) analysis for both DA probes (RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, ACR), and control probes with equivalent accessibility (CCNB1, C9orf66, ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex 5, and 1p36.3 intergenic region). Significant differences in integrated fluorescence intensities between homologs with DA were found relative to probes detecting equivalent hybridization (p <5.0E-02; n = 250 total metaphases, Figure 3C). The normalized intensity ratios between homologs in metaphase cells with DA were more variable (σ2 = 0.111, μ = 0.716) than control probes with equivalent accessibility to homologous targets (σ2 = 0.049, μ = 0.221).

DA is related to differences in internal chromatin accessibility of homologous targets

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

Visualization of metaphase chromosome differential accessibility in 2- and 3-dimensions. A. Epifluorescence image of metaphase cell hybridized with HERC2 single copy probe (1.81 kb) shows a DA pattern. Chromosome 15 homologs are magnified. 3D structured illumination microscopy of hybridized probe volume (panel B) and probe depth (panel C) for the magnified homologs in panel A are presented. B. The left homolog with greater accessibility contains fluorescence embedded within the chromosome and protrudes above the surface. In contrast, the right homolog with less accessibility has a much smaller volume of hybridized probe fluorescence and is mainly embedded within the chromosome. Reconstructed volume view in the left homolog was generated by rotating it clockwise about the z-axis (see orientation schematic). Volume view in the right homolog was generated by up-righting it (arrow 1) and turning it 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 15 homolog, and highlight differences in chromatin accessibility. The axial projection (depth) of the probe fluorescence spans 18 of 21 0.1 μm reconstructed optical sections (white rectangles delineate boundaries along the z axis) in the left more accessible homolog; and only 12 of 21 reconstructed optical sections in the right homolog (white rectangles).

Figure 6

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).

Additional file 3: Movie S1: 3D anaglyph view of single copy FISH probe targets with DA (PMP22:IVS3) between chromosome homologs. Movie in upper left panel shows differences in probe fluorescence depth, dynamically visualized through 0.1 μm optical cross-sections of the hybridized chromosome 17 homologs. Upper right panel is a 3D projection of the DNA probe fluorescence, from which probe volume was obtained. The lower panel shows the same homologs, as in upper left, with occupancy of probe volume in the context of the reconstructed chromosomes, rotated 360° in the X/Y/Z axes and depicting inter-homolog DA from all angles. Reconstructed optical sections were taken over 20 z-stacks, at 0.1 μm per stack with 3D-Structured Illumination Microscopy. (MP4 7 MB)

Epigenetic features of open chromatin are enriched in genomic regions exhibiting equivalent accessibility versus those with DA

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

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.

Discussion

We have demonstrated differences in accessibility of allelic genomic targets in homologous metaphase chromosomes using independent and complementary approaches. First, we have detected and characterized DA with short, single copy FISH probes in genomic regions representative of telomeric, pericentromeric and chromosome arms (RGS7, CACNA1B, PMP22:IVS3, ADORA2B:IVS1, ACR, HERC2, GABRA5, and SNRPN) on cytogenetically distinguishable homologs. Differences in probe accessibility between homologs were non-random, and these findings were unrelated to the presence of chromosomal rearrangements that were used as markers to distinguish the homologs. With the one exception (OPCML), the brighter signal for each of the probes exhibiting non-random DA was biased to the same homolog in the cells from an individual. At the OPCML locus, DA occurred randomly, with either homolog exhibiting greater accessibility.

Aside from non-random hybridization patterns, DA was also found to be heritable. The proximal 15q region showed greater accessibility on the paternally-derived homolog, irrespective of the presence of a small molecular deletion adjacent to these probes. This pattern was stable and preserved across two generations in a family carrying the deletion. While our results do not inform on the degree to which parent-of-origin effects contribute to DA, future studies of additional familial rearrangements of known parental origin (e.g. chromosome 11;22 translocation carriers) for the probes in this study, as well as others, will be useful in demonstrating this.

The three dimensional distribution of probes displaying DA was visualized by 3D-SIM. This technique improves optical resolution by two-fold over conventional imaging, and more precisely delineates probe signals. Imaging at sub-optical diffraction scale occurs at a much higher frame rate, which enabled us to quantify differences in chromatin structure between homologous regions for single copy FISH probes more efficiently relative to other super-resolution techniques [5],[28],[29]. The spatial distributions of fluorescent hybridization to chromosome targets, emitted by single copy probes with DA, varied between homologous metaphase regions. The homolog with a lower hybridization intensity signal exhibited restricted probe occupancy in both the lateral and axial dimensions. The depth of the target sequences on the less accessible chromosome was also found to be an order of magnitude less than its corresponding homolog in the same cell. Finally, the target sequence in the homolog with lower intensity hybridization occupied a smaller volume of metaphase chromatin based on the spatial distribution of its probe fluorescence. The radial chromosome structure hypothesis, suggests that accessibility should be related to the proximity of the target sequence to the chromosome surface [30]. Our results suggest rather, that the differences in the volume and depth of the hybridized target sequence are more likely related to the degree of compaction of corresponding DNA in each of the homologous chromosomes.

