Three alanine substitutions in the CHD of Tf1 IN and one CHD-deleted mutant caused significant reductions in transposition frequency. The results of the immunoblotting showed that, apart from a small reduction caused by V1290A, the mutations did not alter the levels of the IN and RT expressed by Tf1. DNA blots indicated that, except for the 33% reduction caused by W1305A, the levels of cDNA produced were also not significantly changed. By measuring homologous recombination, we determined that the mutations in Tf1 had little or no effect on the accumulation of cDNA in the nucleus. Nevertheless, all four of the mutants exhibited substantial reductions in transposition. Thus, these drastic reductions in transposition likely resulted from defects that occurred specifically at the integration step of the transposition cycle.
The mutation CHDΔ reduced the amounts of cDNA that bound IN. Although this result indicates that the CHD contributed to the binding of cDNA, experiments that tested each domain of Tf1 IN for DNA binding activity revealed that as a recombinant fragment, the CHD itself does not bind DNA (10
). Instead, strong DNA binding activity in the C-terminal domain was mapped to the GPY/F domain, a region of IN just upstream of the CHD. Together, these findings suggest that Tf1 cDNA may have bound the GPY/F domain and that the deletion of the CHD reduced this binding by generating a conformational change. Testing the GPY/F domain in vivo for a role in binding cDNA will be difficult since single amino acid substitutions in the conserved residues caused IN to be degraded (10
). Alternatively, it is also possible that the interaction detected between IN and the cDNA is indirect and that another factor is responsible for binding the cDNA.
The ChIP assays revealed that CHDΔ caused a 2.7-fold reduction in binding of IN to cDNA. Although this defect in cDNA binding was substantial, this does not appear to be sufficient to account for the 14.3-fold reduction in transposition caused by the mutation. Assuming a direct interaction between IN and cDNA, one possibility is that the mutations may have inhibited binding of IN to both ends of the cDNA. Since our ChIP assay measured only binding of IN to the downstream LTR, the cumulative defect in cDNA binding could potentially be the square of what was detected. Thus, the defect in binding cDNA for CHDΔ could be a large as 7.3-fold. Currently, we are unable to test this possibility directly because the ChIP assay as designed with the AI cannot be used to test binding of IN to the upstream LTR. Primers that amplify the upstream LTR of the cDNA would also detect copies of Tf1 in the expression plasmid. It is also possible that the 2.7-fold reduction in binding of CHD-deleted IN to cDNA was an underestimate of the true defect. Since the anti-IN antibody used for ChIP was more efficient in precipitating CHDΔ than wild-type IN (Fig. ), the defect in the interaction between IN and cDNA is likely to be greater than 2.7-fold.
It is possible that the mutations in the CHD reduced the catalytic activity of IN. Such mutations may allow interaction with target DNA but have an indirect effect on the catalytic site. This kind of defect is consistent with the mutations that did not significantly lower levels of IN, cDNA, or the amounts of cDNA available for homologous recombination in the nucleus. However, one result that argues against a defect in catalytic activity is that as a recombinant protein, the CHDΔ form of IN had four to seven times greater strand transfer activity than wild-type IN (19
The point mutations in the CHD and the CHDΔ IN showed comparable reductions in transposition frequencies (Fig. ). However, only the CHDΔ mutation caused a profound effect on target recognition. In the absence of the CHD, Tf1 demonstrated a drastically reduced ability to target the pol II promoter regions of fbp1, ade6, and SPCC4F11.03c. The disparity between the CHDΔ and the point mutations in their ability to direct integration to the promoters indicates that the CHD has two separate functions. One function contributed significantly to the frequency of integration. The other function of the CHD, as revealed by CHDΔ, contributed to the selection of target sites. The role of the CHD in positioning integration is likely due to interactions with a chromatin factor. Our data do not address whether the single amino acid mutations in the CHD may have lowered integration frequencies by partially disrupting an interaction. The deletion of the CHD may have fully disrupted the interaction, resulting in both low integration frequencies and an inability to recognize pol II promoters. Alternatively, the interactions responsible for integration efficiency may be distinct from those required for target selection.
Cellular proteins have been shown to play a role in directing the position of integration of retrotransposons. For example, the Sir4 protein recruits Ty5 IN and promotes integration at heterochromatic regions in the telomere and the silent mating loci of the budding yeast Saccharomyces cerevisiae
). Similarly, Ty3 IN requires transcription factors of RNA Pol III to direct highly selective integration into Pol III transcript start sites (5
). For HIV, integration occurs preferentially in highly active transcription units (32
), and the cellular protein LEDGF binds the IN of HIV type 1 and mediates the selection of target sites (7
It has been recently reported that Tf1 integration is directed to pol II promoters by transcription activators such as Atf1p (28
). That work demonstrates that Atf1p plays a role in recruiting Tf1 IN to the promoter of fbp1
but not to the divergent promoters of bub1
. However, our study shows that the absence of the CHD not only disrupted the targeted integration at the promoter of fbp1
but also caused similar defects in targeting the promoters of bub1-ade6
. From these results, we speculate that the CHD of Tf1 IN may function together with multiple factors of chromatin that are commonly found at promoters where Tf1 integrates. This feature of chromatin could be a variety of transcription factors that together are responsible for many targets of Tf1 integration. Atf1p belongs to the basic leucine zipper family of transcription factors and shares its highly conserved bZIP motif with other transcription factors found in S. pombe
, such as Pap1p, Atf31p, Zip1p, and Atf21p (4
). One or more of these proteins, together with the CHD, could be required for targeting Tf1 to the bub1-ade6
The relatively few insertion events that were observed in the bub1-ade6
promoters in the absence of the CHD were due to residual integration activity of the truncated IN. Although CHDΔ no longer preferred the promoter sequences, the insertions were not randomly positioned. Interestingly, the absence of the CHD caused integration to occur at nucleotide 340 of the β-lactamase gene, as demonstrated by 6 insertion events out of 10 in the plasmid with the bub1-ade6
promoter and 5 insertion events out of 9 in the fbp1
plasmid. Thus, in the absence of the CHD, Tf1 developed a new integration preference. Since the β-lactamase gene is from E. coli
, this preference is likely a result of fortuitous interactions. One possibility is that IN lacking the CHD may retain low levels of binding to some transcription or chromatin factor that binds to the β-lactamase gene. Alternatively, the DNA sequence of the β-lactamase gene at nucleotide 340 may have a structural perturbation that enhances integration. The integration sites of several transposons have structural features that are recognized by the transposases (17
The selection of target sequence for integration by retroviruses such as HIV and murine leukemia virus (MLV) has important consequences for both the virus and the host. Retroviral integration can affect host gene expression due to insertion of viral promoters or enhancers. Because of their target site preferences, the use of retroviral vectors in gene therapy is associated with great risk. MLV vector-based treatment of X-linked severe combined immunodeficiency led to the activation of the cellular proto-oncogene LMO-2 and contributed to the development of leukemia (16
). These setbacks have generated intense interest in understanding integration site selection. Tf1 exhibits a similar preference for insertion as MLV, as both elements target the promoters of genes. The molecular mechanism of MLV integration is still unknown. In light of the role of the Tf1 CHD in mediating target site selection, further studies of Tf1 integration may lead to improved understanding of how retroviruses target integration.