Direct interaction between the RAG1 HD and the HMG boxes of HMG1,2.
HMG1,2 are highly homologous and are functionally interchangeable in several systems (12
). They have been shown to interact directly with the HDs of HOX and OCT proteins (60
). Therefore, we tested the ability of the RAG1 HD to interact with HMG1,2.
Initial experiments showed that full-length purified RAG1 associates with Sepharose beads bearing immobilized HMG1 (Fig. A) but not with Sepharose beads coated with BSA (not shown) or cytochrome c
(Fig. B), which has a pI similar to that of the immobilized form of HMG1. The association was not quantitative, since about half of the input RAG1 did not bind to the beads. If all input RAG1 were active and all of the HMG1 on the beads had the same activity as native, soluble HMG1, the dissociation constant for the RAG1-HMG1 interaction would be on the order of 10−5
M. This estimate is probably conservative, but nonetheless compares favorably with the concentration of HMG1 in the cell nucleus, which is about 10−6
). The in vitro interaction of HMG1 and RAG1 may thus occur in vivo as well.
FIG. 1 RAG1 interacts with HMG1. Purified GST-RAG1 (about 0.2 μg in 160 μl) was incubated with Sepharose beads bearing immobilized, bacterially expressed tailless HMG1 (M1-V176) (A) or control beads bearing immobilized cytochrome c (B). Conversely, (more ...)
The reverse experiment was also performed (Fig. C): an enzymatically active fusion protein formed between GST and a truncated form of RAG1 (RAG1ΔN380), immobilized to glutathione-Sepharose beads, partially retained soluble HMG1 but did not retain HMG-I(Y), a structurally different high-mobility-group protein that facilitates the assembly of nucleoprotein complexes required for the transcription of several lymphoid cell-specific genes (16
). TCF-1b, another HMG box protein with a DNA binding domain structurally similar to that of HMG1 and necessary for T-cell development (55
), did not interact with RAG1 either (not shown).
We next investigated whether the HD of RAG1 is required for the interaction with HMG1. Soluble RAG1ΔN380, which contains the HD, and RAG1Δ456, from which the HD has been deleted (Fig. B), were incubated with immobilized HMG1 or HMG2. Both HMG1 and HMG2 beads retained RAG1ΔN380 but not RAG1ΔN456 (Fig. A). In addition, a polypeptide corresponding to the RAG1 HD alone (aa 377 to 477) also interacted with HMG1,2 (data not shown). In contrast, RAG2 (active core, RAG2ΔC; aa 1 to 387) showed no obvious association with HMG1,2 (Fig. A, lanes 4 to 6 and 13 to 15). In controls, GST, cytochrome c, and BSA failed to retain the RAG1 protein (data not shown). Posttranslational modifications are not required for the RAG1-HMG1,2 interaction, since RAG1 expressed in either bacteria or mammalian cells interacted with HMG1 with equal efficiency (data not shown).
FIG. 2 HMG1,2 directly interact with RAG1 through its HD. (A) Purified, eukaryotically expressed, GST-fused RAG1ΔN330, RAG1ΔN456, or RAG2ΔC (aa 1 to 388) was incubated with Sepharose beads bearing immobilized, bacterially expressed tailless (more ...)
We identified the surface of interaction for RAG1 on HMGs with the reverse experiment. The immobilized HD of RAG1 was able to retain in vitro-translated tailless HMG1 and 2, respectively (Fig. A and B). A more extensively truncated form of HMG1 (M1-K147) was also retained, but truncated versions containing only one HMG box were not. Thus, both HMG boxes of HMG1 or -2 are necessary for interaction with the HD of RAG1.
FIG. 3 The RAG1 HD directly interacts with HMG boxes A and B of HMG1 and -2. In vitro-translated HMG1 (A) and HMG2 (B) derivatives were incubated with Sepharose beads bearing immobilized, eukaryotically expressed RAG1 HD (Fig. B). The input (more ...)
These data establish that HMG1,2 interact via their HMG boxes with the RAG1 HD even in the absence of DNA.
