THE BINDING OF ESTROGEN, PROGESTERONE AND GLUCOCORTICOID RECEPTORS TO THEIR RECOGNITION SITES IN A NUCLEOSOME AND THE EFFECT OF HMGB1 ON THE BINDING AFFINITY
The eukaryotic genome is packaged in a chain of nucleosomes called chromatin. For transcription to take place in chromatin, RNA polymerase II, transcriptional activators and general transcription factors must interact with their binding sites in the regulatory region of specific genes. When these sites occur in nucleosome, the binding of the transcription factors (TF) is generally reduced drastically and in some cases, may not even bind to these regulatory sites. It therefore follows that, for the TFs to bind, the DNA must be remodeled or moved out of the nucleosome, or the contacts between the histone protein and DNA must be weakened by some mechanism. It has been previously reported that estrogen receptor (ER) binds to its recognition site when in a nucleosome in a dose dependent manner. We find, however, that ER does not bind even at the highest ER concentration of 160 nM. We show that estrogen receptor does not bind to estrogen response element (ERE) when DNA is reconstituted in a nucleosome. However, the ubiquitous protein, high mobility group box-1 (HMGB1) protein relieves ER binding repression and allows ER to bind with a dissociation constant (K¬D) of about 60 nM. We also find that ER binds to 2 different translationally positioned ERE on the nucleosome with the same K¬D values in the presence of HMGB1 protein. We suggest that HMGB1 protein does not strip the nucleosome off the DNA but rather makes the contact between the histone and DNA weaker to allow ER to bind. We show that HMGB1 protein does not form a stable part of the complex formed when ER binds to DNA and (or nucleosome). Our data indicate that glucocorticoid receptor (GR) acts more like a “bubble gum” because it binds to all DNAs and nucleosomes that we tested, with a KD of approximately 2.5 nM. HMGB1 protein does not have any significant effect in enhancing the binding affinities for GR. PR binds to glucocorticoid response element (GRE) with a KD value of 2.5 nM. The binding affinity is enhanced in the presence of HMGB1 protein to a low KD of 0.9 nM. However, when GRE was reconstituted into a nucleosome, PR fails to bind even in the presence of HMGB1 protein.
School:Bowling Green State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:receptor binding in nucleosomes and effect of hmgb1
Date of Publication:01/01/2006