Based on our ENCODE analysis of genomic regions with DA or equivalent accessibility (Figure 7 and Table 1), we envision that the differential condensation of homologous chromosomes represents a transition between parental and daughter cell epigenetic states. Histone marks and chromatin binding proteins may potentiate some genomic loci to maintain a less condensed configuration of one or both alleles during metaphase, which might then poise them to restructure open chromatin regions during the subsequent interphase in daughter cells [31]–[36]. This transition state may be akin to a type of chromatin memory that recalls epigenetic marks derived from the preceding interphase so that they can be transmitted and re-established in subsequent daughter cells. To assess DA as a means of storing chromatin memory will be technically challenging. Chromatin modifications catalyze dynamic structural changes that arise over the course of interphase. It would be necessary to score DA at different cell cycle stages (e.g. G1, S, G2) to place these results in context. This would require enriched, synchronized cell populations at the end of G2 still possessing markers of interphase chromatin at the inception of chromosome condensation. Only a small fraction of unsynchronized cells are in G2. Interphase analysis was beyond the scope of the present study which was to demonstrate and characterize DA on mitotic metaphase chromosomes.

Reduced DNA accessibility may affect chromatin structure and histone modification (the most extreme instance being X chromosome inactivation), enabling the cell to maintain control over epigenetic variation in regulatory regions [37],[38]. This mechanism could exclude co-regulation of both allelic regions at a DA locus [39]. Differences in chromatin accessibility may be a way to distinguish and spatially organize homologous loci so that the less accessible locus is separated from its accessible counterpart. To this end, homologous chromosomes are known to be in repulsion, e.g. significantly more distant from one another in the interphase nucleus relative to heterologous pairs [40]. Alternatively, DA could be envisioned as a stepwise process of chromosome condensation that packages DNA into highly condensed polymers in a tightly confined space [41], producing heterogeneous levels of compaction, as we have observed at discrete allelic loci.

Specific epigenetic marks such as histone modifications or topological constraints on chromatin that characterize each allele at the same locus may be a mechanism that underlies DA. Epigenetic marks can be propagated to ensure stability of chromatin memory and cellular identity in daughter cells, following mitosis [42]. Our findings can be interpreted in this context. Previous studies have demonstrated retention of nuclease hypersensitivity, transcription factor occupancy, and selective histone marks on mitotic chromatin [31]–[36]. Tri-methylation of histone H3 on lysine 9 and 27 is stably transmitted through interphase including mature post-replicative chromatin [43]. Differential condensation of homologous chromosomal regions could encode these features in a structural form that effectively memorizes the state of chromatin preceding metaphase. Maintenance of chromatin memory would be important for normal development and disease avoidance [43].

Previous work has demonstrated differences in intrachromosomal compaction using large FISH probes (e.g. cosmids or bacterial artificial chromosome [BAC] based probes) hybridized to a complex mix of chromatin fibers [44]. Reproducible differential hybridization patterns between metaphase homologs over short genomic distances (Table 1) have not been previously reported. The probes used to demonstrate DA are distinct from short single copy oligonucleotide (25–50 basepairs) DNA probes [45], densely tiled along a particular genomic region of ≥25 kb in length, that produce fluorescence signal intensities equivalent to a cosmid or a BAC. The differences in hybridization intensities to homologous chromosome regions of tiled oligonucleotides or large recombinant DNA probes are much less pronounced than the contiguous single copy probes used in the present study. BAC-based FISH probes, therefore, are not as sensitive for detection of DA, as these probes likely contain both genomic intervals with equivalent accessible and DA targets, and their longer target length increases their overall fluorescence intensity.

We have combined single or low copy probes for FISH, which together are on average 10 kb or more in genomic length, to assess boundaries of chromosomal rearrangements in complex genomic architecture [20],[21],[24]. The total length of these genomic targets does not solely dictate signal intensity. Probes of similar length and composition can vary in fluorescence intensity when hybridized to different regions in the human genome [20],[21]. In the present study, a 3.5 kb probe detects DA on chromosome 22 within ACR (Figure 2), whereas a smaller 2.08 kb single copy probe within C9orf66 (Figure 1B) shows equivalent accessibility and bright signals to both homologs. In addition, a low copy probe with 3 distinct genomic targets spanning 8.5 kb within HERC2 segmental duplicons exhibits DA (Additional file 1: Figure S1F). Finally, we did not find any remarkable differences in the GC content of individual single copy probes exhibiting DA relative to those showing equivalent accessibility (Additional file 5: Table S1). Our findings instead suggest that the context of the chromosomal regions themselves and their respective degrees of condensation primarily determine the differences in hybridization signal intensities that we observe.