HMG1,2 promote the interaction of the RAG1 HD with the RSS.
We next tested whether the protein-protein interaction between RAG1 and HMG1,2 promoted the binding of the RAG1/2 complex to DNA. RAG1/2 binding to the 23RSS was examined with serially deleted forms of RAG1 (Fig. B).
Full-length RAG1 associated with the active core of RAG2 (RAG2ΔC) and the RSS DNA to yield a complex (Fig. A, lane 1) whose formation was enhanced about fivefold by HMG1 (Fig. A, lane 2).
FIG. 4 HMG1 stimulates RAG1/2 binding through the HD of RAG1. (A) EMSAs with a radiolabelled 23RSS oligonucleotide probe. RAG1 deletion derivatives and RAG2ΔC (50 ng each) and 40 ng of HMG1 were added as indicated (+). Lanes 11 to 14 represent (more ...)
RAG1ΔN330, where aa 1 to 330 are deleted, formed two complexes with RSS DNA in the presence of RAG2ΔC, a prominent upper one and a minor lower one (Fig. A, lane 3). While the precise compositions of the two complexes are still to be determined, both contain RAG1 and RAG2 (43
). RAG1ΔN380, which represents the active core of the protein (42
), lacks aa 330 to 380, which include the homodimerization helices of RAG1 (41
). RAG1ΔN380/RAG2ΔC bound the RSS DNA with higher efficiency and predominantly formed the lower band (Fig. A, lane 5). HMG1 stimulated the formation of both complexes when RAG1ΔN330 was used and stimulated the formation of the lower one only when RAG1ΔN380 was used (Fig. A, lanes 4 and 6). This suggests that different homo- or heteromultimerized complexes of RAG1/2 are differently affected by HMG1.
Further deletion of RAG1 to aa 456 (which entirely removes the HD) very severely reduced the binding of RAG1ΔN456/RAG2ΔC to the RSS (Fig. A, lane 7); however, some residual binding is apparent after long autoradiography exposures (Fig. A, lanes 11 and 12), and the protein retains weak activity (43
) (Fig. B, lane 7). Deletion to aa 500 (RAG1ΔN500) eliminated binding (Fig. A, lanes 9 and 13). HMG1 did not enhance RSS binding and cleavage by the RAG1 forms lacking the HD (Fig. A, compare lanes 11 and 12 and lanes 13 and 14; Fig. B, compare lanes 7 and 8).
Similar results were obtained when the 12RSS was used, but the binding and cleavage activities of the RAG1/2 complex were enhanced only twofold (data not shown). HMG2, but not HMG-I(Y) or TCF-1b, was able to stimulate RAG1/2 binding in a very similar manner (data not shown). These results were also confirmed in vivo (see below and Fig. ).
FIG. 7 In vivo stimulation of RAG1 and RAG1/2 binding by HMG1,2. The binding of RAG proteins to a multimerized RSS in vivo can be measured by means of a mammalian one-hybrid assay (13). RAG1 and RAG2 proteins were transformed into transcriptional activators (more ...) HMG1,2 do not alter the sequence requirements for RSS recognition by RAG1/2.
RAG1/2 binding to the RSS is dependent on both the nonamer and heptamer motifs (13
). In order to explore whether the stimulatory effect of HMG1 on RAG1/2 binding is also dependent on both motifs, we assayed RAG1ΔN380/RAG2ΔC binding to mutant 23RSS. HMG1 stimulated binding to the heptamer mutants, while it failed to boost binding to the nonamer mutant (Fig. B).
FIG. 5 HMG1 enhances nonamer-dependent binding to the RSS. EMSAs with radiolabelled oligonucleotide probes carrying either wild-type (wt) or mutated RSS sequences. (A) Schematic representation of the RSS and the positions of the mutations employed. The mutations (more ...)
