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. 2003 Apr 1;22(7):1579-87.
doi: 10.1093/emboj/cdg147.

Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor

Marcus Lutz  1 , Les J Burke, Pascal LeFevre, Fiona A Myers, Alan W Thorne, Colyn Crane-Robinson, Constanze Bonifer, Galina N Filippova, Victor Lobanenkov, Rainer Renkawitz
Affiliations

Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor

Marcus Lutz et al. EMBO J. .

Abstract

The highly conserved, ubiquitously expressed, zinc finger protein CTCF is involved in enhancer blocking, a mechanism crucial for shielding genes from illegitimate enhancer effects. Interestingly, CTCF-binding sites are often flanked by thyroid hormone response elements (TREs), as at the chicken lysozyme upstream silencer. Here we identify a similar composite site positioned upstream of the human c-myc gene. For both elements, we demonstrate that thyroid hormone abrogates enhancer blocking. Relief of enhancer blocking occurs even though CTCF remains bound to the lysozyme chromatin. Furthermore, chromatin immunoprecipitation analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF-binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter. Thus, when TREs are adjacent to CTCF-binding sites, thyroid hormone can regulate enhancer blocking, thereby providing a new property for what was previously thought to be constitutive enhancer shielding by CTCF.

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Figures

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Fig. 1. CTCF sites are often found close to binding sites for TR. Five different cases of CTCF/TR composite elements are listed: the human c-myc upstream elements N and TRE(myc-N) (see text), the chicken lysozyme upstream F1 and F2 sequences (Baniahmad et al., 1990), the human 144 CTCF- and TR-binding sites (Bigler and Eisenman, 1995; Awad et al., 1999), the promoter region of the human APP gene (Quitschke et al., 1996; Belandia et al., 1998) and the mouse c-myc site with the CTCF site A and the TRE (Filippova et al., 1996; Perez-Juste et al., 2000). (A) Alignment of the 10 different TRE half sites from the five different CTCF/TR composite elements and their arrangement as direct repeats spaced by 4 or 5 nucleotides (DR4 and DR5), or as inverted palindromes spaced by 3, 6 or 7 nucleotides (IP3, IP6 and IP7) is indicated. (B) Alignment of the five different CTCF-binding sites with their spacing (in bp) from their respective TREs, upstream (left hand side) or downstream (right hand side) of the CTCF binding sequence. (C) Gel retardation with the c-myc upstream composite element N and TRE(myc-N) as probe. TRα, RXRα or CTCF were added as indicated, in the absence or presence of T3. Competitors, DR4 to compete specifically for TR/RXR binding and F1 to compete CTCF binding, were added as indicated.
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Fig. 1. CTCF sites are often found close to binding sites for TR. Five different cases of CTCF/TR composite elements are listed: the human c-myc upstream elements N and TRE(myc-N) (see text), the chicken lysozyme upstream F1 and F2 sequences (Baniahmad et al., 1990), the human 144 CTCF- and TR-binding sites (Bigler and Eisenman, 1995; Awad et al., 1999), the promoter region of the human APP gene (Quitschke et al., 1996; Belandia et al., 1998) and the mouse c-myc site with the CTCF site A and the TRE (Filippova et al., 1996; Perez-Juste et al., 2000). (A) Alignment of the 10 different TRE half sites from the five different CTCF/TR composite elements and their arrangement as direct repeats spaced by 4 or 5 nucleotides (DR4 and DR5), or as inverted palindromes spaced by 3, 6 or 7 nucleotides (IP3, IP6 and IP7) is indicated. (B) Alignment of the five different CTCF-binding sites with their spacing (in bp) from their respective TREs, upstream (left hand side) or downstream (right hand side) of the CTCF binding sequence. (C) Gel retardation with the c-myc upstream composite element N and TRE(myc-N) as probe. TRα, RXRα or CTCF were added as indicated, in the absence or presence of T3. Competitors, DR4 to compete specifically for TR/RXR binding and F1 to compete CTCF binding, were added as indicated.
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Fig. 2. CTCF/TR-mediated enhancer blocking by the chicken lysozyme F1/F2 element is abrogated by thyroid hormone. K562 cells were transfected with the indicated DNA constructs and after neomycin selection in the absence (open bars) or presence of thyroid hormone (filled bars) the number of colonies was determined. (A) The F1/F2 sequences outside of the enhancer (E)/promoter unit in the sense (6xsF1/F2E) or antisense orientation (6xaF1/F2E) do not effect colony numbers. Addition of thyroid hormone causes a general reduction in colony numbers irrespective of the presence of a TR-binding site (F2). (B) Relative enhancer blocking activity (determined by dividing the colony numbers obtained with the control plasmid pNI-MCS by the colony number from the respective DNA constructs) for four constructs. F1/F2 elements placed between the enhancer and promoter in the sense (E6xsF1/F2) or antisense orientation (E6xaF1/F2) mediate enhancer blocking in the absence, but not in the presence of thyroid hormone.
