Research hints at a role of LEDGF/p75 in stress response [See (12
) and references therein]. LEDGF/p75 expression is induced by oxidative stress and LEDGF/p75 itself is believed to activate stress-related genes by binding stress response and heat shock-related elements (4
). In this work, we defined the LEDGF/p75 chromatin binding profile in the ENCODE region using DamID. DamID technology is extensively used in Drosophila
genomics and is as powerful as ChIP-on-chip to determine chromatin binding profiles (50
). While the resolution is the same as for ChIP-on-chip (1–2 kb), DamID has the advantage that neither antibody nor cross-linking is needed. Furthermore, expression of the Dam fusion protein at extremely low level prevents aspecific and saturating methylation levels, as well as disturbance of the normal cellular physiology.
Analysis of the resulting LEDGF/p75 binding islands revealed that 75% of LEDGF/p75 islands were located in TUs although the promoter region itself was disfavored. TUs are not enriched for ‘gatc
’ sites as compared to the whole ENCODE region (P
< 0.05, Mann–Whitney U-test). Hence, enrichment of the LEDGF/p75 binding islands within TUs cannot be attributed to the ‘gatc
’ dependence of the DamID technology, but is indicative of genuine LEDGF/p75 enrichment in these regions. LEDGF/p75 binds downstream of the TSS with a frequency decreasing towards the end of the TU. These results seem at odds with our current understanding of the cellular function of LEDGF/p75, since an activator of expression of stress response genes would be expected to be enriched at promoter sites. Though some studies indicated that LEDGF/p75 binds specific sequence elements associated with the promoter regions of stress response genes (4
), others failed to detect sequence-specific LEDGF/p75 binding activity in vitro
). It can however not be excluded that LEDGF/p75 binds stress response elements during stress conditions. Of note, the ENCODE region does not contain stress-responsive genes. A future DamID experiment with stress-induced cells could shed light on this issue. Nevertheless, at this stage our results demonstrate that LEDGF/p75 chromatin binding is not limited to stress response genes since it amounts to 28% of the ENCODE RefSeq genes. Although the proportion of LEDGF/p75-bound genes might be underestimated due to the strict threshold used to analyze the microarray data, this is significantly less than the 37% for the corresponding control set, suggesting that LEDGF/p75 targets only a specific subset of genes. Though we have to take into account the relatively small amount of genes present in the ENCODE region, Gene Ontology analysis did not support any significant functional enrichment for certain gene product characteristics. Twenty-five percent of the LEDGF/p75 islands located outside of TUs. The mean score and size of this fraction is indistinguishable from that of all islands suggesting that these represent genuine LEDGF/p75 islands. In addition, the mean distance of this fraction to TUs does not differ from random (data not shown). The function of those islands remains to be investigated. At this stage, one can only speculate that these islands may have an important function in the establishment of latent lentiviral proviruses.
As expected and based on the known association between LEDGF/p75 binding and HIV-1 integration, the LEDGF/p75 chromatin interaction profile is reminiscent of that of HIV-1 integration (28
). Much alike HIV-1 integration, LEDGF/p75 binding prefers the body of genes, disfavoring the promoter regions and correlating with transcriptional activity. Compared to 75% of the LEDGF/p75 islands, 86% of the ENCODE region-associated HIV integration sites were found in a TU and 30% of all integration sites were found in a window of 3.5 kb around the center of an LEDGF/p75 island, which amounts to a more than 3-fold enrichment over control integration sites. These data corroborate that LEDGF/p75 plays a role in HIV-1 targeting. This apparent window might however be influenced by the resolution and sensitivity of the DamID technology. Moreover, our DamID experiments were carried out in HeLaP4 cells, while the integration data set was derived from a Jurkat T cell line. Nevertheless, controlling our analysis for different expression profiles or chromosomal content between both cell lines did not significantly change the obtained results.
The correlation of the majority of the more than 200 studied ENCODE tracks with the LEDGF/p75 binding profile often mirrors that of HIV integration [this article and (28
)]. Overall, LEDGF/p75 binding was associated with markers of active transcription like H3 and H4 acetylation, H3K4 monomethylation and RNA polymerase II binding, but correlated negatively with markers of heterochromatin. The cross-correlation curves with LEDGF/p75 binding sites revealed interesting patterns and some striking differences with HIV integration sites. In most cases, ENCODE tracks with a strong correlation with LEDGF/p75 chromatin binding also showed a high coefficient of correlation with HIV integration. While the correlation with LEDGF/p75 peaked over a relatively small window, that with HIV integration was more spread out around the LEDGF/p75 peak, in line with the window of enrichment of HIV-1 integration straddling LEDGF/p75 binding spots. These results indicate that the DamID resolution is high enough for comparison of LEDGF/p75 chromatin interaction with HIV integration and again suggests that HIV integrates in the wide neighborhood of LEDGF/p75 binding.
Interestingly, our data point out that not all chromatin bound LEDGF/p75 supports effective HIV integration. Indeed, the transcription factors Stat1, Hnf4a, Hnf3b and Usf1 correlated well with LEDGF/p75 binding but not with HIV-1 integration. The chromatin binding profile of this integration incompatible LEDGF/p75 fraction is indistinguishable from that of the complete LEDGF/p75 track (data not shown). HIV-1 integrase interacts with the integrase binding domain of LEDGF/p75, which is known to bind as well to other proteins like Jpo2, pogZ, MLL/menin and Cdc7-ASK (7
). It will be of interest to verify whether the chromatin binding of the LEDGF/p75 fraction that is incompatible for HIV integration correlates with the binding profile of one of these alternative partners. Competition in the binding with LEDGF/p75 may abrogate efficient integration.
In conclusion, the LEDGF/p75 chromatin binding profile corroborates the previously claimed association between LEDGF/p75 binding and HIV-1 integration. Still, other determinants seem to play a role since not all LEDGF/p75 sites support HIV integration, and integration can occur at some distance from the actual LEDGF/p75 chromatin interaction spot. Moreover, our data challenge the current concept on the role of LEDGF/p75 in cell metabolism. It is clear that the function of LEDGF/p75 is not restricted to stress response. The more general chromatin interaction profile is compatible with a global role in transcriptional regulation. Of note, LEDGF/p75 was originally identified as a component of the general transcriptional machinery (1
). In this regard, it will be of interest to analyze the expression and chromatin interaction profile of LEDGF/p75 in response to cellular stress.