Conclusions

We have previously designed and tested [20],[21] novel single copy DNA probes to precisely ascertain small pathogenic chromosome copy number changes and complex genomic architecture in the human genome [24]. In this study, we have expanded the utility of single copy DNA sequences to investigate chromatin accessibility differences between metaphase chromosome homologs. We demonstrate that chromatin accessibility differences are non-random with respect to specific homologous loci, they occur within exons, introns and intergenic regions, and these regions are not enriched for epigenetic marks of accessible interphase chromatin. Examination of allelic regions with DA, by super-resolution 3D-SIM, further showed that the internal chromatin structure of the accessible locus is less condensed relative to its inaccessible counterpart. Expanding the analysis of DA on a genomic scale to larger chromosomal domains containing allelic regions can help generate a high resolution map of chromatin accessibility during metaphase. Relating this information to epigenetic modifications during interphase may provide possible insight into how higher order chromatin structure is remodeled during mitosis.

Methods

Probe selection and scoring of differential accessibility (DA) on hybridized metaphase chromosomes

Single copy genome-coordinate defined DNA probes were previously developed and used with FISH to precisely localize breakpoints in rearranged metaphase chromosomes for many different diseases and disorders [20]–[25]. All single copy probes are devoid of repetitive elements and their nucleotide composition and genomic coordinates are precisely known. They map to a single location and can be developed from any unique region in the genome (e.g. exons, introns, intergenic, regulatory). As part of the development and validation of these single copy probes for FISH, they were hybridized to normal human chromosomes from the lymphocytes of at least one male and one female to confirm mapping of the probes to the expected genomic location [20]–[25]. Genomic locations of single copy probes were also compared to locations of common CNVs (≥1% of general population) from blood derived DNA in two independent sample sets from healthy individuals. Common CNVs on both sample sets were identified on Affymetrix CytoScan HD array using ChAS (Chromosome Analysis Suite) software. These population CNV data were obtained from Ontario Population Genomics Platform (873 individuals of European ancestry with minimum of 25 probes per CNV; Database of Genomic Variants) and Healthy sample track (~400 individuals with minimum of 35 probes per CNV; obtained from Affymetrix). During our validation studies, it was observed that while most single probes hybridized with similar affinity to both homologs within a cell, there were some probes in the validation samples with consistent, striking probe hybridization fluorescence intensity differences (or differential accessibility [DA]) between homologs. These probes were not pursued for clinical applications. In this study, we revisited some of these probes to begin to characterize the disparate fluorescence intensity differences between homologs. In order to determine if the hybridization intensity patterns were non-random, we selected DA probes based on availability of patient samples with cytogenetically distinguishable homologs (one normal, one rearranged) and the specific chromosomes involved in the rearrangements. Table 2 lists the FISH probes, their chromosomal location and the karyotypic findings of the 10 cell lines used to assess chromatin accessibility. These DA FISH probes were euploid and did not overlap the rearranged chromosomal regions. Parental origin of the chromosome rearrangement was known for 4 cell lines. Three cell lines (II-1 [mother], III-1 and III-2 [children]) were from a family carrying a microdeletion within the chromosome 15q12 imprinted region [13],[26]. The remaining cells lines were from unrelated individuals.

Chromosome preparations and fluorescence In situ hybridization

Peripheral blood lymphocytes or lymphoblastoid cell lines were cultured and chromosomes harvested using routine cytogenetic methods that included 0.075 M KCl hypotonic solution and 3:1 methanol:acetic acid fixation (Carnoy’s fixative) (also see Additional file 6: Supplementary methods) [46]. With the exception of single copy FISH probe designed from within CCNB1 (2.47 kb) on chromosome 5q13.2 (genomic coordinates, Table 2), all probes were previously developed [20],[25]. The CCNB1 probe was specifically designed from a genomic region with hallmarks of open chromatin [31]–[36]. Single copy FISH probes used in this study ranged from 1.78 kb to 3.55 kb in length. Details of probe amplification, purification, labeling, hybridization, and detection are provided in supplementary material and have been previously described [47]. To identify the chromosome 15q12 submicroscopic deletion (II2, III-1 and III-2), different biotin-labeled and digoxigenin-labeled single copy probes (one probe from within the deletion and one adjacent to the deletion), were hybridized simultaneously and detected in different colors to distinguish the deleted homolog from the normal one. For the other cell lines, the normal and rearranged homologs were distinguishable by DAPI staining and single copy probe hybridizations were performed.