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Fig. 2. CTCF/TR-mediated enhancer blocking by the chicken lysozyme F1/F2 element is abrogated by thyroid hormone. K562 cells were transfected with the indicated DNA constructs and after neomycin selection in the absence (open bars) or presence of thyroid hormone (filled bars) the number of colonies was determined. (A) The F1/F2 sequences outside of the enhancer (E)/promoter unit in the sense (6xsF1/F2E) or antisense orientation (6xaF1/F2E) do not effect colony numbers. Addition of thyroid hormone causes a general reduction in colony numbers irrespective of the presence of a TR-binding site (F2). (B) Relative enhancer blocking activity (determined by dividing the colony numbers obtained with the control plasmid pNI-MCS by the colony number from the respective DNA constructs) for four constructs. F1/F2 elements placed between the enhancer and promoter in the sense (E6xsF1/F2) or antisense orientation (E6xaF1/F2) mediate enhancer blocking in the absence, but not in the presence of thyroid hormone.
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Fig. 3. CTCF/TR-mediated enhancer blocking by the c-myc upstream composite element N and TRE(myc-N) is abrogated by thyroid hormone. Relative enhancer blocking activity in the absence (open bars) or presence of thyroid hormone (filled bars) was determined as in Figure 2. The composite element was tested between the enhancer and the promoter as a single wild-type sequence (Emyc-N/TRE) or as a single mutated sequence with a mutation in the CTCF-binding site (Emyc-Nmut/TRE).
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Fig. 4. CTCF remains bound to the chromatin upstream of the chicken lysozyme gene even after thyroid hormone-induced relief of enhancer blocking. HD37 cells (non-expressing) and HD11 cells (lysozyme expressing) were grown in the absence or presence of thyroid hormone. (A) DMS in vivo footprinting shows protection over the CTCF-binding site under all conditions when compared with the G-ladder (G-DNA) prepared from purified DNA. (B) The scan of the autoradiograph shown in (A) demonstrates that the footprint is not changed upon incubation with T3. (C and D) Chromatin immunoprecipitation with antibodies against CTCF shows CTCF binding on the endogeneous F1 element (–2.38kb) in HD37 cells and HD11 cells. Absence (–T3) or presence of thyroid hormone (+T3) does not change CTCF binding on the –2.38kb sequence. Fold enrichment or fold depletion is plotted relative to non-specific precipitation at CTCF free sites (–3.9 kb, –4.8 kb and –6.1 kb; see Materials and methods).
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Fig. 4. CTCF remains bound to the chromatin upstream of the chicken lysozyme gene even after thyroid hormone-induced relief of enhancer blocking. HD37 cells (non-expressing) and HD11 cells (lysozyme expressing) were grown in the absence or presence of thyroid hormone. (A) DMS in vivo footprinting shows protection over the CTCF-binding site under all conditions when compared with the G-ladder (G-DNA) prepared from purified DNA. (B) The scan of the autoradiograph shown in (A) demonstrates that the footprint is not changed upon incubation with T3. (C and D) Chromatin immunoprecipitation with antibodies against CTCF shows CTCF binding on the endogeneous F1 element (–2.38kb) in HD37 cells and HD11 cells. Absence (–T3) or presence of thyroid hormone (+T3) does not change CTCF binding on the –2.38kb sequence. Fold enrichment or fold depletion is plotted relative to non-specific precipitation at CTCF free sites (–3.9 kb, –4.8 kb and –6.1 kb; see Materials and methods).
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Fig. 4. CTCF remains bound to the chromatin upstream of the chicken lysozyme gene even after thyroid hormone-induced relief of enhancer blocking. HD37 cells (non-expressing) and HD11 cells (lysozyme expressing) were grown in the absence or presence of thyroid hormone. (A) DMS in vivo footprinting shows protection over the CTCF-binding site under all conditions when compared with the G-ladder (G-DNA) prepared from purified DNA. (B) The scan of the autoradiograph shown in (A) demonstrates that the footprint is not changed upon incubation with T3. (C and D) Chromatin immunoprecipitation with antibodies against CTCF shows CTCF binding on the endogeneous F1 element (–2.38kb) in HD37 cells and HD11 cells. Absence (–T3) or presence of thyroid hormone (+T3) does not change CTCF binding on the –2.38kb sequence. Fold enrichment or fold depletion is plotted relative to non-specific precipitation at CTCF free sites (–3.9 kb, –4.8 kb and –6.1 kb; see Materials and methods).