Using surface plasmon resonance (BIAcore), we previously showed that the RAG1 HD establishes specific interactions with the nonamer motif of the RSS, even in the absence of RAG2 (47
). To study the effect of HMG1 on the binding of RAG1 alone, we assayed the binding of RAG1ΔN380 alone to 12RSS mutants in the presence or the absence of HMG1 (Fig. C). Mutation of the first 3 or last 3 nucleotides of the heptamer (7mers m1 and m2, respectively) reduced RAG1ΔN380 binding, but HMG1 was still incorporated in the RAG-RSS complex, as shown by the slight supershift of the band corresponding to the complex. The HMG1-dependent stimulation of RAG1 binding to the mutated heptamer was slight (less than twofold) but reproducible (Fig. C, lanes 4 to 9). Mutation of positions 5 to 7 of the nonamer abolished binding of RAG1ΔN380 (Fig. C, lane 11), and addition of HMG1 failed to rescue it (lane 12). Essentially the same results were obtained when 23RSS mutants were used, except that RAG1 binding was significantly lower, as expected (data not shown). These results were confirmed in vivo (see below and Fig. ).
Thus, HMG1,2 stimulate RSS binding by RAG1 alone and in combination with RAG2 but do not alter the relative dependence of RAG1/2 binding on the heptamer and the nonamer.
Bending of RSS DNA by the RAG1/2-HMG1 complex.
RAG1/2 binds with greater affinity on the 12RSS than on the 23RSS (references 43
, and 52
and data not shown). Conversely, HMG1,2 have a more pronounced effect on the binding of RAG1/2 to the 23RSS. Based on this, it has been suggested that HMG1,2 bind and bend the spacer region of the 23RSS to bring the heptamer and nonamer motifs into close proximity (44
). This is in accordance with the DNA-flexing function of HMG1,2, which are known to bind to irregular or prebent DNA structures (8
) and to mediate bending of normal B-form DNA in ring closure assays (37
). Hence, we investigated by circular-permutation analysis whether HMG1 enhanced binding of RAG1/2 to the 23RSS through DNA bending. In this assay, proteins that induce DNA distortions show differential electrophoretic migration when bound to isomeric DNA probes containing their cognate DNA binding site placed at different sites along the probe (59
The 12RSS and 23RSS motifs were subcloned into the bending vector pBend2 (28
) and used as probes in EMSAs (Fig. A). In the absence of RAGs, HMG1 failed to interact with the pBend2-RSS probes (data not shown). RAG1/2 bound to the isomeric pBend2-RSS DNA with reduced overall efficiency compared to oligonucleotide probes. Moreover, due to the nonspecific DNA binding affinity of RAG1/2, binding to the large (150- to 161-bp) isomeric probes produced increased background levels compared to those with oligonucleotide probes (43 to 54 bp).
Unexpectedly, the RAG1/2 complex showed an intrinsic ability to bend the 12RSS DNA, even in the absence of HMG1,2 (Fig. B, lanes 1 to 6). The 12RSS probe was deflected by an angle that was estimated at between 43 and 49°, with the site of bending corresponding to the 12RSS itself (several gels were used to estimate the angle; Fig. C shows an example of the data from one such gel). Addition of HMG1 increased the deflection to between 55 and 60° without changing the site of bending (Fig. B; compare lanes 7 to 12 to lanes 1 to 6).
Binding and bending of the 23RSS probes by the RAG1/2 complex was almost undetectable (Fig. D, lanes 1 to 6), but the addition of HMG1 significantly stimulated binding (Fig. D, lanes 7 to 12). The RAG1/2-HMG1 complex bent the 23RSS DNA to a pattern similar to that of the 12RSS.
The DNA-bending properties of RAG1/2 on the 12RSS were also addressed by phasing analysis, where the RSS were placed at increasing distances from an intrinsic DNA bend induced by in-phase AT tracts (61
). The results on the phasing DNA probes (not shown) were comparable to the circular-permutation data.
HMG1,2 stimulate specific RAG1 and RAG1/2 binding in vivo.