DA was scored as differences in FISH probe hybridization intensities between homologous loci by direct examination using epifluorescence microscopy, and subsequently by quantification of hybridized probe epifluorescence images. At the microscope, hybridized probe fluorescence signals for each homolog were scored as bright, intermediate, dim, or nil. For a cell to be scored as DA, one homolog was required to exhibit an intermediate or bright probe signal and the other homolog a different intensity signal (e.g. bright/intermediate, bright/dim, bright/nil, intermediate/dim or intermediate/nil on homologs in a cell). For a cell to be scored as having equivalent accessibility, both homologs were required to exhibit probe hybridization of similar intensities (e.g. bright/bright, intermediate/intermediate). Microscope slides with metaphase cells were coded, hybridized and scored by 2 certified cytogeneticists. Twenty-five to 50 hybridized cells were scored for each sample. To exclude bias resulting from inefficient hybridizations, cells with dim hybridizations on both homologs or in which one homolog had a dim hybridization and the other had no hybridization were not scored. A two proportion Z-test was used to determine whether the fraction of cells showing DA or equivalent accessibility was statistically significant at α = 5.0E-02. Variance in the frequency of cells reported to have DA among different samples was assessed for significance (α = 5.0E-02) using Bartlett’s test for equality of variances.

For DA probes, a two proportion Z-test was also used to determine whether there was non-random preference for one parental homolog to have brighter probe fluorescence intensity (i.e. more accessible hybridization). From the Z-test score, a p-value was obtained to determine whether the proportion of the brighter hybridizations showed a significant bias (α = 5.0E-02) to one homolog. Additionally, probe fluorescence intensities in each cell were quantified by integrated gradient vector flow (GVF) analysis (next section).

Gradient Vector Flow (GVF) analysis to quantify differences in probe intensity between homologs

We previously developed a GVF-based algorithm that determined probe hybridization boundaries and quantified probe fluorescence [5],[48]. The GVF algorithm generated an active binary contour of the gray scale image of the probe fluorescence on each homolog. From the active contour, the integrated intensity values (in pixels) were calculated. The intensity values were normalized for each cell by taking the difference in integrated intensities between homologs, and dividing this difference by the sum of the intensities of both homologs. This converted raw total intensity values into a set of normalized intensity ratios (0 to 1). Values close to 0 confirmed that the probe intensities between the homologs appeared equivalent and ratios close to 1 indicated DA. A bias in hybridization intensities between homologous regions was reported as statistically significant (α = 5.0E-02) using a two-tailed t-test.

Examination of short target hybridized probe features using high resolution 3-D structured illumination super-resolution microscopy

3D-SIM (Nikon Corporation) was used to examine and quantify volume and depth of single and low copy DNA probe fluorescence embedded in metaphase chromatin. Low copy probes recognize multi-target DNA sequences that occur within segmental duplications [24]. 3D-SIM image reconstruction algorithms, for generating high resolution chromosome images, were optimized using a low copy probe from within NOMO1 hybridized to normal metaphase chromosomes. This probe yielded bright fluorescence signals on both homologs as it hybridized to multiple genomic targets on chromosome 16 duplicons, ([GRCh37] genomic coordinates: 16452359–16455837, 15013674–15017156, 16412325–16415807, 18440574–18444056, and 18484058–18487536).

Chromosome image acquisition was performed on a motorized inverted Ti-E microscope equipped with a CFI Apo TIRF 100X oil (NA 1.49) objective (Nikon USA) and SIM illuminator (Nikon Corporation) in stack 3D-SIM mode. The epifluorescence image was captured using total internal reflection fluorescence mode followed by 3D-SIM on the same cell to gain resolution in the X/Y/Z dimensions. Compatible lasers with wavelengths of 457 nm and 561 nm were used to excite DAPI (chromosome counterstain) and Cy3 (probe fluorescence), respectively. Using moiré superimposed pattern formation [28], high frequency signal components were captured and deduced from the image reconstruction algorithms. Fast Fourier transforms were generated to validate that previously irresolvable high frequency signals from the epifluorescence metaphase image had been properly acquired by 3D-SIM (Additional file 7: Figure S3). The NIS-Elements AR software (version 4.13.00, Nikon Canada Inc.) reconstructed 3D-SIM images of hybridized sequence-defined probes demonstrating DA (HERC2, PMP22:IVS3, ACR) or equivalent accessibility (NOMO1) to metaphase chromosome homologs. The lateral fluorescence depth of each probe was calculated from a maximum of 20 reconstructed optical sections. Each section was collected in 0.1 μm steps from a total of 20 metaphase cells for NOMO1, 10 cells each for HERC2 and PMP22: IVS3, and 2 cells for ACR. A threshold on the gray scale image of the DNA probe signal was performed in NIS-elements software using image segmentation, which converted the gray scale image into a binary image contour. Following probe fluorescence thresholding, the volume of bound probe fluorescence was calculated over all reconstructed optical sections. From these data, differences in probe volume and depth between homologs were quantified (NIS-Elements AR software) and analyzed for significance (α = 5.0E-02, two-tailed t test). Movie montages of DNA probe volume and depth were generated as AVI files, using the Movie Maker option (NIS-Elements AR software). Key frames depicting DA between homologs from all angles were added to the movie in order to emphasize the volume view, which built and rotated the metaphase chromosome 360° around the X/Y/Z axis.

