Temperature stress alters TAD border strength

Changes in TAD borders could explain the redistribution of interactions from within TADs to between TADs observed after heat shock. To describe TAD borders in a quantitative manner, we and others have established the concept of TAD border strength: the ratio between inter-TAD and intra-TAD interactions on either side of the border (Sofueva et al., 2013; Van Bortle et al., 2014). Using this definition, every restriction site in the genome can be assigned a quantitative border strength and any specific fragment above a certain threshold will be defined as a TAD border by previously published methods. This strategy allows us to quantitatively evaluate the ability of every restriction fragment in the genome to form a border, independent of whether it is defined as a TAD border by standard methods. To illustrate this concept, we evaluated border strength after heat shock for a ~200 kb region (Figure 1G). Border strength decreases after heat shock at every restriction fragment present in this region, including both standard TAD borders (green arrows) and a non-borders (black arrow). We then extended this analysis of border strength to the whole Drosophila genome in NT and HS samples (Figure 1H). Border strength at most standard TAD borders (blue dots) decreases after heat shock. This is also observed for most HindIII fragments in the genome (black dots), although a subset of fragments show an increase in border strength in cells subjected to temperature stress (Figure 1H). Thus, heat shock reduces border strength at a majority of HindIII fragments and traditional TAD borders, which agrees with the observed increase in inter-TAD interactions.

Inhibition of transcription has a weak effect on TAD border strength and 3D organization

Since TAD borders are present in regions containing highly transcribed genes, the decrease in TAD border strength after heat shock may be a consequence of the absence of transcription. In addition, the decrease in intra-TAD interactions, may represent lost contacts between promoters and regulatory sequences after transcriptional silencing. This is suggested by ChIP-seq analysis of RNA polymerase II (RNAPII) distribution, which shows a dramatic decrease in RNAPII at gene promoters and coding regions in heat shocked cells (Figure 2A). To explore the possibility that transcriptional silencing contributes to the changes in 3D organization observed after heat shock, we inhibited transcription by treating cells with flavopiridol or triptolide. Treatment with flavopiridol inhibits elongation and pauses RNAPII (Jonkers et al., 2014), resulting in lower levels of RNAPII phosphorylated in Ser5 and loss of RNAPII phosphorylated in Ser2 (Figure 2B). Triptolide inhibits transcription initiation and results in degradation of RNAPII (Jonkers et al., 2014), resulting in a further decrease of RNAPII phosphorylated in Ser5 and absence of RNAPII phosphorylated in Ser2 (Figure 2B). We then compared RNAPII occupancy after treatment with transcription inhibitors or heat shock using ChIP-seq (Figure 2A). Flavopiridol depletes RNAPII throughout the coding regions of genes, resulting in an accumulation of total RNAPII at promoters. In contrast, triptolide results in a decrease in RNAPII at promoters and coding regions of genes similar to that observed after heat shock (Figure 2A). Heat shock dramatically decreases RNAPII at most promoters, with most TSSs containing low levels of RNAPII (Figure 2C). Cells treated with flavopiridol show a modest decrease in the number of enhancers and promoters containing high levels of RNAPII, while triptolide treatment exhibits a more pronounced decrease (Figure 2C).

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Figure 2

Effect of transcription inhibition on 3D organization

(A) Screenshot of a region of the Drosophila genome showing ChIP-seq signal for RNAPII in control, flavopiridol-treated, triptolide-treated, and heat shocked cells.

(B) Western analysis of control, flavopiridol-treated and triptolide-treated cells using antibodies to the CTCD domain of RNAPII phosphorylated in serine 5 (left) or serine 2 (right). Antibodies to lamin were used for the loading control (bottom).

(C) Distribution of ChIP-seq normalized reads with respect to sites in the genome at enhancers and promoters in cells grown at normal temperature, heat shocked, and treated with tripdolide or flavopiridol.

(D) Changes in frequency of intra- and inter-arm interactions obtained from Hi-C data in cells treated with triptolide or flavopiridol relative to untreated cells.

(E) Decay curves of intra- and inter-TAD interactions with respect to distance for control and triptolide-treated cells.

(F) Decay curves of intra- and inter-TAD interactions with respect to distance for control and flavopiridol-treated cells.

(G) Two-dimensional contact matrices of Hi-C data obtained with control (upper left) and triptolide-treated (bottom right) cells.

(H) Subtraction heat map in which the contact matrix for a 400 kb of chromosome 2L obtained from Hi-C data in control cells has been subtracted from that obtained in triptolide-treated cells.

