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. 2015 Apr 28:6:7048.
doi: 10.1038/ncomms8048.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

Lars Grøntved  1   2 , Joshua J Waterfall  3 , Dong Wook Kim  4 , Songjoon Baek  1 , Myong-Hee Sung  1 , Li Zhao  4 , Jeong Won Park  4 , Ronni Nielsen  2 , Robert L Walker  3 , Yuelin J Zhu  3 , Paul S Meltzer  3 , Gordon L Hager  1 , Sheue-Yann Cheng  4
Affiliations

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

Lars Grøntved et al. Nat Commun. .

Abstract

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

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Conflict of interest statement

Competing financial interests: The authors declare no competing financial interests.

Figures

Figure 1 |
Figure 1 |. Thyroid hormone induces de novo remodelling of chromatin in the liver.
(a) DNase-seq tag density of accessible chromatin in liver of mice treated with PTU or PTU together with T3. Replicate concordant DHS hotpots were identified based on two biological replicates (average tag densities of DHSs from the two biological replicates are plotted). Red and orange data points represent DHS hotspots increased in accessibility as a consequence of T3 treatment, where red marks de novo DHSs. Dark and light green represent DHS hotspots with decreased accessibility after T3 treatment, where light green marks DHS that disappears as a consequence of hormone treatment. Black shows unchanged accessible regions. Differential DHS accessibility where identified at an adjusted P value <0.05. (b) Distribution of DNase-seq tag density at de novo, remodelled and unchanged accessible regions. (c) Representative genome browser shots of DHS categorized as de novo, remodelled and unchanged. (d) Relative distribution of DHS within promoters (−1 kb to +100 bp of TSS), exons, introns and intergenic regions. (e) Upper panel, de novo motif analysis of all DHS with T3-induced DNase accessibility. The most enriched motif is shown. Lower panel, de novo motif analysis of T3-induced DHS lacking the identified canonical DR4-based thyroid hormone response elements (TRE). The three most enriched motifs are shown. (f) Frequency of the DR4 TRE at T3-induced and repressed accessible regions of chromatin. (g) Relative distribution of DR4 TRE in DHS hotspots, where accessibility is T3 regulated.
Figure 2 |
Figure 2 |. Chromatin remodelling correlates with level of nearby gene transcription.
(a) T3-induced and -repressed genes in the liver identified by mRNA expression microarrays of three independent biological repeats. Blue data points represent differentially expressed genes scored by an FDR of 0.3 and a log2 fold change of 1.5 (genes induced by T3) or −1.5 (genes repressed by T3). Two T3-induced genes (Thrsp and Dio1) and one T3-repressed gene (Agxt2l1) are marked by arrows. (b) Level of DNase accessibility near regions of T3-induced genes (Thrsp and Dio1) and a repressed gene (Agxt2l1). Arrows mark DHS accessibility regulated by T3. (c) Heatmap illustrating quantity of T3-regulated genes with at least one T3-regulated DHS within 50 kb of TSS. Genes are sorted from most induced to most repressed by T3. (d) Cumulative fraction of T3-regulated DHS near TSS (0–100 kb) of T3-induced genes. (e) Cumulative fraction of T3-regulated DHS near TSS (0–100 kb) of T3-repressed genes.
Figure 3 |
Figure 3 |. Thyroid hormone-induced chromatin remodelling correlates with hormone-induced TR occupancy.
ChIPs were performed on livers from mice treated with PTU, PTU + T3 and TR dKO. TR-binding sites (TRBS) were identified using HOMER with the TR ChIP-seq in TR dKO as a background control, a FDR threshold of 0.001 and a tag density threshold of 10 tags per peak. (a) The Venn diagram illustrates the overlap of TRBS in livers of PTU (yellow) and PTU plus T3-treated (blue) mice. Preexisting TR binding was defined by presence of TR peaks in absence and presence of T3, whereas hormone-facilitated TR binding was defined by a TR peak unique to T3 treatment. (b) Top panel, de novo motif analysis of TRBS in presence of T3. The most enriched motif is shown. Bottom panel, frequency of identified TRE in preexisting, hormone-facilitated and hormone-depleted TRBS. Broken line represents frequency of the TRE in all DNase-accessible regions of the genome in liver. (c) Distribution of TRBS within DHS regulated by T3 treatment. (d) Heat map illustrating TR ChIP-seq and DNase-seq tag distribution surrounding TRBS within the different categories of unchanged and T3-induced DHS. (e) Frequency of T3-regulated genes with at least one TRBS within 50 kb of TSS. Two hundred TSS from random genes were chosen as background control. (f) Heat map illustrating quantity of T3-activated genes with at least one TRBS (category 1–5) within 50 kb of TSS. Following TRBS categories are quantified:(1) Preexisting TRBS within unchanged DHS. (2) Preexisting TRBS within remodelled TRBS. (3) Hormone-facilitated TRBS within unchanged DHS.(4) Hormone-facilitated DHS within remodelled DHS. (5) Hormone-facilitated TRBS with de novo remodelled DHS. Genes are sorted from most induced and to most repressed by T3. (g) Number of T3-activated genes with a preexisting TRBS (n = 39, 25%), hormone-facilitated TRBS (n = 68, 44%) or a combination of preexisting and hormone-facilitated TRBS (n = 47, 31%) within 50 kb of TSS.
Figure 4 |
Figure 4 |. Recruiment of co-repressors and co-activators to ligand-dependent and ligand-independent TRBS.
(a–d) Examples of T3-activated genes (Idh3, Gpd2, Dio1 and Pdp2) harbouring nearby TRBS with preexisting TR occupancy (black arrows) and hormone-facilitated TR occupancy (red arrows). (e–g) ChIP against NCoR (e), HDAC3 (f) and CBP (g) at TRBS indicated in a–d. ChIPs were performed on livers from mice treated with PTU and PTU + T3. Error bars indicate s.e.m. with n = 4 in each group. *P <0.05. **P <0.01. ***P <0.001 (t-test).
Figure 5 |
Figure 5 |. Absence of DNase protection at DR4 motifs within TRBS.
(a) Top panel, average DNase cut analysis of DR4 motifs enriched at TRBS within TR-bound (blue) and -unbound (green) regions of chromatin. DNase-seq library is from livers isolated from hyperthyroid mice. Average DNase cut count analysis of DR4 motifs at TR-bound (blue) and within genome-wide naked DNA from human IMR90 cells (bottom panel). (b) Average DNase cut count analysis of DR4 motifs within preexisting pre-accessible TRBS under hypo (PTU, blue) or hyperthyroid (T3, green) condition. (c) Average DNase cut count analysis of motifs within C/EBPα, CTCF, HNF1A, HNF6, HNF4 and E2F4-binding sites identified previously in liver. DNase-seq library is from livers isolated from hyperthyroid mice.
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