Sequence analysis of epigenetic chromatin marks for single copy probes detecting DA or equivalent accessibility

The genomic sequence of the single copy probes, which displayed DA or equivalent hybridization accessibility (asterisks, Table 1) in metaphase were compared with epigenomic DNA features that characterize open chromatin and active regulatory elements during interphase in multiple cell types [27],[49]. The epigenomic features from ENCODE [27] that we examined include DNase1 HS, Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), and histone marks (H3K4me1, H3K9ac, H3K27ac, H3K4me2). The cell line used for ENCODE interphase comparisons, (GM12878, Coriell Cell Repository), was of the same B-cell lineage that we used to characterize DA and equivalent chromatin accessibility on metaphase homologs (Table 2). Furthermore, the cells were grown under the same culture conditions (37°C/5% CO2 in RPMI-1640 complete medium with 15% fetal bovine serum). ENCODE chromatin immuno-precipitation sequencing (ChIP-seq) data generated high resolution, multidimensional view of chromatin accessibility from the above-mentioned epigenomic DNA features [50]. ChIP-seq signal intensities of each open chromatin feature were visualized along the full length of a given single copy interval using the UCSC (University of California Santa Cruz) genome browser. Individual data points of the ChIP-seq signal intensities overlapping the genomic length of each single copy interval (Table 1) were retrieved from the UCSC table browser using the Duke DNase1 HS, University of North Carolina FAIRE seq, and Broad Institute histone modification custom tracks. The data point intensities were summed for each single copy interval (Table 1) and mean integrated single intensity values with standard deviations at 95% confidence were computed and plotted for all open chromatin features within each category (DA or equivalent accessibility). We then determined whether the differences in these values were significant by the analysis of variance test (ANOVA) for DA probes versus those with equivalent accessibility. Significance was determined from the p value of the F ratio following ANOVA.

Additional files

Abbreviations

ANOVA: 

Analysis of variance

AS: 

Angelman syndrome

ChIP-seq: 

Chromatin immuno-precipitation sequencing

CCNV: 

Common copy number variant

DA: 

Differential accessibility

DNase I HS: 

Deoxyribonuclease I hypersensitivity

ENCODE: 

Encyclopedia of DNA elements

FAIRE: 

Formaldehyde-assisted isolation of regulatory elements

FISH: 

Fluorescence in situ hybridization

GVF: 

Gradient vector flow

H3K9Ac: 

Histone 3 lysine 9 acetylation

H3K27ac: 

Histone 3 lysine 27 acetylation

H3K4me1: 

Histone 3 lysine 4 mono-methylation

H3K4me2: 

Histone 3 lysine 4 di-methylation

3D-SIM: 

3-dimensional structured illumination microscopy

Declarations

Acknowledgements

The authors would like to acknowledge support from Canadian Foundation for Innovation (JHMK, PKR), Canada Research Chairs Secretariat (PKR), Queen Elizabeth II-Graduate Scholarship in Science and Technology (WAK), Schulich Graduate Scholarship Fund, Graduate Research Thesis Awards and the Ontario Graduate Scholarship (WAK.). A special thanks to Dr. Kevin Conway (Nikon Canada Inc.) and Nikon affiliates (Marine Biology Lab – Woods Hole, MA and Center for Biological Imaging - University of Pittsburgh) for their expertise on super resolution 3D-SIM and the use of their microscopy facilities. We thank the members of the Image Recognition and Intelligent Systems Laboratory at University of Western Ontario who built the Matlab compiler for GVF. Lymphoblastoid cell lines and lymphocyte fixed cell pellets were obtained from Coriell Cell Repository and the London Health Sciences Center Clinical Cytogenetics Laboratory. Cytognomix Inc. has received support from Ontario Genomics Institute, NRC-IRAP, MaRS Discovery District Market Readiness Program and the Ontario Centers of Excellence.

