Specific histone lysine 4 methylation patterns define TR-binding capacity and differentiate direct T3 responses

Abstract

The diversity of thyroid hormone T(3) effects in vivo makes their molecular analysis particularly challenging. Indeed, the current model of the action of T(3) and its receptors on transcription does not reflect this diversity. Here, T(3)-dependent amphibian metamorphosis was exploited to investigate, in an in vivo developmental context, how T(3) directly regulates gene expression. Two, direct positively regulated T(3)-response genes encoding transcription factors were analyzed: thyroid hormone receptor β (TRβ) and TH/bZIP. Reverse transcription-real-time quantitative PCR analysis on Xenopus tropicalis tadpole brain and tail fin showed differences in expression levels in premetamorphic tadpoles (lower for TH/bZIP than for TRβ) and differences in induction after T(3) treatment (lower for TRβ than for TH/bZIP). To dissect the mechanisms underlying these differences, chromatin immunoprecipitation was used. T(3) differentially induced RNA polymerase II and histone tail acetylation as a function of transcriptional level. Gene-specific patterns of TR binding were found on the different T(3) -responsive elements (higher for TRβ than for TH/bZIP), correlated with gene-specific modifications of H3K4 methylation (higher for TRβ than for TH/bZIP). Moreover, tissue-specific modifications of H3K27 were found (lower in brain than in tail fin). This first in vivo analysis of the association of histone modifications and TR binding/gene activation during vertebrate development for any nuclear receptor indicate that chromatin context of thyroid-responsive elements loci controls the capacity to bind TR through variations in histone H3K4 methylation, and that the histone code, notably H3, contributes to the fine tuning of gene expression that underlies complex physiological T(3) responses.

Fig. 1.

T₃ induces TRβ and TH/bZIP transcription. Tadpoles were treated for 48 h with 10 nm T₃. Total RNA was extracted from brain and tail fin and used for RT and real-time qPCR analysis of TRβ and TH/bZIP (a basic leucine-zipper TH-response gene) expression. Gene expression was normalized against rpl8 RNA. The data plotted are the log of 2^−ΔCT value where ΔCT is the difference between the control gene and the gene of interest. The results represents the mean and sem of three or six independent experiments. Statistical significance is indicated as not significant (ns); **, P < 0.01; or ***, P < 0.001.

Fig. 2.

Effects of T₃ on DNA binding by TR at T₃-response genes. Chromatin isolated from brain or tail fin of T₃-treated X. tropicalis tadpoles (10 nm T₃ for 48 h) was immunoprecipitated, and the products were analyzed by qPCR for the presence of T₃RE or an upstream control region (CZ) as schematically represented in panel A for the TRβ gene and in panel B for the TH/bZIP gene. The distance between the T₃RE area and the CZ area, the position of the primers used for qPCR, and the position of the TSS are also indicated. Sequences are not drawn to scale. C, T₃ increases TR binding to TRβ and TH/bZIP T₃RE in tadpole brain. Chromatin was immunoprecipitated with antibodies against TR (ChIPαTR). D, T₃ induces TR binding to TRβ and TH/bZIP T₃RE in tadpole tail fin. Chromatin was immunoprecipitated with antibodies against TR (ChIPαTR). E, TRβ overexpression and TR recruitment on TRβ and TH/bZIP T₃RE in brain. Chromatin isolated from brain of T₃-treated X. laevis transgenic tadpoles overexpressing GFP fused to TRβ in neurons (10 nm T₃ for 48 h) was immunoprecipitated with antibodies against GFP (ChIPαGFP). The product was analyzed by qPCR for the presence of the conserved between X. tropicalis and X. laevis T₃RE containing region (T₃RE) of TRβ and TH/bZIP gene. TH/bZIP coding locus was used as negative control (Exon1 TH/bZIP). The mean values and sem of four (panels C and D) or three (panel E) independent experiments are expressed as percent of input. Statistical significance as compared with untreated animals is indicated as not significant (ns); *, P < 0.05; **, P < 0.01; or ***, P < 0.001.