(I) Scatter plot comparing border strength (BS) in Hi-C samples from control and triptolide-treated cells. Blue dots indicate TAD borders determined using a probability-based model. Black dots indicate all DpnII restriction fragments in the genome. Dashed lines above and below the diagonal indicate + or − one standard deviation, respectively.

(J) Scatter plot comparing border strength (BS) in Hi-C samples from control and flavopiridol-treated cells. Blue dots indicate TAD borders determined using a probability-based model. Black dots indicate all DpnII restriction fragments in the genome. Dashed lines above and below the diagonal indicate + or − one standard deviation, respectively.

To examine the effects of inhibiting transcription initiation or elongation on the 3D nuclear interactome, we generated Hi-C libraries, using DpnII instead of HindIII, with Kc167 cells treated with triptolide or flavopiridol. We obtained 169 million processed read pairs for the untreated control, 65.8 million read pairs for triptolide-treated and 37.8 million for flavopiridol-treated cells from two biological replicates for each condition (Table S1). The changes in chromatin contact frequencies are very similar for cells treated with either triptolide or flavopiridol. Interactions between centromeres increase after transcription inhibition, contrasting with the marked decrease observed after heat shock (Figure 2D). Decay curves of intra-arm interactions follow a similar but less pronounced pattern as that observed after temperature stress (Figure 2E and 2F), and inter-chromosomal interactions increase with respect to untreated cells (Figure 2D). Intra-TAD interactions decrease slightly whereas inter-TAD interactions increase, most notably at distances above 100 kb. These changes can be visually observed in two-dimensional contact matrices (Figure 2G), and in heat maps in which the contact matrix of control cells has been subtracted from that of treated cells (Figure 2H). Similar to heat shocked cells, chromatin interactions are redistributed, resulting in fewer intra-TAD interactions and more inter-TAD interactions compared to control. In addition, TAD border strength also decreases after treatment with triptolide or flavopiridol (Figure 2I and 2J). These findings suggest that the genome-wide inhibition of transcription that occurs after temperature stress may contribute to the observed changes in TAD organization but that additional factors are required to account for all of the changes observed after heat shock.

The genome-wide distribution of architectural proteins is altered after temperature stress

Since architectural proteins are enriched at TAD borders, it is possible that they also contribute to the changes in 3D organization observed after temperature stress. To determine whether the distribution of architectural proteins is affected by heat shock, we conducted ChIP-seq with antibodies to Su(Hw), CTCF, BEAF-32, TFIIIC, DREF, Rad21, Cap-H2, L(3)mbt, Fs(1)h-L, Mod(mdg4), CP190, Z4, and Chromator in NT and HS cells. The number of sites in the genome containing architectural proteins increases for all proteins except for Cap-H2 and Rad21 (Figure 3A). We next evaluated TAD border strength in relation to architectural protein occupancy. We divided all restriction fragments in the genome into ten groups based on border strength and assigned each group a border strength between 1 and 10. Each group was then subdivided into 12 subgroups based on the number of architectural proteins present (0–2, 3, 4, through 13) and, for each subgroup, we determined the amount of RNAPII present based on the number of normalized ChIP-seq reads. Most TAD borders with the highest strengths contain high amounts of RNAPII and more APBSs highly occupied by architectural proteins, whereas the weakest borders are depleted of RNAPII and contain APBSs with low occupancy (Figure 3B). This relationship is maintained in HS cells (Figure 3C), in which case the border strength is lower for the same number of architectural proteins present at specific borders (Figure 3D).

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Figure 3

Genome-wide redistribution of architectural proteins during heat shock

(A) Number of ChIP-seq peaks for different architectural proteins and RNAPII in control (NT) and heat shocked (HS) cells.

(B) Number of borders found in Hi-C data from control cells for each border strength, which ranges from 1 to 10 in arbitrary units. For each border strength, the number of borders containing different amounts of RNAPII (red to blue) and APBSs occupied with different numbers of architectural proteins (12 columns for each border strength ranging in occupancy from 1 to 12 from left to right). Color bar for RNAPII indicates number of mapped ChIP-seq reads in 50 bp bins normalized relative to the total number of reads.

(C) Number of borders found in Hi-C data from heat shocked cells for each border strength, which ranges from 1 to 10 in arbitrary units. For each border strength, the number of borders containing different amounts of RNAPII (red to blue) and APBSs occupied with different numbers of architectural proteins (12 columns for each border strength ranging in occupancy from 1 to 12 from left to right).

(D) Box plots indicating the relationship between median border strength and the number of architectural proteins present at each border for control (left) and heat shocked (right) cells.

(E) Relationship between the normalized number of sites in the genome and the number of normalized ChIP-seq reads for different architectural proteins at TAD borders and non-borders in control and heat shocked cells.

