Nucleic Acids Research, 2011, Vol. 39, No. 14 5883 Figure 2. Nef and HSF1 proteins are co-recruited on HSE of HSP40 promoter in HIV-1-infected cells. (A) HSF1 is required for Nef-mediated activation of HSP40 promoter. HSF1 and Sp1 depleted 293T cells were co-transfected with HSP40-luc and Nef expression vectors and analyzed for luciferase activity. Gene silencing efﬁciency of HSF1 and Sp1 siRNAs are shown as inset. (B) Both HSF1 and Nef activate HSP40 promoter-driven gene expression in 293T cells as analyzed by luciferase assay. (C) Schematic representation of HSP40 promoter showing the position of primers used in ChIP analysis. (D) Nef is recruited on HSP40 promoter during HIV-1 infection. ChIP analysis was performed with HIV-1-infected CEM-GFP cells using Nef antibody followed by PCR ampliﬁcation using F2 and R1 primers. (E) Nef and HSF1 are co-recruited on HSP40 promoter as analyzed by sequential ChIP. Primary immunoprecipitation was performed with Nef and secondary immunoprecipitaion with HSF1 or Sp1 antibody followed by PCR analysis using F2 and R1 primers. (F) Nef and HSF1 is recruited speciﬁcally at HSE elements on HSP40 promoter. Sequential ChIP analysis was performed as in E above using either F1 and R2 or F2 and R1 primer sets. The error bars represent the mean ± SEM of three independent experiments. Statistical analysis was performed using Student’s t-test, with the levels of signiﬁcance deﬁned as *P < 0.05. promoter-luciferase construct. Our results show that silencing of Sp1 did not signiﬁcantly reduce Nef- mediated induction of HSP40 promoter, whereas HSF1 knockdown resulted in signiﬁcant reduction in promoter activity as compared to control siRNA transfected cells (Figure 2A). When both Sp1 and HSF1 were silenced, the HSP40 promoter activity was the same as that ob- served with HSF1 alone silenced cells. These results sug- gest that HSF1 plays an important role in Nef induced HSP40 expression. As HSF1 was known to be involved in regulation of HSP promoters by interacting with HSE elements and Nef was also observed to mediate its effects through HSE elements above, we then co-transfected HSF1 or Nef along with HSP40 promoter-luciferase con- struct to analyze the promoter activity. The results indicate that although Nef or HSF1 alone can induce the promoter activity but when expressed together, they show signiﬁcantly more effect (Figure 2B). As the inducible effect of Nef on HSP40 promoter seems to be associated with HSF1, we then looked at the recruitment of Nef on HSP40 promoter at HSE elements as shown in Figure 2C by ChIP assay. Cross-linked chro- matin from HIV-1-infected cells was pulled down with Nef antibody followed by PCR ampliﬁcation using primers F2 and R1 (Figure 2C) spanning HSE region of HSP40 promoter. Strong PCR signal was observed only in in- fected cells (Figure 2D, lane 3) indicating recruitment of Nef on HSP40 promoter sequence encompassing HSE elements. Since HSE is the consensus site for HSF1, we then checked the co-occupancy of Nef/HSF1 or Nef/Sp1 on the HSE elements (Figure 2E) by sequential ChIP assay. Co-recruitment of Nef and Sp1 was not detected (Figure 2E, lane 2), whereas recruitment of both Nef and HSF1 on HSE elements was clearly observed in HIV-1-infected cells (Figure 2E, lane 3). Also, no detect- able ampliﬁcation was observed in PCR using F1 and R2 primers encompassing a region upstream to HSE elements (Figure 2F, lane 1) whereas strong PCR signal was observed with primers F2 and R1 encompassing the region containing just HSE elements (Figure 2F, lane 2),