Cyclin-dependent kinase 5 phosphorylates mammalian HMGB1 protein only if acetylated.

High mobility group box 1 (HMGB1) protein is the most abundant chromatin-associated non-histone protein expressed in all nucleated eukaryotic cells. We examined the phosphorylation of mammalian HMGB1 by testing the ability of the cyclin-dependent kinase 5 (Cdk5) to use as substrates native protein, either unmodified or in vivo acetylated and recombinant HMGB1. It turned out that Cdk5 was active on the in vivo acetylated HMGB1 only. We studied the effect of the phosphorylation on the 'architectural' properties of the acetylated HMGB1. The treatment with Cdk5 of the acetylated HMGB1 inhibited its capacity to induce DNA end-joining but had no effect on its ability to recognize distorted DNA structures.

Nucleosome binding properties and Co-remodeling activities of native and in vivo acetylated HMGB-1 and HMGB-2 proteins.

The participation of HMGB-1 and -2 proteins in chromatin remodeling is investigated. Here, the ability of these proteins and their posttranslationally acetylated forms to affect SWI/SNF and RSC-dependent nucleosome mobilization was studied. Both proteins assisted nucleosome sliding induced by the two remodelers. Following acetylation, these proteins acquire the ability to bind to core particles, a property that has not yet been documented with parental proteins. We further report that compared to the nonmodified proteins, acetylated HMGB-1 and -2 exhibited both stronger binding to linker DNA-containing nucleosomes and a higher co-remodeling activity. Acetylation of HMGB-1 and -2 proteins enhanced binding of SWI/SNF to the nucleosome but did not affect its ATPase activity.

Native HMGB1 protein inhibits repair of cisplatin-damaged nucleosomes in vitro.

The high mobility group box (HMGB) 1 protein, one of the most abundant nuclear non-histone proteins has been known for its inhibitory effect on repair of DNA damaged by the antitumor drug cisplatin. Here, we report the first results that link HMGB1 to repair of cisplatin-treated DNA at nucleosome level. Experiments were carried out with three types of reconstituted nucleosomes strongly positioned on the damaged DNA: linker DNA containing nucleosomes (centrally and end-positioned) and core particles. The highest repair synthesis was registered with end-positioned nucleosomes, two and three times more efficient than that with centrally positioned nucleosomes and core particles, respectively. HMGB1 inhibited repair of linker DNA containing nucleosomes more efficiently than that of core particles. Just the opposite was the effect of the in vivo acetylated HMGB1: stronger repair inhibition was obtained with core particles. No inhibition was observed with HMGB1 lacking the acidic tail. Binding of HMGB1 proteins to different nucleosomes was also analysed. HMGB1 bound preferentially to damage nucleosomes containing linker DNA, while the binding of the acetylated protein was linker independent. We show that both the repair of cisplatin-damaged nucleosomes and its inhibition by HMGB1 are nucleosome position-dependent events which are accomplished via the acidic tail and modulated by acetylation.

Interplay between in vitro acetylation and phosphorylation of tailless HMGB1 protein.

The postsynthetic acetylation of HMGB1 protein and its truncated form affects significantly its properties as "architectural" factor - recognition of bent DNA and bending of short DNA fragments. We created mutants at the target sites (lysines 2 and 81) in the tailless HMGB1 modified by the histone acetyltransferase CBP. The results show that there is no preferential site for the enzymatic activity of CBP and both lysine moieties are modified independently. Our findings for the first time demonstrate the link between the acetylation and phosphorylation of HMGB1DeltaC in vitro. The PKC phosphorylation prior to acetylation inhibits the CBP activity 40-60% for the truncated form and its mutants. The effect of the CBP acetylation on the phosphorylation level turns out to be much more prominent. In the case of HMGB1DeltaC modified at Lys 2 and Lys 81 prior to PKC treatment background phosphorylation is detected. If only one of the lysines is modified the inhibitory effect decreases.

Post-synthetic acetylation of HMGB1 protein modulates its interactions with supercoiled DNA.

