Figure 8.

Mutations in DNA intercalating residues of HMGB-1 did not influence enhancement of PR-DNA binding in vitro but reduced the effect of HMGB-1 on PR transcriptional activity in cells. (A) Protein interaction of HMGB-1 with PR DBD was not affected by mutations in the intercalating residues of HMGB-1. Free GST, GST-Wt HMGB-1 or GST-Mut HMGB-1 were immobilized to glutathione–Sepharose resins and incubated with increasing concentrations of purified PR DBD–CTE₆₇₀ (0.25 µM to 1.0 µM). Resins were washed, eluted and bound protein was detected by western blotting with antibody for PR DBD (input is 10%). (B) Effect of Wt HMGB-1 and Mut HMGB-1 on binding of PR to PRE DNA as detected by EMSA. Increasing concentrations of purified PR DBD–CTE₆₄₈ (0–30 nM) were incubated with a single concentration of [³²P]-labeled PRE (0.6 nM). The free and protein bound PRE complexes were separated by native gel electrophoresis, quantitated, and graphed as fraction of bound PRE–DNA. (C) Mut HMGB-1 had reduced ability to enhance transcriptional activity of PR in cells. Cos-1 cells were cotransfected with PRE₂-tk-LUC reporter, PR-B, Wt HMGB-1 or Mut HMGB-1. Cells were treated with 10 nM R5020 (synthetic progesterone analog) or an equal volume of EtOH for 20 h. Luciferase activity was determined and normalized to internal control β-galactosidase activity. Normalized luciferase activity with vehicle-treated cells was set to 1.0 and all other treated groups were calculated as fold >1.0. Error bars indicate standard mean of the error (n = 5) for doses 0–40 and n = 3 for dose 80.