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Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains - PubMed

Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

Sergey V Ulianov et al. Genome Res. 2016 Jan.

Abstract

Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

© 2016 Ulianov et al.; Published by Cold Spring Harbor Laboratory Press.

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Figures

Figure 1.
Figure 1.

Genomic positions of topologically associating domains (TADs) are largely conserved among Drosophila cells of different origins. (A) A fragment of the Hi-C interaction map (heat map) for the BG3 cell line. Gray rectangles below the heat map represent TADs predicted with two different values of the scaling parameter γ. This illustrates a two-step TAD prediction method utilizing a higher γ value for large TADs containing internal self-interacting domains. (B) Heat maps of a 1.6-Mb region of Chromosome 2R with annotated TADs. Positions of boundary bins are indicated by colored rectangles under the TADs map. The color code for the boundary bins is shown at the bottom. Red arrows indicate unidentified weak boundaries. The resolution of all heat maps is 20 kb. (C) Venn diagram showing the numbers of TADs shared between all studied cell lines (with both boundaries located at the same genomic bin or at adjacent bins). Total numbers of TADs in each cell line are shown in parentheses. Numbers of TADs shared between different pairs of the cell lines are shown to the right of the diagram.

Figure 2.
Figure 2.

Partitioning of chromosomes into TADs and inter-TADs reflects distributions of active and repressed chromatin regions. (A) Heat map of a 4-Mb region of Chromosome 2R aligned with modENCODE tracks of RNA polymerase II, H3K27ac, and histone H1 for BG3 cells. TAD boundaries and inter-TAD regions are shaded in blue. Red arrow indicates an unidentified weak TAD boundary. (B) Distribution of chromatin types (colors) near TAD boundaries. Box plots show proportions of chromatin colors in bins located at the same position relative to a TAD boundary, averaged over all TADs. TAD boundaries were aligned so that inter-TAD bins were placed to the left of the boundary bin (bin 0) and TAD bins were placed to the right of the boundary bin. The region from the third bin in the inter-TAD (left) to the fifth bin in the TAD (right) is shown. Plots with longer distances from the boundary are shown in Supplemental Figure S5A. Chromatin colors are taken from Filion et al. (2010) for the Kc167 cells and from Kharchenko et al. (2011) for the S2 and BG3 cells. The OSC cells were not analyzed, as epigenetic data were not available for this cell line. The P-values are presented in Supplemental Table S6. (C) Distribution of the individual chromatin marks and proteins near TAD boundaries. Curves smoothed with LOESS show the median Z-transformed values in the groups of bins described above. Thick rectangles show TAD boundary bins. Box plots are shown in Supplemental Figure S6. P-values are presented in Supplemental Table S6. (D) Prediction of TADs, inter-TADs, and TAD boundaries in the S2 cells using logistic regression models based on the genome distribution of active chromatin marks or architectural proteins dCTCF and Su(Hw). Receiver operating characteristics (ROC curves) and AUC (area under the curve) values are shown.

Figure 3.
Figure 3.

High transcription level and high content of active chromatin interfere with DNA packaging into TADs. (A) The level of poly(A)+ transcripts around TAD boundaries in the BG3 cells (data for the other cell lines are presented in Supplemental Fig. S8C). Box plots show the number of upper-quantile normalized transcriptome read counts over all bins located at the same position relative to a TAD boundary. Blue dots denote average values; medians are shown by thick black lines. (B) Scatter plot demonstrating the transcription level and fraction of active chromatin colors (sum of 1, 2, and 3 chromatin types) in individual TADs (excluding boundary bins) and inter-TADs (including TAD boundaries) in the BG3 cells. Data for the S2 and Kc167 cells are presented in Supplemental Figure S9. (C) Box plots showing inverse dependence between the proportion of active chromatin colors and γt (the minimal value of the scaling parameter γ required to annotate the bin as a TAD boundary or inter-TAD) in the BG3 cells. Average proportions of chromatin colors over all bins with the same γt are shown in each plot. Data for the S2 and Kc167 cells and similar diagrams built with γt ranging from 0 to 10 are shown in Supplemental Figure S5B. P-values are presented in Supplemental Table S6. (D) Box plots showing the inverse dependence between the transcription level within a genomic bin and γt in the BG3 cells. Data for the S2 and Kc167 cells are presented in Supplemental Figure S8D. (E) Box plots demonstrating the dependence between γt and the proportion of active chromatin marks (bottom) or the proportion of architectural proteins (top) in BG3 cells. (F) The Pearson correlation coefficients between the proportions of different chromatin colors (numbered as in C) or transcription level and the TAD density averaged over all size groups of TADs. Bar plots show the mean and variance values for the TAD size groups. (G) Scatter plot demonstrating the negative correlation between the TAD density and the proportion of active chromatin colors (sum of 1, 2, and 5 colors) (Kharchenko et al. 2011) for 140-kb-long TADs in the BG3 cell line. (H) A representative pair of TADs with different proportions of active chromatin display different TAD density.

Figure 4.
Figure 4.

Distribution of housekeeping genes, intensity of transcription, and the presence of active chromatin, as related to TAD profiles. (A) Distribution of housekeeping and tissue-specific genes, housekeeping, and developmental STARR-seq-identified enhancers around TAD boundaries from the S2 cells (red) and OSCs (blue). The proportion of a bin occupied by gene bodies (upper graphs) and the average number of enhancers in a bin (lower graphs) were calculated for all bins located at the same position relative to a TAD boundary. (B) Pie charts showing the distribution of differentially or uniformly transcribed bins in the four groups of bins defined by pairwise comparison of the cell lines (see the description in the text).

Figure 5.
Figure 5.

Inter-TADs correspond to polytene chromosome inter-bands. (A) Our annotation of TADs and inter-TADs at sites of 32 cytologically identified inter-bands (Zhimulev et al. 2014). (B) Distribution of inter-band-related chromatin colors (Zhimulev et al. 2014) near TAD boundaries. The alignment of bins relative to the TAD boundary bin is as in Figure 2B. The P-values are presented in Supplemental Table S6. (C) Bar plot showing the inverse dependence between the proportions of inter-band-specific chromatin colors within a genomic bin and γt. The P-values are presented in Supplemental Table S6. (D) Phase-contrast image of two bands clearly corresponding to TADs in the BG3 cells. Reprinted from Figure 4A from Vatolina et al. (2011) with permission from the publisher.

Figure 6.
Figure 6.

Computer simulation of a linear polymer that folds into a set of TADs supports a key role of hyperacetylated chromatin in separation of TADs. (A) One of the predicted spatial configurations of a polymer composed of 19 blocks of inactive (interacting) nucleosomes (500 nucleosomes each, green) interspaced by shorter blocks of active noninteracting nucleosomes (50 nucleosomes each, black). (B) Spatial proximity map (distance heat map) of the polymer configuration presented in A. Distances are measured in numbers of nucleosomes. A scheme of the model polymer and positions of TADs predicted by the Armatus algorithm are shown below the map. (C) Distance heat maps of the four individual configurations of the model polymer. Configuration 1 is used in A and B. (D) Distance heat map (upper) and contact heat map (simulated Hi-C map constructed at resolution of 4 kb [20 beads] for three consecutive TADs, lower) of the model polymer obtained by the averaging of the heat maps of 12 individual configurations. Notation as in B. (E) A schematic illustrating the proposed model of chromatin folding into TADs/inter-TADs, as directed by the self-association of nucleosomes. A high acetylation level of the chromatin within the genomic regions harboring actively transcribed genes interferes with chromatin packaging into TADs due to decreased inter-nucleosomal interactions.

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