Fig 1.
Identification of T3-regulated hyperacetylated regions in the liver.
(A) Example of increased H3K9 and H3K27 acetylation at DHSs near the Thrsp gene. TR binding sites (TRBSs) identified from TR ChIP-seq data are indicated as black bars. The identified hyperacetylated region is displayed above density graphs of sequenced tags. (B) 1592 regions hyperacetylated at both H3K9 and H3K27 constitute high confidence hyperacetylated regions in hyperthyroid condition. (C) De novo motif analysis of 3778 DHSs within hyperacetylated regions. The most enriched motif is shown. (D) Relative amount of hyperacetylated regions with at least one TRBS based on TR ChIP-seq data. (E) T3-induced H3K27Ac in mice expressing TR-PV mutant lacking a functional activating helix 12. Statistical difference was determined by a Wilcoxon Signed Rank Test, ***p<0.001.
Fig 2.
Configuration of hyperacetylated enhancers in SEs and TADs.
Hyperacetylated regions were divided into regions with TRBS (w/TRBS) and without TRBS (no/TRBS). (A) Size distribution of SE in hyperthyroid condition based on H3K27Ac. (B) Fraction of SE containing T3-responsive hyperacetylated enhancers with TRBS and without TRBS. (C) Fraction of SEs containing a hyperacetylated region without a TRBS together with regions containing a TRBS. ***Fisher’s exact test p< 0.001 using 600 random selected histone acetylated regions. (D) Fraction of hyperacetylated regions without a TRBS that co-occurs in SE with hyperacetylated regions with a TRBS. ***Fisher’s exact test p< 0.001 using 600 random selected histone acetylated regions. (E) Acute H3K27Ac of regions without a TRBS in a SE with and without hyperacetylated regions containing a TRBS. Statistical difference was determined by a Wilcoxon Signed Rank Test, *p<0.05, ***p<0.001. Non significant is marked by n.s. (F) Example of a SE near the T3-regulated Proca1 gene. (G) Distance between hyperacetylated regions w/TRBS and no/TRBS. The median [M] distance is indicated for no/TRBS to w/TRBS containing regions and no/TRBS to random regions. (H) Fraction of hyperacetylated regions without TRBSs within TADs containing a TRBS. ***Fisher’s exact test p< 0.001 using 600 random selected histone acetylated regions. (I) Example of HiC contacts in a TAD harbouring the Thrsp gene. (J) HiC tag count of all detected interactions between hyperacetylated regions with and without TRBS. Interaction between regions in the opposite direction was quantified as a background control. Statistical difference was determined by a Wilcoxon Signed Rank Test (n = 56), ***p<0.001. (K) Frequency of hyperacetylated regions contacting other hyperacetylated regions. Fisher’s exact test ***p<0.001, **p< 0.01, *p<0.05 using 365 random selected histone acetylated regions. (L) Acute H3K27Ac of regions without a TRBS interacting with regions with a TRBS or random selected regions. Statistical difference was determined by a Wilcoxon Signed Rank Test, *p<0.05, ***p<0.001. Non-significant is marked by n.s.
Fig 3.
Occupancy of transcriptional co-regulators at TRBSs within T3 hyperacetylated regions.
(A) Enrichment of HDAC3 occupancy within hyperacetylated regions compared to random selected H3 acetylated regions. Statistical test was performed by a Wilcoxon Signed Rank Test, ***p<0.001. (B) Fraction of hyperacetylated regions containing a TRBS (w/TRBS) occupied by HDAC3. (C) Heatmap illustrating H3K27Ac, H3K9Ac, co-regulator and TR occupancy at TRBSs within hyperacetylated regions (n = 720). TRBSs are sorted according to HDAC3 occupancy in hypothyroid condition. Type 1A TRBSs, poised enhancers, are defined by high occupancy of HDAC3, CBP and H3K4me1. Type 1B TRBSs, T3-established enhancers, are defined by low occupancy of HDAC3, CBP and H3K4me1 in absence of T3. (D) ChIP-seq tag density of histone modifications, DHS, TR and co-regulators at TRBSs associated with poised (type 1A) and T3-established enhancers (type 1B). Statistical difference was determined by a Wilcoxon Signed Rank Test. (E) ChIP-qPCR for four type 1A TRBSs (1–4) and four type 1B TRBSs (5–8). Genomic coordinates can be found in S2 Table. Statistical difference was determined by Student’s t-test (n = 4), *p<0.05, **p<0.01 and ***p<0.001. (F) Example of co-regulator occupancy of hyperacetylated regions near T3-regulated genes Dio1 and Pdp2.
Fig 4.
