The goal of this study was to reexamine and further localize the sites of RAG1–HMGB1 interaction in light of recent structural data showing that the NBD of RAG1 is not a HD (11
). In contrast to a previous study (38
), we did not find a robust interaction between the purified RAG1c and HMGB1 proteins in either pulldown or size exclusion chromatography experiments. We did, however, find a synergistic binding interaction between RAG1, HMGB1 and DNA. The requirement of DNA for a robust HMGB1–RAG1 interaction precluded us from pursuing our intended goal of localizing the sites of RAG1–HMGB1 interaction, as mutation of the RAG1 NBD is likely to affect the DNA-binding ability of RAG1, which would be difficult to distinguish from an effect on the RAG1–HMGB1 protein–protein interaction. Our fluorescence anisotropy experiments and pulldown assays using biotinylated DNA indicate that HMGB1 has a higher binding affinity for a RAG1–DNA complex versus RAG1 or DNA alone. This is clearly illustrated by the stronger signal seen in lanes 4, 5, 8 and 9 compared with lanes 3 and 7 in A. It is also illustrated by the dramatic increase in anisotropy (~75% of the maximal increase observed) seen when the three components were incubated together at low concentrations of 30 nM HMGB1-A488, 50 nM 23RSS and 50 nM RAG1c (B); in contrast, similar concentrations yielded small increases in anisotropy when only two components were present (A and B). These data suggest that the pathway to assembly of the functional recombinase complex is primarily through binding of HMGB1 to a pre-formed RAG1–DNA complex, which may or may not contain RAG2, and not via binding of an RSS by a pre-bound RAG1–HMGB1 or RAG1–RAG2–HMGB1 complex as had been previously proposed (38
). Given the transient interaction of HMGB1 with chromatin (33
) and the weak or transient interaction of HMGB1 with B-form DNA in the absence of RAG1 as found in our biotin pulldown experiments, a pathway of complex assembly involving a pre-formed HMGB1–DNA complex recruiting RAG1 is also less likely. Interestingly, the synergistic HMGB1–RAG1–DNA binding interaction was not found to require an intact heptamer and nonamer sequence. This raises the possibility that a similar series of binding events (RAG1 binding to DNA followed by incorporation of HMGB1) supports the formation of RAG1–HMGB1–non-specific DNA complexes in vivo
(discussed further below).
The tighter binding of HMGB1 to a RAG1–DNA complex over DNA alone also suggests a mechanism by which this notoriously transient DNA-binding and -bending protein can become stably integrated into the V(D)J recombinase complex. Although HMGB1 has been identified as a cofactor in the assembly of a wide variety of nucleoprotein complexes, it is not often found as a stable component of the assembled complex. HMGB1 has been found to interact directly with the HDs of the Hox and Oct proteins, and HMGB1 enhances their DNA-binding activity and transcriptional activation. Nonetheless, there is no evidence of a ternary complex of HMGB1 with either of these proteins and their cognate DNA by electrophoretic mobility shift assay (EMSA) (40
). Similarly, many of the steroid (class I) nuclear receptors have enhanced site-specific binding and transcriptional activity in the presence of HMGB1, and both progesterone receptor and estrogen receptor directly interact with HMGB1 via their DNA-binding domains (58
). However, ternary complexes of HMGB1 with these proteins and their cognate DNA are difficult to isolate (60
), suggesting that a complex containing HMGB1 is a transient intermediate. In contrast, stable integration of HMGB1 appears to be an important element of the functional V(D)J recombinase complex, as HMGB1 has been found in complexes throughout the cleavage reaction, from the signal complex and paired complex to the post-cleavage signal end complex (35–37
). Our finding of high-affinity HMGB1 binding to a RAG1–DNA complex suggests an explanation for its stable incorporation into V(D)J recombinase complexes.
The lack of interaction identified between RAG1c and HMGB1 by pulldown assay in our work is in contrast to the results of the previous study (38
). This may be due to the relative purity of the proteins used in each study. The RAG1c and HMGB1 proteins used here have been purified over several affinity, ion exchange and sizing columns to ensure purity, whereas the previous study used a single affinity column purification of GST-tagged proteins. Our finding that the presence of even small amounts of DNA greatly increase complex formation suggest that the presence of even slight DNA contamination in the protein preparations of Aidinis et al.
) would have led to the detection of a significantly more robust RAG1–HMGB1 interaction than we observe in the absence of any DNA. An alternative explanation for the difference between our findings and those of the previous study is the source of the RAG1c protein, which we purified from E. coli
and Aidinis and colleagues purified from transfected mammalian cells (38
). However, RAG1c from E. coli
is fully functional in both RSS cleavage and binding assays (11
), and is capable of a functional interaction with HMGB1 as evidenced by enhanced RAG-mediated 23RSS cleavage and binding in the presence of HMGB1 (Supplementary Figure S5
). Thus, it is unlikely that a functionally significant RAG1–HMGB1 interaction would require RAG1 purified from a mammalian source. Finally, it is possible that the MBP tag on RAG1 somehow inhibits a RAG1–HMGB1 interaction, as the previous study used GST-tagged RAG1 proteins. However, both MBP and GST are bulky tags, and both were attached to the N-terminus of the RAG1 constructs used, so this is unlikely to be an important difference.
