The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition - PubMed (original) (raw)

The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition

Hatem Zayed et al. Nucleic Acids Res. 2003.

Abstract

Sleeping Beauty (SB) is the most active Tc1/ mariner-type transposon in vertebrates. SB contains two transposase-binding sites (DRs) at the end of each terminal inverted repeat (IR), a feature termed the IR/DR structure. We investigated the involvement of cellular proteins in the regulation of SB transposition. Here, we establish that the DNA-bending, high-mobility group protein, HMGB1 is a host-encoded cofactor of SB transposition. Transposition was severely reduced in mouse cells deficient in HMGB1. This effect was rescued by transient over-expression of HMGB1, and was partially complemented by HMGB2, but not with the HMGA1 protein. Over-expression of HMGB1 in wild-type mouse cells enhanced transposition, indicating that HMGB1 can be a limiting factor of transposition. SB transposase was found to interact with HMGB1 in vivo, suggesting that the transposase may recruit HMGB1 to transposon DNA. HMGB1 stimulated preferential binding of the transposase to the DR further from the cleavage site, and promoted bending of DNA fragments containing the transposon IR. We propose that the role of HMGB1 is to ensure that transposase-transposon complexes are first formed at the internal DRs, and subsequently to promote juxtaposition of functional sites in transposon DNA, thereby assisting the formation of synaptic complexes.

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Figures

Figure 1

Efficient SB transposition requires HMGB1. (A) Schematic representation of the in vivo transposition assay. Constructs expressing HMG proteins are cotransfected with transposon donor and transposase-expressing helper plasmids into cultured cells. In control transfections, a plasmid expressing β-galactosidase is cotransfected. Cells are placed under zeocin selection, and resistant colonies are counted. The ratio of colony numbers in the presence versus in the absence of transposase is a measure of the efficiency of transposition. Arrows flanking the zeocin gene in the transposon donor construct represent the terminal IRs. (B) The effect of HMG proteins for transposition. HMG protein expressing constructs were cotransfected into either wild-type (black columns) or HMGB1-deficient (gray columns) mouse cells. The indicated constructs were used either to complement or to over-express different HMG proteins. The efficiency of transgene integration was estimated by counting zeo-resistant colonies. The numbers on the left represent the numbers of colonies per 105 cells plated.

Figure 2

HMGB1 enhances bending of the transposon IR. Intramolecular ligation (circularization) assay was performed to monitor the effect of HMGB1 on bending of the left IR of the transposon. The probe was ligated by T4 DNA ligase in the absence or presence of HMGB1 for the time periods indicated. Lane 11 is the same as lane 10, except treated with ExoIII. Empty triangles indicate linear ligation products. Filled triangles point to circular ligation products resistant to ExoIII digestion.

Figure 3

Bending effect of HMGB1 on a complete SB transposon. (A) Schematic representation of the circularization assay. The SB transposon contains zeo and a bacterial origin of replication (ORI). Black arrows flanking the element are the terminal IRs, white arrows inside the IRs are the transposase binding sites. T4 ligase circularizes the linear transposon. The effect of HMGB1 on T4 ligase-mediated circularization is measured by transformation into E.coli cells, and counting bacterial colonies. (B) Effect of HMGB1 on circle formation of SB transposon DNA. Shown are numbers of bacterial colonies after transformation of DNA incubated with T4 ligase in the presence and absence of HMGB1, for the time periods indicated. Numbers are the average of three individual experiments.

Figure 4

HMGB1 stimulates specific binding of SB transposase to the transposon IRs. (A) The effect of HMGB1 on transposase binding to the left IR. EMSA was performed using the left IR of SB, containing two binding sites for the transposase, as a probe, and N123, an N‐terminal derivative of SB transposase containing the specific DNA-binding domain. C1 and C2 indicate the two DNA–protein complexes formed in the assay. (B) Comparison of SB transposase binding sites and the RAG1/2 recognition signal sequences. The degrees of similarities to the heptamer and nonamer motifs are indicated. (C) The effect of HMGB1 on binding to either the outer or the inner transposase binding sites in the context of the left IR. EMSA showing the stimulatory effects of HMGB1 on binding of N123 to the ODR and IDR.

Figure 5

SB transposase interacts with HMGB1. Immunoblot of nuclear extracts of HeLa cells expressing SB transposase, and control cells, after incubation with antibodies against human HMGB1, actin and p15 proteins or a preimmune serum, with or without DNase I treatment. The blot was hybridized with an anti-SB antibody.

Figure 6

Formation of a ternary complex of the full-length SB transposase, HMGB1, and transposon DNA. (A) HMGB1 stimulates specific binding of a MBP–SB transposase fusion to the transposon IRs. EMSA was performed using the left IR of SB, containing two binding sites for the transposase, as a probe, and MBP–SB. The radioactively labeled IR fragment was incubated with buffer only (lane 1), or with 20 nM MBP–SB alone (lane 2), or together with 0.1 µM HMGB1 (lane 3). Lane 4 contained 0.1 µM HMGB1 alone. The arrow denotes the free probe. (B) Coimmunoprecipitation of transposon–transposase complexes with SB and HMGB1 antibodies. Purified HMGB1 (1 µM) and purified MBP–SB (0.2 µM) were incubated individually or together with a radioactively labeled IR probe. Anti-SB and anti-HMGB1 antibodies were used to coimmunoprecipitate labeled DNA after incubation with MBP–SB and HMGB1 alone or together. After extensive washing, the radioactivity of DNA bound to immunoabsorbent agarose was measured by scintillation counting. The average c.p.m. values obtained with the anti-SB antibody are the following: MBP–SB, 6946; MBP–SB plus HMGB1, 23 033. The values with the anti-HMGB1 antibody are: MBP–SB, 2010; HMGB1, 2304; and MBP–SB plus HMGB1, 5473.

Figure 7

A proposed model for the role of HMGB1 in SB synaptic complex formation. SB transposase (gray spheres) recruits HMGB1 (dotted hexagons) to the transposon IRs. First, HMGB1 stimulates specific binding of the transposase to the IDRs. Once in contact with DNA, HMGB1 bends the spacer regions between the DRs, thereby assuring correct positioning of the ODRs for binding by the transposase. Cleavage (scissors) proceeds only if complex formation is complete. The complex includes the four binding sites (black boxes), the HDR enhancer sequence (black circle) and a tetramer of the transposase.

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The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition - PubMed (original) (raw)