(F) Change in the number of architectural protein binding sites (APBSs) with different numbers of architectural proteins (APBS occupancy) in heat shocked (HS) with respect to control (NT) cells.

(G) Changes in APBS occupancy with respect to the distance from TAD borders for TAD borders with low (1–4) mid (5–8) and high (9–13) number of architectural proteins in heat shocked cells with respect to control.

We then compared changes in architectural proteins at TAD borders and non-border regions. In all cases, with the exception of Cap-H2, the number of sites containing high amounts of bound architectural proteins increases at non-borders with respect to those at TAD borders after temperature stress (Figure 3E). This reorganization results in a higher number of low and high occupancy APBSs, whereas the number of medium occupancy APBSs is unchanged (Figure 3F). Furthermore, the numbers of low and medium occupancy APBS sites increases at TAD borders after heat shock. However, the number of highly occupied APBSs increases in regions located away from TAD borders (Figure 3G). The decrease in APBS occupancy at TAD borders and its increase inside TADs after heat shock may contribute to the observed decrease in TAD border strength and increase in inter-TAD interactions.

Highly occupied APBSs established after heat shock are enriched at enhancers and promoters

To gain insight into the function of high occupancy APBSs located inside TADs in HS cells, we examined the location of architectural proteins before and after heat shock with respect to enhancers and promoters. We used enhancer sequences identified by two different methods (see supplemental information). The results are very similar for chromatin-based and functionally defined enhancers, so only the results for the latter are shown (Figure 4A). After heat shock, the amount of architectural proteins, with the exception of Cap-H2, bound at enhancers, and the number of enhancer sites bound by architectural proteins, increases. Cap-H2 follows the opposite pattern, decreasing in both the number of enhancers bound and the amount of CAP-H2 bound to enhancers. A similar pattern is observed at gene promoters (Figure 4A), suggesting that the changes in architectural protein distribution observed after heat shock may serve to mediate novel interactions among these sequences.

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Figure 4

Changes in enhancer-promoter interactions in heat shocked cells

(A) Relationship between the number of sites and the ChIP-seq read count for various architectural proteins in control and heat shocked cells at enhancers and promoters.

(B) Changes in the number of interactions among enhancers and promoters in heat shocked (HS) with respect to control (NT) cells.

(C) Number of interactions at different distances for enhancer-promoter contacts in cells grown at normal temperature (NT).

(D) Number of interactions at different distances for enhancer-promoter contacts in heat shocked cells (HS).

(E) Frequency of interactions between enhancers and promoters containing different numbers (1–13) of architectural proteins in cells grown at normal temperature (NT).

(F) Frequency of interactions between enhancers and promoters containing different numbers (1–13) of architectural proteins in cells subjected to temperature stress (HS).

See also Figure S2.

To determine whether increased architectural protein occupancy at enhancers and promoters changes the pattern of their chromatin contacts, we compared the chromatin interaction frequency data from Hi-C in NT and HS samples. Enhancer-promoter, enhancer-enhancer, and promoter-promoter interactions increase dramatically after heat shock, comprising >90% of all the heat shock-specific interactions (Figure 4B). Interactions between enhancers and promoters predominantly occur within relatively short distances (< 300 kb) at normal temperature (Figure 4C), while enhancer-promoter contacts span up to 10 Mb after heat shock (Figure 4D). In agreement with these observations, most enhancer-promoter interactions occur intra-TAD in control cells and inter-TAD after heat shock (Figure S2A). In general, most enhancer-promoter contacts in control cells involve APBSs with an occupancy of 0 to 4 architectural proteins (Figure 4E) and involve sequences located in the same TAD (Figure S2B). However, enhancer-promoter interactions that occur between TADs are mediated by medium occupancy APBSs (Figure S2B). In contrast, heat shock results in an enrichment of APBSs containing 9–13 architectural proteins at sites of enhancer-promoter interactions (Figure 4F) with most interactions occurring between TADs (Figure S2B). These results suggest that redistribution of architectural proteins after heat shock results in the formation of new highly occupied APBSs, which then mediate long-rage inter-TAD enhancer-promoter interactions and contribute to the global 3D chromatin changes observed after temperature stress.