Authors’ Affiliations

(1)
Department of Pathology and Laboratory Medicine, University of Western Ontario
(2)
Departments of Biochemistry and Computer Science, University of Western Ontario
(3)
Cytognomix, Inc

References

  1. Londoño-Vallejo JA, DerSarkissian H, Cazes L, Thomas G: Differences in telomere length between homologous chromosomes in humans. Nucleic Acids Res 2001, 29: 3164–3171. 10.1093/nar/29.15.3164PubMed CentralView ArticlePubMedGoogle Scholar
  2. Ghosh PK, Rani R, Nand R: Lateral asymmetry of constitutive heterochromatin in human chromosomes. Hum Genet 1979, 52: 79–84. 10.1007/BF00284600View ArticlePubMedGoogle Scholar
  3. Wang Y, Maharana S, Wang MD, Shivashankar GV: Super-resolution microscopy reveals decondensed chromatin structure at transcription sites. Sci Rep 2014, 4: 4477.PubMed CentralPubMedGoogle Scholar
  4. Ushiki T, Hoshi O: Atomic force microscopy for imaging human metaphase chromosomes. Chromosome Res 2012, 16: 383–396. 10.1007/s10577-008-1241-7View ArticleGoogle Scholar
  5. Khan WA, Chisholm R, Tadayyon S, Subasinghe A, Norton P, Samarabandu J, Johnston LJ, Knoll JH, Rogan PK: Relating centromeric topography in fixed human chromosomes to α-satellite DNA and CENP-B distribution. Cytogenet Genome Res 2013, 139: 234–242. 10.1159/000348744View ArticlePubMedGoogle Scholar
  6. Craig JM, Bickmore WA: Chromosome bands – flavours to savour. Bioessays 1993, 15: 349–354. 10.1002/bies.950150510View ArticlePubMedGoogle Scholar
  7. Watanabe Y, Maekawa M: R/G-band boundaries: genomic instability and human disease. Clin Chim Acta 2013, 18: 108–112. 10.1016/j.cca.2013.02.011View ArticleGoogle Scholar
  8. Goradia RY, Davis BK: Asymmetry in sister chromatids of human chromosomes. J Hered 1978, 69: 369–372.PubMedGoogle Scholar
  9. Brito-Babapulle V: Lateral asymmetry in human chromosomes 1, 3, 4, 15 and 16. Cytogenet Cell Genet 1981, 29: 198–202. 10.1159/000131570View ArticlePubMedGoogle Scholar
  10. Schmid M, Haaf T, Grunert D: 5-Azacytidine-induced undercondensations in human chromosomes. Hum Genet 1984, 67: 257–263. 10.1007/BF00291352View ArticlePubMedGoogle Scholar
  11. Chambers EV, Bickmore WA, Semple CA: Divergence of mammalian higher order chromatin structure is associated with developmental loci. PLoS Comput Biol 2013, 9: e1003017. 10.1371/journal.pcbi.1003017PubMed CentralView ArticlePubMedGoogle Scholar
  12. Izumikawa Y, Naritomi K, Hirayama K: Replication asynchrony between homologs 15q11.2: cytogenetic evidence for genomic imprinting. Hum Genet 1991, 87: 1–5. 10.1007/BF01213082View ArticlePubMedGoogle Scholar
  13. Knoll JH, Cheng SD, Lalande M: Allele specificity of DNA replication timing in the Angelman/Prader-Willi syndrome imprinted chromosomal region. Nat Genet 1994, 6: 41–46. 10.1038/ng0194-41View ArticlePubMedGoogle Scholar
  14. Dutrillaux B, Couturier J, Richter CL, Viegas-Pequignot E: Sequence of DNA replication in 277 R- and Q-bands of human chromosomes using BrdU treatment. Chromosoma 1976, 58: 51–61. 10.1007/BF00293440View ArticlePubMedGoogle Scholar
  15. Terrenoire E, McRonald F, Halsall JA, Page P, Illingworth RS, Taylor AM, Davison V, O’Neill LP, Turner BM: Immunostaining of modified histones defines high-level features of the human metaphase epigenome. Genome Biol 2010, 11: R110. 10.1186/gb-2010-11-11-r110PubMed CentralView ArticlePubMedGoogle Scholar
  16. Banerjee T, Chakravarti D: A peek into the complex realm of histone phosphorylation. Mol Cell Biol 2011, 31: 4858–4873. 10.1128/MCB.05631-11PubMed CentralView ArticlePubMedGoogle Scholar
  17. Jenuwein T, Allis CD: Translating the histone code. Science 2001, 10: 1074–1080. 10.1126/science.1063127View ArticleGoogle Scholar
  18. Bonenfant D, Towbin H, Coulot M, Schindler P, Mueller DR, van Oostrum J: Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry. Mol Cell Proteomics 2007, 6: 1917–1932. 10.1074/mcp.M700070-MCP200View ArticlePubMedGoogle Scholar