To explore the effect of HMG1,2 on the DNA binding activity of RAG1/2 in vivo, we utilized the previously described one-hybrid assay (13
). Briefly, the RAG1 and RAG2 proteins were converted to transcriptional activators by adding the acidic domain of the herpes simplex virus protein VP16. A reporter construct provides the substrate for binding of RAG1/2 to the RSS: multiple copies of the RSS are cloned in front of a minimal promoter driving expression of the luciferase gene (Fig. E). Cotransfection of RAG-VP16 constructs with the reporter in mammalian cells leads to transactivation of the luciferase gene to a degree directly proportional to RAG binding. Luciferase activity was normalized for transfection efficiency and background levels as described in Materials and Methods. The expression levels of all recombinant proteins were verified by Western analysis to ensure comparability of the results.
Overexpression of HMG1 or HMG2 stimulated binding to the 12RSS of RAG1 and RAG1/2 but not of RAG2 alone (Fig. A). HMG-I(Y) had no major effect, but TCF-1b invariably slightly enhanced the binding of RAG1/2 to the 12RSS (Fig. A).
HMG1,2 increased binding of RAG1 alone to the 12RSS by 3- to 4-fold and enhanced binding to the 23RSS by about 10-fold (Fig. B). Mutation of the nonamer severely reduced binding of RAG1 alone and was not compensated for by addition of HMG1,2, while mutation of the heptamer allowed RAG1 binding and stimulation by HMG1,2 (Fig. B). These findings are in accordance with the in vitro results showing that HMG1,2 did not alter the sequence requirements for RSS recognition by RAG1 (Fig. ).
The lack of effect of HMG1,2 on the sequence requirements of the RAG1/2 complex (as opposed to RAG1 alone) was also verified (Fig. C). It is worth noting that HMG2 stimulated better binding to the 23RSS than HMG1 (Fig. B and C).
As further controls, and to allow direct comparison with the in vitro results, GST fusion deletion mutants (homologous to the ones used for the in vitro DNA binding assays [Fig. B]) were produced as RAG1-VP16 fusions and analyzed in the one-hybrid assay (Fig. D). Only RAG1ΔN330 and -ΔN380 retained specific binding to the RSS DNA, which was stimulated by HMG1,2. Conversely, RAG1ΔN456 and -ΔN500, from which the HD has been deleted, showed no specific binding, which was unaffected by overexpression of HMG1,2.
HMG1,2 stimulate V(D)J recombination in vivo.
We tested the overall effect of HMGs on V(D)J recombination by conducting the extrachromosomal substrate recombination assay in the presence of overexpressed HMGs (Fig. ). RAG1, RAG2, the recombination substrate pJH299, and vectors expressing the various HMGs were cotransfected in 293T cells, and recombined products (signal joints) were detected by PCR analysis (56
). The amounts of PCR products were strictly proportional to the input material, as indicated by titration experiments (not shown) and by 10-fold dilution of the template (compare Fig. B and B′).
FIG. 8 HMG1,2 increase the yield of V(D)J recombination products in vivo. 293T cells were cotransfected with the recombination substrate pJH299 and the expression constructs for the indicated proteins. The RAGs were GST tagged, while HMG1, HMG2, HMG-I(Y), and (more ...)
We directly estimated the amounts of protein expression directed from the transfected plasmids by Western blotting with anti-GST or anti-HA antibodies and judged them to be comparable for different HMGs and very similar for RAGs in all samples (Fig. C). Finally, we estimated the amounts of HMG1 and HMG2 overexpression by Western blotting with anti-HMG1 (Fig. C′) or anti-HMG2 (Fig. C") antibodies. The proteins expressed from transfected plasmids contain a tag and run with slightly lower mobility than the natural HMG1,2. Our results indicate that the cellular pool of HMG1,2 can be transiently increased about threefold in 293 cells.
HMG1,2 drastically stimulated V(D)J recombination efficiency, in contrast to HMG-I(Y) and TCF-1b (Fig. D). We obtained identical results with assays conducted in 293 and 3T3 cells, using either the deletional substrate pJH200 or the inversional substrate pJH299 (data not shown). Thus, the ability of HMG1,2 to boost the DNA binding properties of RAG1/2 has a clear effect on the overall efficiency of V(D)J recombination in vivo.