Progesterone receptor-induced bending of its target DNA: distinct effects of the A and B receptor forms

Abstract

We have used circular permutation and phasing electrophoretic mobility shift assays to determine the ability of the A and B forms of human progesterone receptor (PR) to bend target DNA. Studies were done with baculovirus-expressed full-length receptors purified to apparent homogeneity. By circular permutation analysis, both forms of PR induced substantial distortions in the structure of target DNA with calculated distortion angles (alpha D) of 57 degrees for PR-A and 84 degrees for PR-B. The apparent bend centers for both forms of PR were similarly located a few base pairs (-4 to -2 bp) from the middle of the progesterone response element. No differences were detected in the magnitude of distortion or apparent bend centers when PR was bound to hormone agonist (R5020) or the antagonist RU486. Phasing analysis, which can determine the orientation of a DNA bend, revealed that both forms of PR mediated directional bends toward the major groove of the DNA helix. Calculated directed bend angles (alpha B) were 40 degrees for PR-B and 31 degrees for PR-A. The chromatin high mobility group protein HMG-1, which acts as an accessory factor to enhance the binding affinity of purified PR for progesterone response elements, had minimal influence on PR-mediated DNA bending. This result, taken together with the fact that HMG-1 can form a ternary complex with PR and DNA, is consistent with the conclusion that HMG-1 facilitates PR binding by stabilizing a receptor-induced DNA conformation that is required for assembly of a high affinity PR-DNA complex. The results of this study also suggest that DNA bending may be coupled to transcriptional regulation since PR-B is generally a stronger transcriptional activator than PR-A and also mediates a larger bend in target DNA than PR-A.

DNA-looping by RXR Tetramers Permits Transcriptional Regulation “at a Distance”

“…For example, DNA-binding by estrogen receptor a, heterodimers of RXR with the vitamin D receptor, and the progesterone receptor results in distortion angles ranging from 78 to 758. [19][20][21][22] For a 162 bp oligonucleotide that contains an appropriate response element in its center, it can be approximated that a 758 distortion will shorten the distance between the DNA ends from 590 Å to 468 Å . In contrast, tetrameric RXR places the ends of a 162 bp oligonucleotide at a 76 Å distance ( Figure 4).…”

Structural and functional analysis of domains of the progesterone receptor

“…Whether differences in minor groove width reflect intrinsic abilities of different steroid receptors to distort DNA structure is not known. Receptor-induced distortion and directional bending of DNA has been detected for different steroid receptors in solution by circular permutation gel shift assays (Petz et al, 1997; Prendergast et al, 1996; Schultz et al, 2002). An effect on DNA conformation appears to be a general property of steroid receptor-DNA binding that may have a functional role in facilitating the assembly of multi-protein complexes at enhancers or promoters of target genes.…”

Thermodynamic Dissection of Progesterone Receptor Interactions at the Mouse Mammary Tumor Virus Promoter: Monomer Binding and Strong Cooperativity Dominate the Assembly Reaction

“…The exact basis for this phenomenon is unclear, but previous observations suggest that it is due to receptorinduced distortion of the DNA. 15,16,21,22 Quantitation of sites 1-4 ( Fig. 2b, plotted in units of total protein concentration) generated steep individual-site binding isotherms indicative of cooperative interactions among all the sites.…”