  19. Vagnarelli P: Chromatin reorganization through mitosis. Adv Protein Chem Struct Biol 2013, 90: 179–224. 10.1016/B978-0-12-410523-2.00006-7View ArticlePubMedGoogle Scholar
  20. Rogan PK, Cazcarro PM, Knoll JH: Sequence based design of single-copy genomic DNA probes for fluorescence in situ hybridization. Genome Res 2001, 11: 1086–1094. 10.1101/gr.171701PubMed CentralView ArticlePubMedGoogle Scholar
  21. Knoll JH, Rogan PK: Sequence-based, in situ detection of chromosomal abnormalities at high resolution. Am J Med Genet 2003, 121A: 245–257. 10.1002/ajmg.a.20123View ArticlePubMedGoogle Scholar
  22. Knoll J, Rogan P, inventors; Knoll J, Rogan P, original assignee: Single copy genomic hybridization probes and method of generating same. United States patent US 20030224356 A1. 2003 Dec 4Google Scholar
  23. Knoll J, Rogan PK, inventors; Children’s Mercy Hospital, Knoll J, Rogan P, assignee: Subtelomeric DNA probes and method of producing the same. International Patent WO2004029283 A2. 2004 Apr 8Google Scholar
  24. Khan WA, Knoll JH, Rogan PK: Context-based FISH localization of genomic rearrangements within chromosome 15q11.2q13 duplicons. Mol Cytogenet 2011, 4: 15. 10.1186/1755-8166-4-15PubMed CentralView ArticlePubMedGoogle Scholar
  25. Dorman SN, Shirley BC, Knoll JH, Rogan PK: Expanding probe repertoire and improving reproducibility in human genomic hybridization. Nucleic Acids Res 2013, 41: e81. 10.1093/nar/gkt048PubMed CentralView ArticlePubMedGoogle Scholar
  26. Hamabe J, Kuroki Y, Imaizumi K, Sugimoto T, Fukushima Y, Yamaguchi A, Izumikawa Y, Niikawa N: DNA deletion and its parental origin in Angelman syndrome patients. Am J Med Genet 1991, 41: 64–68. 10.1002/ajmg.1320410117View ArticlePubMedGoogle Scholar
  27. Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, Snyder M: ENCODE Project Consortium: An integrated encyclopedia of DNA elements in the human genome. Nature 2012, 489: 57–74. 10.1038/nature11247View ArticleGoogle Scholar
  28. Flors C, Earnshaw WC: Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes. Curr Opin Chem Biol 2011, 15: 838–844. 10.1016/j.cbpa.2011.10.004View ArticlePubMedGoogle Scholar
  29. Kner P, Chhun BB, Griffis ER, Winoto L, Gustafsson MG: Super-resolution video microscopy of live cells by structured illumination. Nat Methods 2009, 6: 339–342. 10.1038/nmeth.1324PubMed CentralView ArticlePubMedGoogle Scholar
  30. Strukov YG, Sural TH, Kuroda MI, Sedat JW: Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila. PLoS Biol 2011, 9: e1000574. 10.1371/journal.pbio.1000574PubMed CentralView ArticlePubMedGoogle Scholar
  31. John S, Workman JL: Bookmarking genes for activation in condensed mitotic chromosomes. Bioessays 1998, 20: 275–279. 10.1002/(SICI)1521-1878(199804)20:4<275::AID-BIES1>3.0.CO;2-PView ArticlePubMedGoogle Scholar
  32. Xing H, Wilkerson DC, Mayhew CN, Lubert EJ, Skaggs HS, Goodson ML, Hong Y, Park-Sarge OK, Sarge KD: Mechanism of hsp70i gene bookmarking. Science 2005, 307: 421–423. 10.1126/science.1106478View ArticlePubMedGoogle Scholar
  33. Xing H, Vanderford NL, Sarge KD: The TBP-PP2A mitotic complex bookmarks genes by preventing condensin action. Nat Cell Biol 2008, 10: 1318–1323. 10.1038/ncb1790PubMed CentralView ArticlePubMedGoogle Scholar
  34. Zhao R, Nakamura T, Fu Y, Lazar Z, Spector DL: Gene bookmarking accelerates the kinetics of post-mitotic transcriptional re-activation. Nat Cell Biol 2011, 13: 1295–1304. 10.1038/ncb2341PubMed CentralView ArticlePubMedGoogle Scholar
  35. Yan J, Enge M, Whitington T, Dave K, Liu J, Sur I, Schmierer B, Jolma A, Kivioja T, Taipale M, Taipale J: Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites. Cell 2013, 154: 801–813. 10.1016/j.cell.2013.07.034View ArticlePubMedGoogle Scholar
  36. Zaidi SK, Young DW, Montecino M, Lian JB, Stein JL, van Wijnen AJ, Stein GS: Architectural epigenetics: mitotic retention of mammalian transcriptional regulatory information. Mol Cell Biol 2010, 30: 4758–4766. 10.1128/MCB.00646-10PubMed CentralView ArticlePubMedGoogle Scholar
