Modular insulators: genome wide search for composite CTCF/thyroid hormone receptor binding sites

Abstract

The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, including insulation and enhancer blocking. We had previously identified two composite elements consisting of a CTCF and a TR binding site at the chicken lysozyme and the human c-myc genes. Using these it has been demonstrated that thyroid hormone mediates the relief of enhancer blocking even though CTCF remains bound to its binding site. Here we wished to determine whether CTCF and TR combined sites are representative of a general feature of the genome, and whether such sites are functional in regulating enhancer blocking. Genome wide analysis revealed that about 18% of the CTCF regions harbored at least one of the four different palindromic or repeated sequence arrangements typical for the binding of TR homodimers or TR/RXR heterodimers. Functional analysis of 10 different composite elements of thyroid hormone responsive genes was performed using episomal constructs. The episomal system allowed recapitulating CTCF mediated enhancer blocking function to be dependent on poly (ADP)-ribose modification and to mediate histone deacetylation. Furthermore, thyroid hormone sensitive enhancer blocking could be shown for one of these new composite elements. Remarkably, not only did the regulation of enhancer blocking require functional TR binding, but also the basal enhancer blocking activity of CTCF was dependent on the binding of the unliganded TR. Thus, a number of composite CTCF/TR binding sites may represent a subset of other modular CTCF composite sites, such as groups of multiple CTCF sites or of CTCF/Oct4, CTCF/Kaiso or CTCF/Yy1 combinations.

Figure 1. Frequency of TREs at CTCF binding regions.

13720 CTCF binding regions from −500 to +500 bp relative to the respective peak centers were scanned with the PSSMs (DR4, DR0, IR4, ER6 and Oct4/Sox2) downloaded from TRANSFAC and Li et al. . (A) Depicted is the number of 1000 bp regions that were marked by one or more of the respective motifs (found). Those results were compared to 3500 sets of 13720 random 1000 bp sequences from the repeat masked human genome (random). Shown is the mean number of sites marked by the respective motif. To control for potential sequence bias in the CTCF bound regions we shuffled the matrices keeping the information content constant. Shuffled matrices were used to scan the 13720 CTCF binding sequences (yellow). Error bars indicate the maximum deviation from the mean. (B) Venn diagram presentation of all TREs identified in CTCF regions, indicating the frequency of single, double and triple TRE occurrences at CTCF regions. (C) The positive control for this procedure shows the highly significant detection of the CTCF consensus (found) in contrast to random sequences (random) as in (A).

Figure 2. Schematic representation of genes with a composite element.

(A) 94 T3 responsive genes conserved in mice and man searched for CTCF composite elements (see main text). (B) Each diagram presents a region of the indicated gene. Putative binding sites for CTCF (CTS), binding sites for TR (IR4, DR4, DR5, ER6 or DR0) and the transcriptional start site (arrow) are shown. Elements being shown to bind TR or CTCF (see Fig. 3) are indicated in green.

Figure 3. In vitro binding assays (EMSA) show direct binding of CTCF and TRβ to predicted target sites.

EMSA experiments were performed using E.coli expressed GST, GST-CTCF-ZF (A) or GST-TR (B) with the indicated radioactively labeled probes. For competition experiments a 50-fold molar excess of non-labeled probes were used. These were either an unrelated CTCF binding site (F1) or an unrelated TR binding site (F2). Arrows mark the CTCF or TR specific shift.

Figure 4. F1F2 and ESRRα composite elements mediate hormone sensitive enhancer blocking on episomes.

(A) Schematic representation of the insulator-enhancer-reporter arrangement of the episomal vectors. The composite insulator sequence (CTCF/TR) is usually integrated as a single insert, or as 4x or 5x multimers as indicated for the constructs used. Cells transfected with the indicated episomes were incubated for 48 h in the absence or presence of T3. (B) Efficient enhancer blocking depends on the presence of the enhancer. The repressive effects cannot be attributed to direct promoter repression. Transfection was in N2aβ cells. (C) TSA and 3-ABA were added 8 h or 28 h after transfection, respectively. Transfection was in N2aβ cells. (D–F) HepG2 cells were additionally cotransfected with 0.2 µg TRβ expression vector. (F) Comparison of the enhancer-less constructs with the enhancer constructs demonstrate that the enhancer blocking activity is dependent on the enhancer.

Figure 5. CTCF and TR synergize in enhancer blocking.

Cells transfected with episomes were incubated for 48 h in the absence or presence of T3. ESRRA  =  abbr. ERR. (A) 293T cells were transfected with the indicated episomes, a TRβ expression vector, and with either the CTCF expression vector (CTCF) or an empty vector (vector). (B) A 293T cell clone containing a stably integrated vector with Tet-inducible expression of shRNA against CTCF was transfected with the indicated reporter episome and the TRβ expression vector. The cells were incubated with tetracycline for 0, 24 or 48 h. (C) Indicated cell lines expressing either a low amount of or no TRβ were transfected with reporter episomes and either empty vector (vector) or TRβ-expression (TRβ). (D) EMSA experiments were performed using E.coli expressed GST, GST-CTCF-ZF and GST-TR and radioactively labeled F1/F2 probe. For competition experiments, non-labeled probes (N) were used in amounts as indicated. (E) Either the mutated F1 (F1mutF2) or the mutated F2 (F1F2mut) were tested in N2a cells in the presence of TR-expression vector.

Figure 6. TR requires more than just the CTCF interaction domain to mediate enhancer blocking.

(A) GST-pulldown experiments were carried out using the indicated E.coli expressed GST-fusion proteins. (B) N2a cells were transfected with the indicated episomes and expression vector for full length TRβ or for TRβ DBD and incubated in the absence or presence of T3.

Figure 7. Modular CTCF binding sites.

Enhancer blocking elements may require multiple CTCF sites for optimal function, as exemplified at the H19 locus or the Xist and Tsix locus , , (top). The composite elements of the lysozyme and ESRRA genes require binding of RXR and/or TR for enhancer blocking (second row). Long range chromatin interaction is not only required for enhancer blocking, but for X chromosomal pairing and HLA locus interaction as well. In addition to CTCF, this involves Yy1 and Oct4 , , (third row) or CIITA at the CTCF site and RFX at the interacting site , (bottom row).