Histone acetylation of TR occupied regions due to disrupted interaction between TR and NCoR.
(A) HDAC3 occupancy of chromatin in WT mice and mice expressing a mutated NCOR1 (NCOR1ΔID) unable to interact with TR. TRBSs occupied by HDAC3 are marked with grey. (B) Differential H3K9Ac of hyperacetylated regions in WT hypothyroid mice compared to hypothyroid NCOR1ΔID mice. (C) Differential H3K27Ac of hyperacetylated regions in WT hypothyroid mice compared to hypothyroid NCOR1ΔID mice. (D) Genomic overlap of hyperacetylated regions occupied by TR (w/TRBS) and low or high occupancy of NCOR1-HDAC3. (E) Fraction of hyperacetylated regions with increased H3K9Ac and H3K27Ac in livers from hypothyroid L-NCOR1ΔID mice compared to hypothyroid WT mice. ***Fisher’s exact test p< 0.001. (F) Fold change of H3K9/27Ac at hyperacetylated regions occupied by TR and associated with high and low occupancy of NCOR1-HDAC3 in livers from hypothyroid L-NCOR1ΔID mice compared to hypothyroid WT mice. Statistical difference was determined by a Wilcoxon Signed Rank Test, ***p<0.001. (G) Fold change of H3K27Ac at hyperacetylated regions occupied by TR and associated with high and low occupancy of NCOR1-HDAC3 in livers from hypothyroid NCOR1 KO mice compared to hypothyroid WT mice. Statistical difference was determined by a Wilcoxon Signed Rank Test, ***p<0.001. (H) Examples of hyperacetylated regions occupied by type 1A TRBS (left, chr16:21,817,000–21,842,000) and type 1B TRBS (right, chr2:167,527,000–167,558,000).
Fig 5.
Expression of genes associated with hyperacetylated regions with and without occupancy of HDAC3.
(A) Differentially expressed genes in hyperthyroid condition. Genes associated with hyperacetylated regions are marked by red. Genes are scored positive if one or more hyperacetylated regions are within 100kb of the transcriptional start site of the gene. The number of induced and repressed genes at FDR<0.01 and log2FC of one are indicated. (B) Fraction of T3-induced genes with nearby hyperacetylated regions with and without TRBSs. (C) Expression of genes associated with hyperacetylated regions as sequenced reads per kilo base per million total reads (RPKM). Statistical difference was determined by a Wilcoxon Signed Rank Test, ***p<0.001. (D) Differential expression of T3-induced genes associated with hyperacetylated regions in hypothyroid WT mice compared to hypothyroid L-NCOR1ΔID mice. Red marks induced genes in L-NCOR1ΔID mice at FDR<0.01 and log2FC>0. (E) Number of T3-induced genes associated with hyperacetylated type 1A (brown) or type 1B (orange) TRBSs. (F) Differential expression of T3-induced genes associated with type 1A TRBSs in hypothyroid WT mice compared to hypothyroid L-NCOR1ΔID mice. Brown marks induced genes in L-NCOR1ΔID mice at FDR<0.01 and log2FC>0. (G) Differential expression of T3-induced genes associated with hyperacetylated regions occupied by type 1B TRBSs in hypothyroid WT compared to hypothyroid L-NCOR1ΔID mice. Yellow marks induced genes in L-NCOR1ΔID mice at FDR<0.01 and log2FC> 0. (H) Enrichment of genes induced in hypothyroid L-NCOR1ΔID mice relative to random selected genes. Genes are binned according to association with hyperacetylated type 1A and/or type 1B TRBSs. Ten different sets of 1000 randomly selected genes from all annotated genes were used as control. Statistical difference is calculated using a Student’s t-test (n = 10), **p<0.01.
Fig 6.
Models describing T3-dependent H3 hyperacetylation and activation of enhancers.
(A) Poised enhancers (type 1A) are hyperacetylated as a result of HDAC3 disassociation from the TRBS. As HDAC3 occupancy is reduced, histone acetylation increases as a result of pre-occupied HATs. In addition, MED1 is recruited to the enhancers in a T3-dependent manner. (B) Dormant enhancers (type 1B) are hyperacetylated as a result of T3-dependent TR and HAT recruitment to chromatin. Activation of these enhancers is associated with increased chromatin accessibility, H3K4me1 and MED1 recruitment. (C) A subset of T3-activated enhancers is not occupied by TR yet activated in a TR-dependent manner. These enhancers are connected to TR occupied enhancers either though long-range interaction within TADs or local interaction within SEs. The TRE illustrates the TR response element, which predominantly consists of DR4 motifs.