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Genome-wide binding patterns of thyroid hormone receptor beta.

Ayers S, Switnicki MP, Angajala A, Lammel J, Arumanayagam AS, Webb P - PLoS ONE (2014)

Bottom Line: In contrast, no significant enrichment of TRβ is seen at negatively regulated genes or genes that respond to unliganded TRs in this system.There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks.We conclude that T3-dependent gene induction commonly involves proximal TRβ binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions.

View Article: PubMed Central - PubMed

Affiliation: The Methodist Hospital Research Institute, Genomic Medicine Program, Houston, Texas, United States of America.

ABSTRACT
Thyroid hormone (TH) receptors (TRs) play central roles in metabolism and are major targets for pharmaceutical intervention. Presently, however, there is limited information about genome wide localizations of TR binding sites. Thus, complexities of TR genomic distribution and links between TRβ binding events and gene regulation are not fully appreciated. Here, we employ a BioChIP approach to capture TR genome-wide binding events in a liver cell line (HepG2). Like other NRs, TRβ appears widely distributed throughout the genome. Nevertheless, there is striking enrichment of TRβ binding sites immediately 5' and 3' of transcribed genes and TRβ can be detected near 50% of T3 induced genes. In contrast, no significant enrichment of TRβ is seen at negatively regulated genes or genes that respond to unliganded TRs in this system. Canonical TRE half-sites are present in more than 90% of TRβ peaks and classical TREs are also greatly enriched, but individual TRE organization appears highly variable with diverse half-site orientation and spacing. There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks. We conclude that T3-dependent gene induction commonly involves proximal TRβ binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions. Better understanding of genomic context of TR binding sites will help us determine why TR regulates genes in different ways and determine possibilities for selective modulation of TR action.

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Characterization of TRβ binding near induced genes.A. Patterns of TRβ binding depicted at representations of individual target gene loci (LDLR, BCL3, NCOR2,ADSSL1 and SOX7). Blue bars represent genomic binding regions, and the vertical red lines represent peaks, as classified by QuEST. The horizontal black bars are regions analyzed by ChIP-PCR (locations of primer amplification). Observed binding patterns included 5′, 3′ and intronic binding events, as shown in genomic data tracks (UCSC Genome Browser). Putative regulatory elements, identified through sequence analysis of the genomic regions indicated, are depicted below bound regions in which they occur. B. QPCR of ChIP analysis confirming DNA binding in regulatory regions of genes. C. Realtime PCR analysis depicting enhancement of transcription of individual loci in Fig. 4A by T3 in the presence of TRβ. (*P<0.05 by Student's T-Test).
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pone-0081186-g004: Characterization of TRβ binding near induced genes.A. Patterns of TRβ binding depicted at representations of individual target gene loci (LDLR, BCL3, NCOR2,ADSSL1 and SOX7). Blue bars represent genomic binding regions, and the vertical red lines represent peaks, as classified by QuEST. The horizontal black bars are regions analyzed by ChIP-PCR (locations of primer amplification). Observed binding patterns included 5′, 3′ and intronic binding events, as shown in genomic data tracks (UCSC Genome Browser). Putative regulatory elements, identified through sequence analysis of the genomic regions indicated, are depicted below bound regions in which they occur. B. QPCR of ChIP analysis confirming DNA binding in regulatory regions of genes. C. Realtime PCR analysis depicting enhancement of transcription of individual loci in Fig. 4A by T3 in the presence of TRβ. (*P<0.05 by Student's T-Test).

Mentions: Closer analysis of patterns of TRβ binding near selected genes revealed clusters of TRβ binding sites in diverse distributions. In Figure 4, TRβ binding events are represented by blue bars defining the extent of sequences precipitated by the BioChIP experiment and red bars representing TRβ peak positions defined by QuEST. We observed TRβ binding near both verified human TRβ target genes LDLR and BCL3 (Fig. S1C) and this mapped to the 5′ region of the transcription unit for LDL-R and to the 5′ and 3′ regions for BCL3 (Fig. 4A). Similar “5′ only” and 5′+3′ distributions were also seen for other genes (not shown and see Fig. S4). Interestingly, for both genes, TRβ peaks detected by BioChIP were close to known TREs in the proximal promoter region [55], [56]). We also observed other types of TRβ binding site distributions near highly induced genes; these included intronic (NCOR2 and ADSSL1) and 3′ only (SOX7). We confirmed TRβ binding at predicted sites by conventional ChIP with an antibody specific for human TRβ (Fig. 4B, amplified regions are represented by black bars in Fig. 4A) and verified that there was increased T3-dependent gene induction versus control HepG2 cells with qPCR (Fig. 4C). Thus, TRβ binds at multiple locations in varied patterns near strongly T3 induced genes and this correlates with enhancement of T3 response.


Genome-wide binding patterns of thyroid hormone receptor beta.