Fig. 3.

Effects of T₃ on RNA PolII recruitment and Me3H3K36 occupancy at T₃-response genes. Chromatin isolated from tail fins of T₃-treated X. tropicalis tadpoles (10 nm T₃ for 48 h) was immunoprecipitated, and the product was analyzed by qPCR for the presence of the TSS-containing region, which is also the T₃RE-containing region, an upstream control region (CZ), or a coding region (TZ) as schematically represented in panel A for the TRβ gene and in panel B for the TH/bZIP gene. The distance between each areas and the position of the primers used for qPCR are also indicated. Exonic sequences are boxed and intronic or upstream sequences are indicated with solid line. Sequences are not drawn to scale. C, T₃ increases RNA PolII recruitment to TRβ TSS. Chromatin was immunoprecipitated with antibodies against RNA PolII (ChIPαRNA PolII). D, T₃ induces RNA PolII recruitment to TH/bZIP promoter. ChIP was done as described in panel C, but the product was analyzed with primers for TH/bZIP genomic regions. E, T₃ increases Me3H3K36 deposition at the TRβ transcribed genomic locus. ChIP was done using antibodies against Me3H3K36 (ChIPαMe3H3K36) and primers on TRβ genomic locus. F, T₃ induces H3K36 trimethylation at TH/bZIP. ChIP was done using antibodies against Me3H3K36 (ChIPαMe3H3K36) and primers for TH/bZIP genomic locus. The average values and sem of four independent experiments are expressed as percent of input. Statistical significance as compared with untreated animals is indicated as not significant (ns); *, P < 0.05; or **, P < 0.01.

Fig. 4.

T₃ treatment effects on histone H3 and H4 acetylation at T₃-response genes. Chromatin isolated from brains or tail fins of T₃-treated tadpoles (10 nm T₃ for 48 h) was immunoprecipitated with antibodies against pan-acetylated histone H4 (AcH4), acetylated histone H3 (AcH3) at lysine 9 (K9) or lysine 18 (K18). ChIP products were analyzed by qPCR for the presence of T₃RE-containing region and CZ region as presented in Fig. 2A for TRβ gene and Fig. 2B for TH/bZIP gene. A, T₃ increases pan-AcH4 to T₃RE in brain. Chromatin was immunoprecipitated with antibodies against pan-AcH4 (ChIPαAcH4). B, T₃ increases pan-AcH4 to T₃RE in tail fin. ChIP was done with antibodies against pan-AcH4 (ChIPαAcH4). C, T₃ does not affect H3K9 acetylation at T₃RE in brain. ChIP was done using antibodies against AcH3K9 (ChIPαAcH3K9). D, T₃ increases H3K9 acetylation on TH/bZIP T₃RE but has no effect on TRβ T₃RE in tail fin. Antibodies against AcH3K9 (ChIPαAcH3K9) were used. E, T₃ increases H3K18 acetylation at T₃RE in brain. ChIP was done using antibodies against AcH3K18 (ChIPαAcH3K18). F, T₃ increases T₃RE H3K18 acetylation in tail fin. Chromatin was immunoprecipitated with antibodies against AcH3K18 (ChIPαAcH3K18). The average values and sem of four independent experiments are expressed as percent of input. Statistical significance as compared with untreated animals is indicated as not significant (ns); *, P < 0.05; **, P < 0.01; or ***, P < 0.001. CZ at least 2000 bp upstream T₃RE area.

Fig. 5.