Cohesin and Condensin II have opposite effects on enhancer-promoter interactions

Contrary to other architectural proteins, redistribution of Cap-H2 after heat shock leads to fewer sites and lower levels at enhancers and promoters (Figure 4A). Cap-H2 has been shown to disrupt transvection by antagonizing inter-chromosome contacts required for enhancer-promoter interactions in trans {Bauer, 2012 #270;Hartl, 2008 #276}. Thus, the normal role of Cap-H2 may include the negative regulation of enhancer-promoter contacts in cis. Downregulation of Cap-H2 after heat shock may then be required for disruption of short-range intra-TAD interactions. To determine the contribution of Cap-H2 downregulation to dynamic 3D chromatin remodeling after heat shock, we examined the effect of Cap-H2 depletion on chromatin interactions (Figure S2C). ChIP-seq analysis demonstrates that RNAi treatment results in depletion of Cap-H2 from chromatin (Figure S3A). We then generated Hi-C libraries from Cap-H2 depleted cells using DpnII and obtained a total of 139 million processed read pairs from two biological replicates (Table S1) and we subtracted two dimensional contact matrices of control cells from Cap-H2-depleted cells. Contrary to observations in heat shocked cells, intra-chromosome arm interactions are reduced with respect to control (Figure 5A). However, as is the case in cells subjected to temperature stress, interactions between pericentromeric regions, telomeres and chromosome arms increase, such that chromosomes 2 and 3 appear to acquire a Rabl configuration (Figure 5A and S3B). These data suggest that Cap-H2 downregulation reproduces some aspects of the global changes in chromatin architecture observed after heat shock.

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Figure 5

Effect of Cap-H2 and Rad21 depletion on 3D chromatin organization

(A) Whole genome heat map obtained by subtracting the two dimensional contact matrix of Hi-C data from control cells from that of Cap-H2 depleted cells. Black vertical and horizontal lines indicate chromosome boundaries.

(B) Heat map for a 400 kb region of chromosome 2L obtained by subtracting the two dimensional contact matrix of Hi-C data from control cells from that of Cap-H2 depleted cells.

(C) Scatter plot comparing border strength (BS) in Hi-C samples from control and Cap-H2 depleted cells. Blue dots indicate TAD borders determined using a probability-based model. Black dots indicate all DpnII restriction fragments in the genome. Dashed lines above and below the diagonal indicate + or − one standard deviation, respectively.

(D) Number of interactions among enhancers and promoters specific for normal (WT) and Cap-H2-depleted (CapH2) cells.

(E) Scatter plot comparing border strength (BS) in Hi-C samples from control and Rad21 depleted cells. Symbols are as in panel C.

(F) Number of interactions among enhancers and promoters specific for normal (WT) and Rad21-depleted (Rad21) cells.

(G) Scatter plot comparing border strength in Hi-C samples from control and Rad21 depleted cells subjected to heat shock. Symbols are as in panel C.

(H) Scatter plot comparing border strength in Hi-C samples from normal control and Rad21 depleted cells, both subjected to heat shock. Symbols are as in panel C.

(I) Number of interactions among enhancers and promoters specific for heat shocked (HS) and Rad21-depleted cells subjected to heat shock (RaHS).

See also Figure S3.

At a higher resolution, changes in 3D interactions are similar but less pronounced after Cap-H2 knockdown than after heat shock (Figure 5B and S3C). Intra-TAD interactions decrease after Cap-H2 depletion (Figure 5B), but regions containing Cap-H2 in control cells show an increase of short-range intra-TAD interactions after heat shock (Figure S3D). The redistribution of chromatin interactions after Cap-H2 depletion can be illustrated by changes in border strength, which is slightly reduced, both at TAD borders determined in control cells and at most DpnII fragments in the genome (Figure 5C). Interestingly, interactions among enhancers and promoters increase in Cap-H2 depleted cells when compared to control, suggesting that Cap-H2 antagonizes enhancer-promoter interactions in normal cells (Figure 5D).

The antagonizing effects of Cap-H2 on enhancer-promoter interactions contrast with the function of another architectural protein, Rad21. In mammals, Rad21 interacts with CTCF or Mediator to facilitate interactions between regulatory sequences (DeMare et al., 2013; Dowen et al., 2014; Kagey et al., 2010; Phillips-Cremins et al., 2013). To investigate the possible contribution of Rad21 to heat shock-induced changes in enhancer-promoter interaction, we depleted Rad21 in Kc167 cells using dsRNAs (Figure S2D) and generated Hi-C libraries using DpnII. We obtained a total of 103 million processed read pairs from two biological replicates (Table S1) and compared the chromatin interactions to controls. Downregulation of Rad21 causes a small but detectable reduction in border strength at TAD borders defined in control cells and at most DpnII fragments in the genome (Figure 5E). Contrary to Cap-H2, depletion of Rad21 results in a decrease in enhancer-promoter, enhancer-enhancer and promoter-promoter interactions, in agreement with studies in mammals suggesting that Rad21 facilitates interactions between regulatory regions (Figure 5F).