Ayers S, Switnicki MP, Angajala A, Lammel J, Arumanayagam AS, Webb P - PLoS ONE (2014)

Characterization of TRβ binding near induced genes.A. Patterns of TRβ binding depicted at representations of individual target gene loci (LDLR, BCL3, NCOR2,ADSSL1 and SOX7). Blue bars represent genomic binding regions, and the vertical red lines represent peaks, as classified by QuEST. The horizontal black bars are regions analyzed by ChIP-PCR (locations of primer amplification). Observed binding patterns included 5′, 3′ and intronic binding events, as shown in genomic data tracks (UCSC Genome Browser). Putative regulatory elements, identified through sequence analysis of the genomic regions indicated, are depicted below bound regions in which they occur. B. QPCR of ChIP analysis confirming DNA binding in regulatory regions of genes. C. Realtime PCR analysis depicting enhancement of transcription of individual loci in Fig. 4A by T3 in the presence of TRβ. (*P<0.05 by Student's T-Test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928038&req=5

pone-0081186-g004: Characterization of TRβ binding near induced genes.A. Patterns of TRβ binding depicted at representations of individual target gene loci (LDLR, BCL3, NCOR2,ADSSL1 and SOX7). Blue bars represent genomic binding regions, and the vertical red lines represent peaks, as classified by QuEST. The horizontal black bars are regions analyzed by ChIP-PCR (locations of primer amplification). Observed binding patterns included 5′, 3′ and intronic binding events, as shown in genomic data tracks (UCSC Genome Browser). Putative regulatory elements, identified through sequence analysis of the genomic regions indicated, are depicted below bound regions in which they occur. B. QPCR of ChIP analysis confirming DNA binding in regulatory regions of genes. C. Realtime PCR analysis depicting enhancement of transcription of individual loci in Fig. 4A by T3 in the presence of TRβ. (*P<0.05 by Student's T-Test).
Mentions: Closer analysis of patterns of TRβ binding near selected genes revealed clusters of TRβ binding sites in diverse distributions. In Figure 4, TRβ binding events are represented by blue bars defining the extent of sequences precipitated by the BioChIP experiment and red bars representing TRβ peak positions defined by QuEST. We observed TRβ binding near both verified human TRβ target genes LDLR and BCL3 (Fig. S1C) and this mapped to the 5′ region of the transcription unit for LDL-R and to the 5′ and 3′ regions for BCL3 (Fig. 4A). Similar “5′ only” and 5′+3′ distributions were also seen for other genes (not shown and see Fig. S4). Interestingly, for both genes, TRβ peaks detected by BioChIP were close to known TREs in the proximal promoter region [55], [56]). We also observed other types of TRβ binding site distributions near highly induced genes; these included intronic (NCOR2 and ADSSL1) and 3′ only (SOX7). We confirmed TRβ binding at predicted sites by conventional ChIP with an antibody specific for human TRβ (Fig. 4B, amplified regions are represented by black bars in Fig. 4A) and verified that there was increased T3-dependent gene induction versus control HepG2 cells with qPCR (Fig. 4C). Thus, TRβ binds at multiple locations in varied patterns near strongly T3 induced genes and this correlates with enhancement of T3 response.

Bottom Line: In contrast, no significant enrichment of TRβ is seen at negatively regulated genes or genes that respond to unliganded TRs in this system.There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks.We conclude that T3-dependent gene induction commonly involves proximal TRβ binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions.

View Article: PubMed Central - PubMed

Affiliation: The Methodist Hospital Research Institute, Genomic Medicine Program, Houston, Texas, United States of America.

ABSTRACT
Thyroid hormone (TH) receptors (TRs) play central roles in metabolism and are major targets for pharmaceutical intervention. Presently, however, there is limited information about genome wide localizations of TR binding sites. Thus, complexities of TR genomic distribution and links between TRβ binding events and gene regulation are not fully appreciated. Here, we employ a BioChIP approach to capture TR genome-wide binding events in a liver cell line (HepG2). Like other NRs, TRβ appears widely distributed throughout the genome. Nevertheless, there is striking enrichment of TRβ binding sites immediately 5' and 3' of transcribed genes and TRβ can be detected near 50% of T3 induced genes. In contrast, no significant enrichment of TRβ is seen at negatively regulated genes or genes that respond to unliganded TRs in this system. Canonical TRE half-sites are present in more than 90% of TRβ peaks and classical TREs are also greatly enriched, but individual TRE organization appears highly variable with diverse half-site orientation and spacing. There is also significant enrichment of binding sites for TR associated transcription factors, including AP-1 and CTCF, near TR peaks. We conclude that T3-dependent gene induction commonly involves proximal TRβ binding events but that far-distant binding events are needed for T3 induction of some genes and that distinct, indirect, mechanisms are often at play in negative regulation and unliganded TR actions. Better understanding of genomic context of TR binding sites will help us determine why TR regulates genes in different ways and determine possibilities for selective modulation of TR action.

Show MeSH
Related in: MedlinePlus