T₃ treatment effects on histone H3 methylation at T₃-response genes. Chromatin isolated from brains or tail fins of T₃-treated tadpoles (10 nm T₃ for 48 h) was immunoprecipitated with antibodies against dimethyl histone H3 at lysine 4 (Me2H3K4), trimethyl histone H3 at lysine 4 (Me3H3K4), or trimethyl histone H3 at lysine 27 (Me3H3K27). ChIP products were analyzed by qPCR for the presence of T₃RE-containing region and CZ region as presented in Fig. 2A for TRβ gene and Fig. 2B for TH/bZIP gene. A, In brain, T₃ decreases Me2H3K4 at TRβ T₃RE and increases Me2H3K4 at TH/bZIP T₃RE. ChIP was done using antibodies against Me2H3K4 (ChIPαMe2H3K4). B, As observed in brain, T₃ decreases Me2H3K4 at TRβ T₃RE and increases Me2H3K4 at TH/bZIP T₃RE in tail fin. ChIP was done using antibodies against Me2H3K4. C, In brain, Me3H3K4 is present on TRβ T₃RE and not on TH/bZIP T₃RE with no T₃ effect. ChIP was done using antibodies against Me3H3K4 (ChIPαMe3H3K4). D, In tail fin, Me3H3K4 is present on TRβ T₃RE and slightly detected on TH/bZIP T₃RE with no T₃ effect. ChIP used antibodies against Me3H3K4. E, In brain, T₃ decreases Me3H3K27 at TRβ T₃RE but not on TH/bZIP T₃RE. Chromatin was immunoprecipitated with antibodies against Me3H3K27 (ChIPαMe3H3K27). F, T₃ strongly decreases Me3H3K27 occupancy at T₃RE. ChIP was done using antibodies against Me3H3K27. The average values and sem of four independent experiments are expressed as percent of input. Statistical significance as compared with untreated animals is indicated as not significant (ns); *, P < 0.05; or **, P < 0.01; or ***, P < 0.001. CZ at least 2000 bp upstream from T₃RE area.

Fig. 6.

Pargylin, an inhibitor of HDM, increases T₃-response gene expression and increases TR binding to T₃RE. The tail tips of stage NF 52–53 tadpoles were isolated and placed in cultures dishes. After 48 h with or without 10 nm T₃ and/or 100 nm pargylin (P) for 48 h, tail fins were isolated for RNA extraction or ChIP studies. A, Pargylin treatment increases T₃-response genes expression. The RNA were analyzed by RT-qPCR for the presence of TRβ and TH/bZIP expression. Gene expressions were normalized against the value for rpl8 RNA. The average values and sem of three independent experiments are expressed as multiples of induction, where 1 is equal to expression in the absence of T₃ or pargylin treatment (Ct). B, Effect of T₃ and pargylin treatment on Me2H3K4 occupancy at TRβ locus. After 2 d of culture, tail fins were isolated from tail explants for chromatin extraction. The presence of Me2H3K4 on T₃RE located near the TSS and 2000 bp upstream the TSS of TRβ were analyzed by ChIP (ChIPαMe2H3K4). The average values and sem of at least four independent experiments are expressed as percent of input. C, T₃ and pargylin effect on TH/bZIP T₃RE H3K4 dimethylation. ChIP was done using antibodies against Me2H3K4 and primers around TH/bZIP T₃RE and 3000 bp upstream from its TSS. D, T₃ and pargylin increases AcH4 at TRβ T₃RE. ChIP was done using antibodies against pan-AcH4 (ChIPαACH4) and primers on TRβ T₃RE region and 2000 bp upstream from its TSS. E, T₃ and pargylin effect on AcH4 occupancy at TH/bZIP T₃RE. ChIP was done using antibodies against pan-AcH4 and primers around TH/bZIP T₃RE and 3000 bp upstream from the TSS. F, Pargylin increases TR recruitment at TRβ T₃RE. ChIP was done as using antibodies against TR (ChIPαTR) and primers on TRβ T₃RE region. G, Pargylin treatment increases TR binding at TH/bZIP T₃RE. ChIP was done using antibodies against TR and primers around TH/bZIP T₃RE. Statistical significance as compared with untreated animals is indicated as *, P < 0.05; **, P < 0.01; or ***, P < 0.001. P, Pargylin; Ct, Control tadpoles corresponding to untreated animal.