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Fig. 4. CTCF remains bound to the chromatin upstream of the chicken lysozyme gene even after thyroid hormone-induced relief of enhancer blocking. HD37 cells (non-expressing) and HD11 cells (lysozyme expressing) were grown in the absence or presence of thyroid hormone. (A) DMS in vivo footprinting shows protection over the CTCF-binding site under all conditions when compared with the G-ladder (G-DNA) prepared from purified DNA. (B) The scan of the autoradiograph shown in (A) demonstrates that the footprint is not changed upon incubation with T3. (C and D) Chromatin immunoprecipitation with antibodies against CTCF shows CTCF binding on the endogeneous F1 element (–2.38kb) in HD37 cells and HD11 cells. Absence (–T3) or presence of thyroid hormone (+T3) does not change CTCF binding on the –2.38kb sequence. Fold enrichment or fold depletion is plotted relative to non-specific precipitation at CTCF free sites (–3.9 kb, –4.8 kb and –6.1 kb; see Materials and methods).
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Fig. 5. H4 acetylation at the enhancer blocker, the enhancer and the promoter is present only in lysozyme-expressing cells and is further induced by T3 additon. (A) Nucleosomal structure in the upstream lysozyme chromatin (Huber et al., 1996) is indicated by the position of hypersensitive sites for micrococcal nuclease digestion (arrows). Enhancer factor binding sites for PU.1 and C/EBP (Faust et al., 1999) and binding sites for CTCF and TR on the F1/F2 element are indicated. Nucleotide positions for the amplicons 1 to 5 used for the ChIP assay in (B) and (C) are indicated. Amplicon 6 covers the promoter region (–182/–77; data not shown). Chromatin immunoprecipitation of acetylated histone H4 from HD37 cells and HD11 cells are given in (B) and (C), respectively. Fold enrichment or fold depletion is the ratio of the PCR signal from the Ab-precipitated chromatin relative to that from the input chromatin (see Materials and methods). The x-axis shows the amplicon positions relative to the lysozyme upstream region, as indicated at the bottom of the graph.
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Fig. 5. H4 acetylation at the enhancer blocker, the enhancer and the promoter is present only in lysozyme-expressing cells and is further induced by T3 additon. (A) Nucleosomal structure in the upstream lysozyme chromatin (Huber et al., 1996) is indicated by the position of hypersensitive sites for micrococcal nuclease digestion (arrows). Enhancer factor binding sites for PU.1 and C/EBP (Faust et al., 1999) and binding sites for CTCF and TR on the F1/F2 element are indicated. Nucleotide positions for the amplicons 1 to 5 used for the ChIP assay in (B) and (C) are indicated. Amplicon 6 covers the promoter region (–182/–77; data not shown). Chromatin immunoprecipitation of acetylated histone H4 from HD37 cells and HD11 cells are given in (B) and (C), respectively. Fold enrichment or fold depletion is the ratio of the PCR signal from the Ab-precipitated chromatin relative to that from the input chromatin (see Materials and methods). The x-axis shows the amplicon positions relative to the lysozyme upstream region, as indicated at the bottom of the graph.
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Fig. 5. H4 acetylation at the enhancer blocker, the enhancer and the promoter is present only in lysozyme-expressing cells and is further induced by T3 additon. (A) Nucleosomal structure in the upstream lysozyme chromatin (Huber et al., 1996) is indicated by the position of hypersensitive sites for micrococcal nuclease digestion (arrows). Enhancer factor binding sites for PU.1 and C/EBP (Faust et al., 1999) and binding sites for CTCF and TR on the F1/F2 element are indicated. Nucleotide positions for the amplicons 1 to 5 used for the ChIP assay in (B) and (C) are indicated. Amplicon 6 covers the promoter region (–182/–77; data not shown). Chromatin immunoprecipitation of acetylated histone H4 from HD37 cells and HD11 cells are given in (B) and (C), respectively. Fold enrichment or fold depletion is the ratio of the PCR signal from the Ab-precipitated chromatin relative to that from the input chromatin (see Materials and methods). The x-axis shows the amplicon positions relative to the lysozyme upstream region, as indicated at the bottom of the graph.
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