FIG. 6.

Depletion of chromatin reassembly factors reactivates HIV concomitantly with increased chromatin accessibility at the HIV promoter. (A) Increased sensitivity of chromatin to MNase digestion upon Spt6 depletion. J-Lat E27 cells expressing control or Spt6 shRNA were submitted to controlled MNase digestion as indicated in Materials and Methods after nucleus preparation. After purification of mononucleosomal DNA, DNA resistance to MNase was quantified using real-time PCR and oligonucleotides covering HIV regions depicted in the schematic drawing. Signals were normalized to naked DNA purified from the same cells before MNase digestion, PCR amplified with the same set of primers, and to the values of PCR amplification with primers for a region considered to correspond to linker DNA free of nucleosomes. Data are expressed as relative units of DNA resistance to MNase, i.e., (PCR amplification/linker)/gDNA. As a control, MNase resistance of chromatin in J-Lat E27 cells treated with PMA (10 nM), TSA (400 nM), and TNF-α (10 ng/ml) for 4 h, compared to that in untreated cells, was also assayed and is shown in the right panel. (B) UBXD8 chromatin sensitivity to MNase. DNA resistance to MNase at UBXD8 exon 8-intron 8 regions in J-Lat E27 cells expressing control or Spt6 shRNA was determined as described for panel A with three pairs of specific oligonucleotides. (C) Effect of Spt6 depletion on cellular gene expression. RNA was extracted from J-Lat E27 cells expressing control or Spt6 shRNA, and expression of several genes or the indicated exons of the UBXD8 gene was measured by RT-qPCR using specific oligonucleotides. RPL31 expression was measured for normalization. In order to compare different amplicons, qPCR was performed in parallel from genomic DNA (gDNA). Data are expressed as relative units (RU) of (cDNA amplification/RPL31)/gDNA amplification. (D) HIV reactivation occurs earlier than UBXD8 inhibition upon Spt6 depletion. J-Lat E27 cells were infected with control or Spt6 shRNA expression lentiviruses and selected in puromycin. At 1 or 2 days after puromycin addition, RNA was extracted and Spt6, HIV, and UBXD8 gene expression was measured by RT-qPCR. RPL31 expression was measured for normalization. Data are expressed as fold change (FC) of relative units (RU) of cDNA amplification/RPL31 in shSpt6 compared to that in shControl.