Influence of HMGB1 on Nucleosome Structure and Estrogen Receptor Binding Affinity to Concensus Estrogen Response Element on Nucleosomal DNA

Date of Award


Document Type


Degree Name

Doctor of Philosophy (Ph.D.)


Biological Sciences

First Advisor

William Scovell, PhD

Second Advisor

George Bullerjahn, PhD (Committee Member)

Third Advisor

Carmen Fioravanti, PhD (Committee Co-Chair)

Fourth Advisor

Carol Heckman, PhD (Committee Member)

Fifth Advisor

Gary Silverman, PhD (Committee Member)


Previous work from our lab has shown the high mobility group 1 protein (HMGB1) facilitates the binding of estrogen receptor (ER) to DNAs that contains consensus estrogen response element (cERE), consensus half-sites (cHERE), direct repeats of the cHERE and cEREs with different number of base pairs in the spacer region. This serves as the basis for a new paradigm for estrogen binding. HMGB1 is a dynamic, ubiquitous, “architectural” protein that binds nonspecifically in the minor groove of DNA and has been shown to enhance transcription factor binding to their target sequences and the transcription of a subset of genes by RNA pol II.

This work extends these findings to the effect of HMGB1 on remodeling of nucleosomes, ER binding to HMGB1-remodeled nucleosomes and accessibility of DNase I and the restriction enzyme, Ava I, to nucleosomes. Although the binding of ER to cERE within a nucleosome effectively does not occur in comparison to that on free DNA, the presence of HMGB1 facilitates ER binding within the nucleosome. A 161 bp DNA was constructed to include four nucleosome positioning sequences and a single, rotationally phased and translationally positioned cERE at the dyad axis or 20 or 40 bps from the dyad axis. Nucleosomes were reconstituted by the salt dilution procedure of the DNA with histones from chicken erythrocyte (CE) oligonucleosomes, which was then purified by sucrose gradient centrifugation. The cERE is positioned so that the major groove containing the cERE is facing outward for optimum ER binding.

Using electrophoretic mobility shift assay (EMSA), we show that there is no significant ER binding to cERE (at either position) in the nucleosome up to ca. 150 nM ER. However, in the presence of 400 nM HMGB1, ER binds to either position with a KD value ca. 50 nM. This is ca. 25-fold weaker than its binding affinity to free DNA. We show that the DNase I 10 bp patterns and the Exo III digestion is unchanged by the presence of 400 nM HMGB1. Increasing levels of HMGB1, however, reduces the mobility of the nucleosome band and suggests that HMGB1, by a non-enzymatic mechanism, interacts with the nucleosome to generate a new population of “nucleosomes of altered conformation”. Thus, HMGB1 provides an alternate, ATP-independent mechanism by which a subset of transcription factors can gain access to their recognition sites within a nucleosome. It also suggests that HMGB1 may cooperate with ATP-dependent chromatin remodeling complexes to enhance their activity. The HMGB1-remodeled nucleosome were isolated on a 5-30% sucrose gradient and characterized. We show that the HMGB1-remodeled nucleosomes are more accessible to DNase I and restriction enzyme digestion. The HMGB1-remodeled nucleosomes show additional DNase I sensitive bands and the pattern resembles that obtained for DNase I on free DNA. Restriction enzyme digest shows that the HMGB1-remodeled nucleosomes (N’/N”) are very accessible to Ava I compared to canonical nucleosomes.

Cleavage of the histone tail domains also enhances binding of ER to the nucleosomes. The KD of ER binding to tailless nucleosome (without the addition of HMGB1) was 50 nM and was further reduced to 25 nM in the presence of 400 nM HMGB1. Ava I digest shows that these HMGB1-remodeled tailless nucleosomes (N*) are the most accessible to Ava I compared to the canonical nucleosomes or tailless nucleosome. The HMGB1-remodeled tailless nucleosomes also show additional DNase I sensitive bands and the pattern resemble that obtained for DNase I on free DNA.

The HMGB1-remodeled nucleosomes (with tails and tailless) were, however unstable in high salt, higher temperature and excess DNA, and revert to canonical nucleosomes. There is however, no evidence of dissociation of free DNA or the core histone from the nucleosome complex.