Based on an experiment with a single concentration of RAG1 pulled down by a single concentration of immobilized HMGB1, Aidinis and colleagues estimated that the RAG1–HMGB1 interaction had a Kd
on the order of 10−5
). Our data suggest that this interaction is even less robust. For example, given an estimated Kd
M and the assumption of 1:1 binding, in our size exclusion chromatography experiments using 7.3 μM HMGB1 and 2.1 μM RAG1, we would expect to see ~11% of the HMGB1 shifted into earlier fractions, but far less was observed (E). Our pulldown and size exclusion chromatography data are more consistent with a RAG1–HMGB1 interaction with a Kd
on the order of 10−4
M or higher, though this may underestimate the true interaction due to complex dissociation during the course of the experiment. Whether the RAG1–HMGB1 interaction is on the order of 10−4
M, however, may not be of significant consequence. While this protein–protein interaction could be physiologically relevant, as many biologically significant interactions have similarly high dissociation constants (e.g. some enzyme–substrate interactions), these weak interactions are typically transient and require less than a few seconds to achieve a biologically significant effect (63
). This weak protein–protein interaction is unlikely to provide the primary path for HMGB1 recruitment to the functional V(D)J recombinase, as the interaction between HMGB1 and a RAG1–DNA complex is significantly more robust; whereas the interaction between HMGB1 and RAG1 alone is difficult to detect, ternary complex formation is readily detectable by fluorescence anisotropy and biotin pulldown experiments at nanomolar concentrations of all components.
It has previously been shown that HMGB2 increases the affinity of RAG1 for the 23RSS (18
), and our work has identified the reciprocal effect on HMGB1. While the precise mechanism of the recruitment of HMGB1 to a RAG1–DNA complex is not known, it is appealing to think that HMGB1 is recruited by a combination of its high affinity for bent DNA and its weak affinity for RAG1. HMGB1 has significant intrinsic affinity for bent or distorted DNA, and RAG1 alone has been shown to bend DNA (64
). In addition, HMGB1 might bind RSS-bound RAG1 more robustly than free RAG1, as RAG1 undergoes significant conformational changes upon binding to the RSS (19
). Notably, recent experiments demonstrate that the 23RSS adopts a strongly bent ‘U’ shape in the paired complex, with bending nearly as strong when RAG2 is omitted from the reaction, indicating that RAG1 and HMBG1 are sufficient to induce a large bend in the 23RSS (65
). We do not know if the RAG1–HMGB1 protein–protein interaction in the presence of DNA is mediated by the RAG1 NBD, but it would be interesting to identify the sites of protein–protein interaction, if any, in this ternary complex. It is possible that the high affinity of HMGB1 for a RAG1–DNA complex helps explain how HMGB2 increases the affinity of RAG1 for the RSS. HMGB1 (or HMGB2) can stabilize a DNA bend such as the one created by RAG1 binding, potentially decreasing the off rate of RAG1. This might be the primary mechanism by which the DNA-binding/-bending protein increases RAG1 binding affinity, as opposed to recruiting RAG1 by pre-bending DNA or by causing a conformational change in RAG1 to stabilize its DNA binding, mechanisms suggested for HMGB1 enhancement of DNA-binding by p53 and progesterone receptor, respectively (59
The interplay between HMGB1 and RAG1 in the context of DNA has interesting implications for the DNA-binding activity of both proteins in vivo
. Although the functional V(D)J recombinase complex requires RAG2, there are several phases during the cell cycle when RAG1 might function independently, as RAG2 is degraded at the G1/S boundary and is absent during S/G2/M (22
). When expressed together, RAG1 and RAG2 bind in a largely coincident pattern within ‘recombination centers’ in the immunoglobulin or T-cell receptor loci, strongly suggesting that they bind as a RAG1–RAG2–RSS complex. When expressed alone, RAG1 still binds to RSSs in the majority of these loci (69
), perhaps stabilized by HMGB1. Given the known non-specific DNA-binding activity of RAG1, particularly in the absence of RAG2 (20
), it is reasonable to consider the possibility that binding of RAG1 is not limited to these antigen receptor loci. Based on our in vitro
studies and the high concentration of HMGB1 in the nucleus, we postulate that the majority of chromatin-bound RAG1 is present in the form of cooperatively bound RAG1–HMGB1–DNA complexes. It remains to be seen whether these complexes, which would be predicted to exhibit only low levels of RSS specificity, would be forced into a more sequence-specific mode of binding in the presence of RAG2 (20
). Alternatively, upon RAG2 expression after M phase, a RAG1–HMGB1–DNA complex might be capable of recruiting RAG2 to non-RSS locations throughout the genome, thereby creating the functional V(D)J recombinase complex at off-target sites and providing a pathway for chromosomal translocations.