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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.
Highlights
The conserved 11 zinc-finger protein can be multimers of identical factors (CTCF) is involved in several transcriptional mechanisms, such as gene activation [1], gene repression [2,3] and enhancer blocking [4,5,6,7,8,9,10,11,12,13]
In order to analyze whether the occurrence of composite CTCF and TR binding sites is a common feature for many or most CTCF binding sites, or whether this is limited to a subset of CTCF sites, we examined the distribution of CTCF and thyroid hormone receptor binding elements in a genome-wide fashion
The latter can be found in different arrangements, such as direct repeats with 4 or 0 nucleotides spacing (DR4 or DR0), inverted repeats spaced by 4 nucleotides (IR4) and everted repeats with a 6 nucleotide spacing (ER6) [21]
Summary
The conserved 11 zinc-finger protein CTCF is involved in several transcriptional mechanisms, such as gene activation [1], gene repression [2,3] and enhancer blocking [4,5,6,7,8,9,10,11,12,13]. This leads to inhibition of DNA-binding of CTCF and thereby to the relief of enhancer blocking [5,6,7,9,14,15,16,17] Another mechanism in the regulation of DNA binding of CTCF is in the context of transcription, when RNA polymerase transcribes through the CTCF binding site. For the composite element consisting of a CTCF and a TR binding site within the chicken lysozyme upstream silencer, CTCF binds to footprint 1 (F1), and TR binds as a homodimer or heterodimer with the retinoid-X-receptor (RXR) to footprint 2 (F2) For this element it has been demonstrated that T3 mediates relief of enhancer blocking and that activation occurs even though CTCF remains bound to its binding site [8]. Nuclear hormone receptor binding sites next to important CTCF sites at the Igf2/H19 locus have been found, but no functional effects with respect to insulation, enhancer blocking or imprinting could be shown [19]
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