Abstract
Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is followed by a weaker one with degenerated sequence.
Highlights
The binding of DNA by nuclear receptors (NRs) is essential for the proper transcriptional regulation of the expression of target genes involved in crucial physiological and metabolic pathways [1,2,3]
Estrogen-related receptor binds to inverted repeat 3 (IR3) response elements (REs) encompassing two 6 bps DNA-binding sites separated by 3 bps, as well as to 9 bps extended half-site REs composed of a single specific DNA-binding site of the type TNAAGGTCA (ERRE), the latter REs representing most of the natural REs found in target gene promoters
When considering a composite element made of an extended half-site estrogenrelated response element (ERREs) embedded into an IR3 RE, we observed a delayed migration of the complex into the gel (Figure 1C, lanes 3, 5)
Summary
The binding of DNA by nuclear receptors (NRs) is essential for the proper transcriptional regulation of the expression of target genes involved in crucial physiological and metabolic pathways [1,2,3]. Specific DNA recognition encompasses several levels of complexity and involves at first place a direct readout mechanism, where the NRs bind specific genomic sequences, known as response elements (REs) and make sequence-specific contacts with the major groove by the formation of base- and amino acid-specific interactions [4,5,6,7]. The ERRs can bind in vitro with high affinity to the inverted repeat 3 (IR3) REs, which are bound by the ERs and are composed of two hexanucleotide half-sites of consensus sequence AGGTCA organized as inverted repeats separated by a 3-bp spacer [15, 16]. The ERRs represent major transcriptional regulators of energy metabolism in response to physiological and/or environmental challenges [17,18,19,20,21,22,23]
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