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HMGB1 and HMGB2 cell-specifically down-regulate the p53- and p73-dependent sequence-specific transactivation from the
Michal Stros1, Toshinori Ozaki, Alena Bacikova
1Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolská 135, 612 65 Brno, Czech Republic. stros@ibp.cs
Abstract:
The recently cloned gene p73 is a close homologue of p53, which is a crucial tumor suppressor gene for preventing the malignant transformation of cells by inducing cell cycle arrest and apoptosis. Previous reports have shown that architectural DNA-bending/looping chromosomal proteins HMGB1 and HMGB2 (formerly known as HMG1 and HMG2), which function in a number of biological processes including transcription and DNA repair, interact in vitro with p53 and stimulate p53 binding to DNA containing p53 consensus sites. Here, we report that HMGB1 physically interacts with two splicing variants of p73, alpha and beta (pull-down assay), and enhances binding of p73 to specific cognate DNA sites (gel-shift assay). Both HMG box domains of HMGB1, A and B, interact with p73alpha. Association of HMGB1 with p73, like the demonstrated ability of HMGB1 to stimulate p73 binding to different p53-responsive elements, requires the oligomerization region and/or region between DNA-binding domain and oligomerization domain of p73 (residues 312-381). Transient transfections revealed that ectopically expressed or endogenous HMGB1 and HMGB2 (antisense strategy) significantly inhibit in vivo both p73alpha/beta- and p53-dependent transactivation from the Bax gene promoter (and much less from Mdm2 and p21(waf1) promoters) in p53-deficient SAOS-2 cells. In contrast, HMGB1 and HGMB2 stimulate p73- or p53-dependent transactivation in p53-deficient H1299 cells, irrespective of the promoter used. Our results suggest that ubiquitously expressed HMGB1 and HMGB2 have potential to cell- and promoter-specifically down- or up-regulate in vivo transcriptional activity of different members of the p53 family. A possible mechanism of HMGB1-mediated modulation of p73- and p53-dependent transactivation is discussed.
Insights
High-mobility group box proteins HMGB1 and HMGB2 interact with p73, a p53 homolog. These proteins modulate p73 and p53 transcriptional activity in a cell- and promoter-specific manner.
Area of Science:
- Molecular Biology
- Cancer Biology
- Gene Regulation
Background:
- The p73 gene is a homolog of the tumor suppressor p53, involved in cell cycle arrest and apoptosis.
- High-mobility group box proteins HMGB1 and HMGB2 are known to interact with p53 and influence its DNA binding.
- HMGB proteins are architectural DNA-binding proteins involved in transcription and DNA repair.
Purpose of the Study:
- To investigate the interaction between HMGB1 and p73 splicing variants (p73alpha and p73beta).
- To determine the effect of HMGB1 and HMGB2 on the transcriptional activity of p73 and p53.
- To elucidate the role of HMGB proteins in regulating p53 family member-dependent gene expression.
Main Methods:
- Pull-down assays to confirm physical interaction between HMGB1 and p73alpha/beta.
- Gel-shift assays to assess the effect of HMGB1 on p73 DNA binding.
- Transient transfections in p53-deficient cell lines (SAOS-2 and H1299) to study in vivo transcriptional activity.
- Antisense strategies to inhibit endogenous HMGB1 and HMGB2 expression.
Main Results:
- HMGB1 physically interacts with p73alpha and p73beta.
- HMGB1 enhances the binding of p73 to specific DNA sites.
- HMGB1 and HMGB2 can either inhibit or stimulate p73/p53-dependent transactivation, depending on the cell type and promoter.
- The interaction and functional modulation require specific regions within p73 (residues 312-381).
Conclusions:
- HMGB1 and HMGB2 interact with p73 and modulate its DNA-binding activity.
- HMGB1 and HMGB2 exhibit cell- and promoter-specific regulation of p73 and p53 transcriptional activity.
- These findings suggest a complex role for HMGB proteins in regulating the function of the p53 family in gene expression and potentially in cancer development.