  37. Gimelbrant A, Hutchinson JN, Thompson BR, Chess A: Widespread monoallelic expression on human autosomes. Science 2007, 318: 1136–1140. 10.1126/science.1148910View ArticlePubMedGoogle Scholar
  38. Wagner JR, Ge B, Pokholok D, Gunderson KL, Pastinen T, Blanchette M: Computational analysis of whole-genome differential allelic expression data in human. PLoS Comput Biol 2010, 6: e1000849. 10.1371/journal.pcbi.1000849PubMed CentralView ArticlePubMedGoogle Scholar
  39. Koeman JM, Russell RC, Tan MH, Petillo D, Westphal M, Koelzer K, Metcalf JL, Zhang Z, Matsuda D, Dykema KJ, Houseman HL, Kort EJ, Furge LL, Kahnoski RJ, Richard S, Vieillefond A, Swiatek PJ, Teh BT, Ohh M, Furge KA: Somatic pairing of chromosome 19 in renal oncocytoma is associated with deregulated EGLN2- mediated [corrected] oxygen-sensing response. PLoS Genet 2008, 4: e1000176. 10.1371/journal.pgen.1000176PubMed CentralView ArticlePubMedGoogle Scholar
  40. Heride C, Ricoul M, Kiêu K, von Hase J, Guillemot V, Cremer C, Dubrana K, Sabatier L: Distance between homologous chromosomes results from chromosome positioning constraints. J Cell Sci 2010, 123: 4063–4075. 10.1242/jcs.066498View ArticlePubMedGoogle Scholar
  41. Berndsen ZT, Keller N, Grimes S, Jardine PJ, Smith DE: Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging. Proc Natl Acad Sci U S A 2014, 111: 8345–8350. 10.1073/pnas.1405109111PubMed CentralView ArticlePubMedGoogle Scholar
  42. Margueron R, Reinberg D: Chromatin structure and the inheritance of epigenetic information. Nat Rev Genet 2010, 11: 285–296. 10.1038/nrg2752PubMed CentralView ArticlePubMedGoogle Scholar
  43. Alabert C, Bukowski-Wills JC, Lee SB, Kustatscher G, Nakamura K, de Lima AF, Menard P, Mejlvang J, Rappsilber J, Groth A: Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat Cell Biol 2014, 16: 281–293. 10.1038/ncb2918PubMed CentralView ArticlePubMedGoogle Scholar
  44. Gilbert N, Boyle S, Fiegler H, Woodfine K, Carter NP, Bickmore WA: Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers. Cell 2004, 118: 555–566. 10.1016/j.cell.2004.08.011View ArticlePubMedGoogle Scholar
  45. Yamada NA, Rector LS, Tsang P, Carr E, Scheffer A, Sederberg MC, Aston ME, Ach RA, Tsalenko A, Sampas N, Peter B, Bruhn L, Brothman AR: Visualization of fine scale genomic structure by oligonucleotide-based high-resolution FISH. Cytogenet Genome Res 2011, 132: 248–254. 10.1159/000322717View ArticlePubMedGoogle Scholar
  46. Bangs CD, Donlon TA: Metaphase Chromosome Preparation from Cultured Peripheral Blood Cells. In Current Protocols in Human Genetics. Edited by: Haines JL, Korf BR, Morton CC. John Wiley & Sons,Inc, Massachusetts; 2005:4.1.1–4.1.19.Google Scholar
  47. Knoll JH, Lichter P, Bakdounes K, Eltoum IE: In situ hybridization and detection using nonisotopic probes. Curr Protoc Mol Biol 2007, 79: 14.7.1–14.7.17.Google Scholar
  48. Subasinghe A, Samarabandu J, Knoll JHM, Khan W, Rogan P: An image processing algorithm for accurate extraction of the centerline from human metaphase chromosomes. In Proceedings of IEEE 17 th International Conference on Image Processing; 26–29 September; Hong Kong. Edited by Wai-Kuen Cham; 2010:3613–3616Google Scholar
  49. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K: High-resolution profiling of histone methylations in the human genome. Cell 2007, 129: 823–837. 10.1016/j.cell.2007.05.009View ArticlePubMedGoogle Scholar
  50. Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, Sheffield NC, Stergachis AB, Wang H, Vernot B, Garg K, John S, Sandstrom R, Bates D, Boatman L, Canfield TK, Diegel M, Dunn D, Ebersol AK, Frum T, Giste E, Johnson AK, Johnson EM, Kutyavin T, Lajoie B, Lee BK, Lee K, London D, Lotakis D, Neph S, et al.: The accessible chromatin landscape of the human genome. Nature 2012, 489: 75–82. 10.1038/nature11232PubMed CentralView ArticlePubMedGoogle Scholar

Copyright

© Khan et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Advertisement