High mobility group box (HMGB) proteins 1 and 2 are abundant non-histone nuclear proteins that regulate chromatin structure because of their structure-specific binding to DNA. Here, we have investigated how the post-synthetic acetylation of HMGB1 affects its interaction with negatively supercoiled DNA by employing monoacetylated at Lys2 protein, isolated from butyrate-treated cells. Our data reveal that this modification enhances three reaction parameters: binding affinity, supercoiling activity and capacity to protect the supercoiled DNA from relaxation by topoisomerase I. We show that monoacetylation at Lys2 mimics the effect of acidic tail removal but to a lesser extent thus demonstrating that in vivo acetylated HMGB1 is capable of modulating its interaction with negatively supercoiled DNA.

HMGB1 protein inhibits DNA replication in vitro: a role of the acetylation and the acidic tail.

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.

The inhibitory effect of HMGB-1 protein on the repair of cisplatin-damaged DNA is accomplished through the acidic domain.

The well established inhibitory effect of HMGB-1 on repair of cisplatin-damaged DNA has been studied with two modified forms of the protein, shown to bind platinated DNA with higher affinity than the original protein: in vivo acetylated HMGB-1 and HMGB-1 lacking its C-terminal domain. The native and the modified proteins were assayed for their effects on adduct removal by using cell-free extract capable of repairing cisplatinated DNA in vitro. The inhibition observed with the native HMGB-1 was reduced in the presence of acetylated HMGB-1 and completely abolished when the assay was carried out with the truncated protein. When the repair assay was performed in the presence of a synthetic polypeptide identical to the C-terminal tail, either alone or together with the truncated protein, the inhibitory effect was partially recovered in a concentration-dependent manner. These findings strongly suggest that the HMGB-1-induced inhibition of cisplatin-DNA adduct repair is accomplished through the acidic domain. The results obtained are discussed in terms of the repair events that may occur in the presence of HMGB-1 protein.

In vitro acetylation of HMGB-1 and -2 proteins by CBP: the role of the acidic tail.

Histone acetyltransferases CBP, PCAF, and Tip60 have been tested for their ability to in vitro acetylate HMGB-1 and -2 proteins and their truncated forms lacking the C-terminal tail. It was found that these proteins were substrates for CBP only. Analyses of modified proteins by electrophoresis, amino acid sequencing, and mass spectrometry showed that full-length HMGB-1 and -2 were monoacetylated at Lys2. Removal of the C terminus resulted in (i) an increased incorporation of radiolabeled acetate within the proteins to a level close to that observed with histones H3/H4 and (ii) creation of a novel target site at Lys81. Acetylated and nonmodified HMGB-1 and -2 protein lacking the acidic tail were compared relative to their binding affinity to distorted DNA and the ability to bend linear DNA. Both proteins showed similar affinities to cisplatin-damaged DNA; the acetylated protein, however, was 3-fold more effective in inducing ligase-mediated circularization of a 111-bp DNA fragment. The alterations in the acetylation pattern of HMGB-1 and -2 upon removal of the C-terminal tail are regarded as a means by which the acidic domain modulates some properties of these proteins.

The binding affinity of HMG1 protein to DNA modified by cis-platin and its analogs correlates with their antitumor activity.

The antitumor activity of cis-platin is believed to result from its interaction with cellular DNA and subsequent processing of DNA adducts by damage recognition proteins. Among them are the high mobility group (HMG) proteins 1 and 2, which have been hypothesized to mediate the effect of cis-platin. One possibility suggests that the tight binding of HMG1 to DNA adducts blocks the repair of damaged DNA. In order to further evaluate such a mechanism, several cis-platinum complexes with known antitumor activity have been used to treat DNA and the affinity of HMG1 to the DNA adduct induced by each drug was determined. The dissociation constants for the complexes of HMG1 with the platinated probe were obtained by gel mobility shift assays. The antitumor activity of the tested platinum compounds was found to correlate with the binding affinity of HMG1 to the respective drug-DNA adduct. These findings support the view that HMG1 contributes to cytotoxicity of cis-platin by shielding damaged DNA from repair. In addition, they offer a fast test for screening new platinum compounds for antitumor activity.