High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression.

High mobility group protein 1 (HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N' and N'') remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed (1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (2) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome.

Nucleosome dynamics: HMGB1 relaxes canonical nucleosome structure to facilitate estrogen receptor binding.

High mobility group protein 1 (HMGB1) interacts with DNA and chromatin to influence the regulation of transcription, DNA repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, ATP-independent manner. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N' and N″) remain stable and exhibit characteristics distinctly different from the canonical nucleosome. These findings complement previous studies that showed (i) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (ii) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that one aspect of the mechanism of HMGB1 action involves a restructuring of the nucleosome that appears to relax structural constraints within the nucleosome.

High mobility group B proteins facilitate strong estrogen receptor binding to classical and half-site estrogen response elements and relax binding selectivity.

The estrogen receptor alpha (ER) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes. A key step in the activation process is the initial binding of the ER dimer to the estrogen response element (ERE). We examined the effect of the coactivator proteins, HMGB1 and HMGB2, in enhancing ER binding affinity to single and tandem EREs. Using EMSAs, both HMGB proteins are shown to enhance ER binding and induce cooperative ER binding on tandem ERE elements. We demonstrate that HMGB proteins facilitate strong ER binding to ERE consensus half-sites, exhibiting binding affinities comparable with ER binding to consensus ERE in the absence of HMGB proteins. These findings reveal that although HMGB proteins enhance binding affinity, they also relax ER binding specificity. Deoxyribonuclease I footprinting demonstrates that ER binds very differently to consensus ERE and ERE consensus half-sites, whereas both deoxyribonuclease I and exonuclease III digestions show that the presence of HMGB1/2 does not alter the DNA protection in ER/ERE complexes. Protease digestions of the complexes support this conclusion and show that a global conformation change occurs in ER when bound to the different ER binding sites. Models for these interactions are discussed, together with a hit-and-run mechanism that HMGB proteins may utilize to produce these effects.