To further test the role of Rad21 during heat shock, we subjected Rad21-depleted cells to temperature stress and we generated Hi-C libraries. We obtained a total of 83 million processed read pairs (Table S1). Contrary to the effects observed after heat shock alone, Rad21-depleted cells subjected to temperature stress show a slight increase in border strength with respect to cells grown at normal temperature (Figure 5G). This increase in TAD border strength is more dramatic when compared to normal cells subjected to heat shock (Figure 5H). Furthermore, Rad21-depleted cells subjected to heat shock show a decrease in interactions among enhancers and promoters with respect to normal heat shocked cells (Figure 5I). These results suggest that Rad21 plays an important role in mediating changes in enhancer-promoter interactions during the heat shock response.

Changes in chromatin structure in heat shocked cells

It is possible that the widespread silencing of most of the genome in heat shocked cells involves not only changes in the 3D enhancer-promoter interactomes but also alterations in covalent histone modifications. To investigate this possibility we conducted ChIP-seq in control and heat shocked cells with antibodies to H3K4me1, H3K27ac, H3K4me3, H3K27me3, H3K9me2, CBP, and Polycomb (Pc). Consistent with global downregulation of transcription, histone modifications characteristic of actively transcribed genes decrease at enhancers and promoters in heat shocked cells, H3K27me3 increases at these two types of regulatory sequences, and H3K9me2 remains the same (Figure 6A, S4A and S4B). Interestingly, CBP levels do not change at enhancers and promoters after heat shock, in spite of the decrease in H3K27ac, whereas levels of Pc increase dramatically at these sequences (Figure 6A, S4A and S4B). Furthermore, there is an increase in both the number of sites and the amount of protein present at Pc sites in both enhancers and promoters after heat shock (Figure 6B).

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Figure 6

Changes in histone modifications and chromatin proteins in heat shocked cells

(A) Box plots indicating changes in the levels of various histone modifications and chromatin proteins at enhancers and promoters of control (blue) versus heat shocked (red) cells. ** indicates p<0.001

(B) Changes in the number of Pc sites at enhancers and promoters and the read number at each site in control (NT) versus heat shocked (HS) cells.

(C) Scatter plot showing changes in intra- and inter-TAD interactions between DNA fragments containing Pc in control (NT) versus heat shocked (HS) cells.

(D) Changes in the number of interactions and the distance between the interacting Pc-containing DNA fragments in control (NT) versus heat shocked (HS) cells.

See also Figure S4

To further analyze the possible involvement of Pc in the heat shock-induced silencing of most previously active genes, we examined contacts between fragments containing Pc at both ends of the interacting sites. In normal control cells, most contacts between Pc-containing fragments take place within TADs, with a few inter-TAD contacts, whereas most Pc-containing interactions in heat shocked cells occur between fragments located in different TADs (Figure 6C). Furthermore, cells subjected to temperature stress display a dramatic increase in interactions between Pc-containing fragments (Figure 6D). In addition, whereas Pc-containing interactions are short-range in control cells, these interactions take place between fragments located at long distances from each other in cells undergoing heat shock (Figure 6D).

Hi-C, sequencing, mapping and normalization

Hi-C experiments were performed using cells subjected to the various treatments described in the Results. Hi-C libraries of two biological replicates were generated as described (Hou et al., 2012; Lieberman-Aiden et al., 2009). Paired reads were aligned to the dm3 reference genome using Bowtie 0.12.7. GC content, mappability, and fragment length effects were normalized as described (Hou et al., 2012). A summary of the results and the quality control steps is shown in Table S1. Additional details on the computational analyses can be found in the Extended Experimental Procedures.

ChIP-seq and data analysis

Chromatin immunoprecipitation on control or heat shocked Kc167 cells was performed as described with slight modifications (Van Bortle et al., 2012). Antibodies to architectural proteins have been previously described (Van Bortle et al., 2014). ChIPed and input DNA was amplified following standard protocols using indexed primers and sequenced as above. Paired reads were aligned using Bowtie and used for peak calling using MACS (Langmead, 2010; Zhang et al., 2008). Peaks with p values lower than 1×10⁻¹⁰ were used for further analysis.

We thank Drs. John T. Lis for providing antibodies against RNAPII, and Vincent Pirrotta and Richard Jones for providing antibodies against Pc. We would like to thank the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology, and specially Braden Boone, Angela Jones and Terri Pointer, for their help in performing Illumina sequencing of ChIP-Seq samples. This work was supported by U.S. Public Health Service Awards R01 GM035463 to V.G.C., R01 HG005119 to Z.S.Q., R01 GM069462 to G.B, and funds from the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases DK015602 to E.P.L.