LSF represents a novel family of homo-oligomeric transcription factors that bind direct DNA repeats. In the human α-globin and γ-fibrinogen sites and chicken α-crystallin gene, LSF binds preferentially as a homotetramer (29
). Our findings suggest a model in which LSF binds to the RCS as a homotetramer, recognizing two of the LSF motifs within the RCS, including the most 3′ motif. We find that LSF binding to the HIV LTR is regulated by phosphorylation of LSF.
DNase I footprinting and gel shift experiments show that LSF binds with high affinity to the LTR (19
). The LSF binding site is characterized by a 4-bp motif separated by a linker sequence composed of five or six nucleotides (CTGG-N5/6
). This motif is repeated in the LTR three times, within the −10 to +27 sequences.
Using bacterially expressed LSF and radiolabeled oligonucleotides, we characterized LSF binding to the LTR in greater detail. Mutation of any two of the three 4-bp binding sequences reduces but does not abolish LSF binding. Interestingly, the mutant oligonucleotide M2, which has the two CTGG motifs separated by 15 bp, can bind LSF to the same extent as the mutants M1 or M3, in which the CTGG motifs are separated by five or six nucleotides. This suggests that the altered linker length destabilizes binding but does not eliminate the ability of LSF to bind its cognate sequence. Moreover, the binding of LSF to single site 3 seems to be as efficient as LSF binding to any combination of two 4-bp motifs. It is possible that LSF binds first to site 3, and this interaction allows the protein to recognize the second and first binding 4-bp motif, possibly because site 3 in this context is more accessible to LSF.
Although these results were obtained using oligonucleotides and so do not fully represent the interactions of transcription factors and DNA in the native chromatinized context, it is interesting that our results echo those of previous studies. Using DNase footprinting assays, Jones et al. found that out of all the sets of clustered point mutations in each LSF binding motif at the LTR, only mutations in all three motifs eliminated the binding of LSF (19
By analyzing the DNA-protein complexes from mixtures of LSF derivatives of different sizes, we conclude that LSF binds to the HIV LTR as a tetramer, in agreement with Shirra and Hansen (38
). However, our results differ from those obtained by Zhong et al. (57
). In this article, the authors suggest that LSF binds to the LTR as a dimer. This difference can be explained by the fact that the authors used bacterially produced LSF isolated by glycerol gradient sedimentation and that LSF as a dimer in solution can bind DNA. However, dimers formed in solution could form tetramers upon interaction with DNA, as we observed. In gel shift experiments using LSF and GST-LSF, the authors did not explain the presence of other complexes with intermediate mobility between LSF and GST-LSF-HIV DNA (57
We have previously proposed that LSF plays a role in maintaining or establishing transcriptional repression of the LTR (8
). This effect is mediated by the formation of the repressor complex at the LTR, consisting of LSF binding to HIV DNA, YY1 interacting with LSF via its zinc fingers, and histone deacetylase interacting with YY1. We have also shown that repression is blocked when LSF binding to the LTR is inhibited (8
). We therefore sought to understand the signaling pathways that regulate LSF binding to the LTR and consequently regulate formation of repressor complex.
LSF can be phosphorylated by the cellular kinases Erk, p38, and Jnk. The LSF residues phosphorylated by Erk have been mapped and appear to be distinct from those phosphorylated by p38; Jnk appears to act on both sets of residues. These findings suggest that the in vivo regulation of LSF binding may be quite complex (42
). We have shown in vitro that phosphorylation of recombinant LSF by the activated kinase Erk or p38 is sufficient to regulate its binding to the HIV LTR but not to the SV40 late promoter. Phosphorylated LSF using activated Erk decreased its binding to the RCS, and conversely, LSF binding to the RCS is increased by p38 phosphorylation. These findings suggest that LSF binding to the HIV RCS is subject to counterregulation by MAP kinases.
It has been shown that LSF is phosphorylated by Erk in vitro on the same residues that are phosphorylated in vivo, with serine 291 being the major site of phosphorylation (43
). However, in chromatin immunoprecipitation assays, we were unable to observe a significant increase in LSF occupancy after Erk inhibition by U1026. Either Erk phosphorylation of LSF does not play a role in vivo or there is little phosphorylation at Erk-responsive residues in LSF within the model system that we studied and so no change in LSF occupancy can be detected upon Erk inhibition.
The in vitro effect of p38 on LSF binding to the LTR site was confirmed in chromatin IP experiments. The specific inhibition of p38 decreases LSF occupancy at the LTR, and an agonist of p38, anisomycin, increases LSF occupancy. This provides strong evidence that p38 regulates LSF binding to the HIV LTR. Moreover, this decrease of LSF occupancy is accompanied by increased histone acetylation at nucleosome 1 of the LTR, an event that is correlated with transcriptional activation (5
) and consistent with the requirement for LSF for recruitment of HDAC1 to this site.
Further, phosphorylation of LSF regulates the ability of the LTR promoter to be repressed. The p38 antagonist ablated repression of Tat activation of the integrated LTR. These experiments confirmed other studies showing that LTR activity increases when LSF binding is blocked (54
Several investigators have studied the role of MAPK, such as p38 and Erk, in the activation of LTR in different cells lines and in primary PBMCs (7
). These studies, performed in the context of activation of LTR by cytokines and stress, found that p38 inhibition blocks the activation of the HIV LTR in HeLa-LTR indicator cells in response to IL-1, tumor necrosis factor, UV radiation, or osmotic stress (23
). Similar results were reported for the monocytic cell line U1 and for PBMCs (36
However, we found that specific inhibition of p38 activity induced the outgrowth of latent HIV when the resting CD4 cells of aviremic patients were exposed ex vivo. Maintaining these CD4+ lymphocytes ex vivo with IL-2 (20 U/ml) did not result in virus outgrowth. Global inhibition of p38 activity is likely to alter many gene expression programs within a cell. This complexity likely explains the contradictory findings observed in different experimental systems; the effect of a biological signal in vivo is likely to result from the integration of many such opposing and synergizing pathways.
Nevertheless, when exposed to the specific inhibitor of p38, CD4+
cells did not become activated or more permissive for viral replication. Therefore, p38 inhibitors or future reagents with greater specificity for the cascade of factors that regulates HDAC1 recruitment to the HIV LTR might be considered as future therapeutic candidates to disrupt viral latency in patients on suppressive antiviral therapy (22