Title

Influence of Hmgb1 on Estrogen Responsive Gene Expression and Nucleosome Structure

Date of Award

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Dr. William Scovell, PhD

Second Advisor

Dr. Carmen Fioravanti, PhD (Committee Co-Chair)

Third Advisor

Dr. Carol Heckman, PhD (Committee Member)

Fourth Advisor

Dr. George Bullerjahn, PhD (Committee Member)

Fifth Advisor

Dr. Bruno Ullrich, PhD (Committee Member)

Abstract

High mobility group box-1 protein (HMGB1) increases the in vitro binding affinity of estrogen receptor (ER) to the various estrogen response elements (EREs) such as consensus ERE (cERE), tandem cEREs, consensus half-site ERE (cHERE), tandem cHEREs, and variant spacer cEREn, n= 0-4bp), while decreasing the binding specificity. To test if this in vitro binding characteristic translates to the functional activity in the cell, firefly luciferase reporter vectors were constructed with different EREs noted above at 5- to the TATA box upstream of the luciferase gene. Transient transfection of these constructs was performed in estrogen treated, ER negative human osteosarcoma cell lines, U2OS, along with the co-transfection of mammalian expression vectors pERα and pHMGB1. The in vitro binding activity of HMGB1 was correlated with the functional activity in cells, but there was no linear correlation between in vitro and in vivo. HMGB1 stimulated the transcriptional synergy for tandem cEREs and cHEREs which is reflected in its influence on in vitro binding co-operativity. cEREns with variant spacers, to which the HMGB1 had increased the ER binding affinity very strongly, did not produce a strong transcriptional activity even though it enhanced the activity. HMGB1 knock down experiments with siRNA in determining the influence of HMGB1 on estrogen (E2) responsive gene expression clearly showed that HMGB1 is involved in E2 responsive gene expression.

ER binds strongly to cERE on free DNA but does not bind (KD >200nM) to cERE in nucleosomal DNA. However, the presence of HMGB1 greatly facilitates ER binding (KD~50nM) to cERE in the nucleosome. To determine the influence of HMGB1 on nucleosome structure which could have facilitated enhanced binding of ER to cERE in nucleosomal DNA, characteristic of the nucleosome were studied after treating it with increasing levels of HMGB1 (up to 1600nM). The structure of the nucleosome is markedly altered by HMGB1 interaction, with the population of remodeled nucleosomes increasing with increasing levels of HMGB1. However, HMGB1 is not a stable component of the remodeled nucleosome. The HMGB1-remodeling is ATP-independent. Two different forms of remodeled nucleosomes (N' and N") can be isolated after removal of HMGB1 which are stable in low salt buffer and low temperature. The remodeled nucleosomes (N'/N") can be converted back to the canonical nucleosomes by changing solution conditions. At high salt (100 mM NaCl), N' converts to N, while N' remains stable; increasing the temperature (37°C), converts N" to N' and N' remains stable. However, on challenging the remodeled nucleosomes with excess “cold” competitor DNA (up to 1000 ng), both N' and N" reverts back to the canonical from, while at 100 ng of excess DNA, N" coverts to N, and N' remains stable. This suggests that the HMGB1-remodeled nucleosomes exhibit an altered structure stabilized at low salt buffer with electrostatic interactions. The formation of N' and N" by possible interaction of histone tails with DNA was excluded as these two population can also be isolated by treating tailless nucleosomes with 1600 nM HMGB1. However, removal of histone tails enhance the activity of HMGB1 to distinctly reduce the EMSA mobility of nucleosome even at low concentrations ca. 400 nM, suggesting the HMGB1 needs to interact with histone tails prior to interact with DNA in the nucleosome. Furthermore, HMGB1 at 1600 nM can increase the DNase I sensitivity to alter the 10bp pattern on nucleosomes. Altogether, we find that HMGB1-remodeled nucleosomes exhibit many of the same characteristics of nucleosomes remodeled by ATP-dependent chromatin remodeling complexes. The presence of HMGB1 appears to provide an alternate or complementary mechanism for nucleosome remodeling in which HMGB1 reduces interaction within the nucleosome to reorganize the nucleosome structure and/or in...