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. 2015 Nov 5;163(4):934-46.
doi: 10.1016/j.cell.2015.10.026.

Stable Chromosome Condensation Revealed by Chromosome Conformation Capture

Kyle P Eagen  1 , Tom A Hartl  2 , Roger D Kornberg  3
Affiliations

Stable Chromosome Condensation Revealed by Chromosome Conformation Capture

Kyle P Eagen et al. Cell. .

Abstract

Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.

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Figures

Figure 1
Figure 1. Lack of Regular, Long-range Contacts in Polytene Chromosomes
(A) Genome-wide Hi-C heatmap from polytene cells. Black circles and squares represent where centromeres and telomeres intersect, respectively. (B) Hi-C heatmap from Drosophila embryos (Sexton et al., 2012) of a 17 Mb region of chromosome 3R encompassing the ANT-C and BX-C loci. Black circles represent where the ANT-C and BX-C loci intersect. (C) Hi-C heatmap from polytene cells of a 17 Mb region of chromosome 3R encompassing the ANT-C and BX-C loci. Black circles represent where the ANT-C and BX-C loci intersect. Heatmaps were normalized and divided into 100 kb bins. See also Figures S1 and S2.
Figure 2
Figure 2. Polytene Bands are TADs
(A) Normalized Hi-C heatmap (15 kb bins) of a 3 Mb region of polytene chromosome 2L. In the left panel, areas bounded by black boxes represent locations of polytene bands for which reliable DNA sequence coordinates are available (Belyaeva et al., 2012; Vatolina et al., 2011). In the right panel, areas bounded by black boxes represent TADs. (B) Photographic image (bottom) of the region of polytene chromosome 2L from (A) and the same region of Bridges’s chromosome map (top). Arrows indicate bands represented by black boxes in (A). The DNA sequence coordinates of other bands in this region are not known and therefore cannot be compared with the Hi-C data. Adapted from (Lefevre, 1976). (C) Mean directionality index (DI) of polytene bands (upper panel; n = 61) and heatmap of the directionality index of each band along its length (lower panel). Bands were normalized to the same length and 50 kb of flanking DNA is shown next to each normalized band. (D) Heatmap of the agreement between polytene bands (n = 61) and TADs. Each band is represented by a row. Bands were normalized to the same length and 50 kb of flanking DNA is shown next to each normalized band. Orange segments overlap with polytene TADs, black segments overlap with regions between TADs. (E) Fraction of band boundaries (n = 122) at the distance indicated on the abscissa from the closest TAD boundary (calculated in 20 kb windows). See also Figures S3, S4, and S5 and Table S1.
Figure 3
Figure 3. Hi-C of Polytene Puffs
(A) Normalized Hi-C heatmaps (15 kb bins) of puff stage 5–8 polytene puffs on chromosome 3L. Areas bounded by black boxes correspond to the indicated puff. (B) Mean directionality index (DI) of polytene puffs (left) and heatmap of the directionality index of each puff along its length (right). Puffs were normalized to the same length and 50 kb of flanking DNA is shown next to each normalized puff.
Figure 4
Figure 4. Hi-C Predicts the Location of Polytene Bands
The locations of TADs (normalized Hi-C heatmaps, 15 kb bins; left column) were used to generate FISH probes against TAD centers (red diamonds) or TAD borders (green diamonds), which were hybridized to polytene chromosome spreads counterstained with DAPI (middle-left column). The TAD border FISH signal and the TAD center FISH signal are pseudocolored green and red, respectively, in the merged images (rightmost two columns) and the identity of the polytene band is indicated (right column). White arrows indicate the polytene band of interest. (A) FISH against a region from chromosome 2L. White arrowhead indicates a small interband between bands 22A1-2 and 22A3 in Bridges’s map. (B) FISH against a region from chromosome 3L. (C) FISH against a region from chromosome 3R. Loci presented here are independent of those analyzed in Figure 1. Scale bars, 2 µm.
Figure 5
Figure 5. Conservation of Polytene and Diploid TADs
Normalized Hi-C heatmaps (15 kb bins) of a 3 Mb region of the X chromosome. In the left panels, areas bounded by black boxes represent locations of polytene bands for which reliable DNA sequence coordinates are available (Belyaeva et al., 2012; Vatolina et al., 2011). In the right panel, areas bounded by black boxes represent TADs. (A) Hi-C heatmap from polytene cells. (B) Hi-C heatmap from diploid Kc167 cultured cells. (C) Photographic image (bottom) of the region of polytene chromosome X from (A) and (B) and the same region of Bridges’s chromosome map (top). Arrows indicate cytological bands represented by black boxes in (A) and (B). The DNA sequence coordinates of other bands in this region are not known and therefore cannot be compared with the Hi-C data. Adapted from (Lefevre, 1976). (D) Heatmap of the agreement between polytene TADs and diploid TADs. Each polytene TAD is represented by a row. Green segments overlap with diploid TADs, black segments overlap with regions between diploid TADs. TADs were normalized to the same length and 50 kb of flanking DNA is shown next to each normalized TAD. (E) Fraction of polytene TAD boundaries (n = 692) at the distance indicated on the abscissa from the closest diploid TAD boundary (calculated in 20 kb windows). See also Figures S4, S5, and S6.
Figure 6
Figure 6. Lack of Compartments in the Polytene Nucleus
Normalized observed (left column), observed/expected (middle column), and Pearson correlation (right column) Hi-C heatmaps (100 kb bins) for embryonic (top row), diploid Kc167 cell (middle row), and polytene (bottom row) chromosome 3R. In the observed/expected heatmaps, interactions less than the expected genome-wide average are blue, those greater than the expected genome-wide average are red. A plaid pattern in a Pearson correlation heatmap indicates the presence of compartments. See also Figure S7.
Figure 7
Figure 7. Chromosome Condensation in the Interphase Nucleus
At left, thin section electron micrograph of a nucleus (Cross and Mercer, 1993), with lightly staining euchromatin in the nuclear interior, surrounded by darkly staining heterochromatin, concentrated at the nuclear periphery. At right, cartoon representation of white, grey, and black chromatin, showing proposed relationships to heterochromatin, euchromatin, and the nuclear envelope (yellow). Active TADs in the euchromatin are nearby other active TADs and inactive TADs in the heterochromatin are nearby other inactive TADs, resulting in grey-grey and black-black TAD-TAD interactions. The actual pattern of chromatin folding is unknown and indicated only schematically.
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