The Role Of LEDGF/p75 In Transcriptional Regulation

The Lens Epithelial Derived Growth Factor p75 (LEDGF/p75) is a chromatin bound protein whose cellular function is not yet clearly known. A role in transcriptional regulation had been previously proposed based on its interaction with the basal transcriptional machinery and on its effects on the expression of genes involved in the cellular response to environmental stresses. To further elucidate the function of LEDGF/p75, we conducted a global and unbiased evaluation of the role of this protein in gene expression. To that aim, we performed a microarray analysis of cellular gene expression in cells that are severely depleted of LEDGF/p75. To minimize cell type-specific observations, we used three different LEDGF/p75 deficient human cell lines: embryonic kidney epithelial cells (HEK293) and two different lines of CD4+ T cells (SupT1 and Jurkat). By taking the intersection of the three data sets, using a fold-change of greater than two and a student t-test confidence value of 90% we have identified a group of genes whose expression is dysregulated in at least two of the cell types tested. The potential role of LEDGF/p75 in transcriptional regulation of responsive genes has been described as operating at the level of their promoters as a general co-activator, connecting members of the basal transcriptional apparatus to DNA sequence specific transcriptional activators. However, the observation that LEDGF/p75 is bound to sites within the chromatin across the genome suggests that this protein is also present inside actively transcribed genes. In correlation with this observation, LEDGF/p75 has been demonstrated to promote integration of cDNA copies of the HIV-1 genome within actively transcribed genes. This localization of LEDGF/p75 inside actively transcribed genes suggests a possible role of LEDGF/p75 in transcriptional elongation. To further investigate whether LEDGF/p75 has a role in transcriptional elongation, we performed a transcriptional profile analysis of genes we previously identified as dysregulated in SupT1 and HEK293 cells. We used chromatin immunoprecipitation and quantitative real time PCR analysis to demonstrate that LEDGF/p75 is located on the intragenic regions of these genes as well as their promoters. LEDGF/p75 occupancy on these genomic locations correlated with the gene transcriptional activity. We also demonstrate that LEDGF/p75 coimmunoprecipitates with CDK9 and the FACT complex, two separate components of the transcriptional elongation complex, and corroborated this association by quantitative confocal colocalization. In order to understand the mechanism of LEDGF/p75 on transcriptional regulation at the promoter level, we studied the effect of LEDGF/p75 on the shared bi-directional promoter of the LEDGF/p75-regulated genes DTX3L and PARP9. Our data indicate that LEDGF/p75 negatively regulates the expression of this promoter. Two known motifs within LEDGF/p75, the HIV-1 Integrase binding domain and the PWWP domain, were both required for this inhibitory effect. These data suggest that the effect of LEDGF/p75 on the transcription of the DTX3L/PARP9 genes at the chromatin level may involve not only the promoter regions but other genetic elements as well. Alternatively, LEDGF/p75 could require the interaction with other cellular factors that are limiting in cells that are over expressing LEDGF/p75. LEDGF/p75 has been reported to be involved in the adaptation to cellular stress in cells over expressing this protein; however, our data indicate that under basal endogenous conditions LEDGF/p75 does not significantly affect the regulation of genes involved in stress response. To clarify this potential protective effect we studied the capability of LEDGF/p75-deficient cells to adapt to environmental stresses. Our data indicate a protective effect of LEDGF/p75 to these stresses being necessary for cell viability under these conditions. To further understand this effect of LEDGF/p75 on cellular response to stress we determined the differential distribution of LEDGF/p75-interactor proteins involved in transcriptional regulation in response to cellular insults. We demonstrate that LEDGF/p75 influences the subcellular distribution of SSRP1 and CDK9 in response to environmental stresses. LEDGF/p75 was required to mediate recruitment of both SSRP1 and CDK9 to transcriptionally active complexes following these cellular insults. In summary, our data indicate that LEDGF/p75 interacts with components of the basal transcription machinery and of the transcriptional elongation complex, suggesting that this protein regulates the transcription of LEDGF/p75-responsive genes at the initiation and elongation steps of transcription. Additionally, LEDGF/p75 participates in the recruitment of the transcriptional elongation factors SSRP1 and CDK9 to chromatin under basal conditions and in response to cellular stresses