Eva Polanská

HMGB1 gene knockout in mouse embryonic fibroblasts results in reduced telomerase activity and telomere dysfunction

Telomere repeats are added onto chromosome ends by telomerase, consisting of two main core components: a catalytic protein subunit (telomerase reverse trancriptase, TERT), and an RNA subunit (telomerase RNA, TR). Here, we report for the first time evidence that HMGB1 (a chromatin-associated protein in mammals, acting as a DNA chaperone in transcription, replication, recombination, and repair) can modulate cellular activity of mammalian telomerase. Knockout of the HMGB1 gene (HMGB1 KO) in mouse embryonic fibroblasts (MEFs) results in chromosomal abnormalities, enhanced colocalization of γ-H2AX foci at telomeres, and a moderate shortening of telomere lengths. HMGB1 KO MEFs also exhibit significantly (>5-fold) lower telomerase activity than the wild-type MEFs. Correspondingly, enhanced telomerase activity is observed upon overexpression of HMGB1 in MEFs. HMGB1 physically interacts with both TERT and TR, as well as with active telomerase complex in vitro. However, direct interaction of HMGB1 with telomerase is most likely not accountable for the observed higher telomerase activity in HMGB1-containing cells, as revealed from the inability of purified HMGB1 protein to stimulate telomerase activity in vitro. While no transcriptional silencing of TERT is observed in HMGB1 KO MEFs, levels of TR are diminished (~3-fold), providing possible explanation for the observed lower telomerase activity in HMGB1 KO cells. Interestingly, knockout of the HMGB2 gene elevates telomerase activity (~3-fold) in MEFs, suggesting that the two closely related proteins of the HMGB family, HMGB1 and HMGB2, have opposite effects on telomerase activity in the cell. The ability of HMGB1 to modulate cellular activity of telomerase and to maintain telomere integrity can help to understand some aspects of the protein involvement in chromosome stability and cancer.

HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase IIα

Topoisomerase IIα (topo IIα) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIα, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIα gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIα promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIα promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIα gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIα mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIα by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIα promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIα in tumors.

HMGB1 interacts with human topoisomerase IIα and stimulates its catalytic activity

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme and its unique decatenation activity has been implicated in many aspects of chromosome dynamics such as chromosome replication and segregation during mitosis. Here we show that chromatin-associated protein HMGB1 (a member of the large family of HMG-box proteins with possible functions in DNA replication, transcription, recombination and DNA repair) promotes topo IIα-mediated catenation of circular DNA, relaxation of negatively supercoiled DNA and decatenation of kinetoplast DNA. HMGB1 interacts with topo IIα and this interaction, like the stimulation of the catalytic activity of the enzyme, requires both HMG-box domains of HMGB1. A mutant of HMGB1, which cannot change DNA topology stimulates DNA decatenation by topo IIα indistinguishably from the wild-type protein. Although HMGB1 stimulates ATP hydrolysis by topo IIα, the DNA cleavage is much more enhanced. The observed abilities of HMGB1 to interact with topo IIα and promote topo IIα binding to DNA suggest a mechanism by which HMGB1 stimulates the catalytic activity of the enzyme via enhancement of DNA cleavage.

Binding of histone H1 to DNA is differentially modulated by redox state of HMGB1.

HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein.

HMGB1 protein and linker histone H1 have overlapping binding sites in the nucleosome. HMGB1 has been implicated in many DNA-dependent processes in chromatin involving binding of specific proteins, including transcription factors, to DNA sites pre-bent by HMGB1. HMGB1 can also act as an extracellular signaling molecule by promoting inflammation, tumor growth a metastasis. Many of the intra- and extracellular functions of HMGB1 depend on redox-sensitive cysteine residues of the protein. Here we report that mild oxidization of HMGB1 (and much less mutation of cysteines involved in disulphide bond formation) can severely compromise the functioning of the protein as a DNA chaperone by inhibiting its ability to unwind or bend DNA. Histone H1 (via the highly basic C-terminal domain) significantly inhibits DNA bending by the full-length HMGB1, and the inhibition is further enhanced upon oxidization of HMGB1. Interestingly, DNA bending by HMGB1 lacking the acidic C-tail (HMGB1ΔC) is much less affected by histone H1, but oxidization rendered DNA bending by HMGB1ΔC and HMGB1 equally prone for inhibition by histone H1. Possible consequences of histone H1-mediated inhibition of DNA bending by HMGB1 of different redox state for the functioning of chromatin are discussed.

HMGB1-mediated DNA bending: Distinct roles in increasing p53 binding to DNA and the transactivation of p53-responsive gene promoters

HMGB1 is a chromatin-associated protein that has been implicated in many important biological processes such as transcription, recombination, DNA repair, and genome stability. These functions include the enhancement of binding of a number of transcription factors, including the tumor suppressor protein p53, to their specific DNA-binding sites. HMGB1 is composed of two highly conserved HMG boxes, linked to an intrinsically disordered acidic C-terminal tail. Previous reports have suggested that the ability of HMGB1 to bend DNA may explain the in vitro HMGB1-mediated increase in sequence-specific DNA binding by p53. The aim of this study was to reinvestigate the importance of HMGB1-induced DNA bending in relationship to the ability of the protein to promote the specific binding of p53 to short DNA duplexes in vitro, and to transactivate two major p53-regulated human genes: Mdm2 and p21/WAF1. Using a number of HMGB1 mutants, we report that the HMGB1-mediated increase in sequence-specific p53 binding to DNA duplexes in vitro depends very little on HMGB1-mediated DNA bending. The presence of the acidic C-terminal tail of HMGB1 and/or the oxidation of the protein can reduce the HMGB1-mediated p53 binding. Interestingly, the induction of transactivation of p53-responsive gene promoters by HMGB1 requires both the ability of the protein to bend DNA and the acidic C-terminal tail, and is promoter-specific. We propose that the efficient transactivation of p53-responsive gene promoters by HMGB1 depends on complex events, rather than solely on the promotion of p53 binding to its DNA cognate sites.