Possible requirement of histone H3 Ser10 phosphorylation for the occupancy of the Sp1 site at the LDL receptor promoter. It is important to know whether basal transcriptional machinery binds to the LDL receptor promoter in vivo in the presence of SP600125. To answer this question, ChIP analysis was performed to compare the association of transcription factor Sp1 and RNA Pol II to the LDL receptor promoter BAY 73-4506 in untreated and SP600125 treated cells. As shown in Fig. 6A, the level of Sp1 decreased at the LDL receptor promoter, and maximal suppression was observed at 3 h of SP600125 treatment, followed by a slight increase at 6 h. A comparison of the kinetics of reductions in histone H3 Ser10 phosphorylation and Sp1 occupancy at the LDL receptor promoter with the decrease in LDL receptor expression indicates that the SP600125 dependent decrease in LDL receptor expression correlates with the status of histone H3 Ser10 phosphorylation and the Sp1 occupancy.
Importantly, repression of the LDL receptor expression reached ZSTK474 significant levels at concentrations that inhibited histone H3 Ser10 phosphorylation and Sp1 recruitment. This suggests involvement of histone H3 Ser10 phosphorylation in the recruitment of Sp1 at the LDL receptor promoter in vivo. We also considered the possibility that the mechanisms underlying the action of transcription factors and coactivators are their effect on the initiation and or elongation of transcription by RNA Pol II. We extended the ChIP assays to examine the effect of SP600125 on RNA Pol II occupancy at the LDL receptor promoter and the downstream coding region .
Interestingly, similar efficiencies of association were observed at 1 h and 3 h, whereas a reproducible decrease in the occupancy of RNA Pol II was observed following SP600125 treatment for 6 h at both the promoter and the downstream coding region. The occupancy of the actin promoter was not significantly different under identical conditions. These results raise an intriguing possibility that loss of histone H3 Ser10 phosphorylation and or Sp1 occupancy slightly impairs recruitment of RNA Pol II, without affecting transcriptional elongation by Pol II. As a result, the maximal reduction in LDL receptor expression by SP600125 at 6 h may be due to a reduction in the recruitment of both Sp1 and RNA Pol II.
Using in vivo genomic footprinting assays, we also examined the occupancy of the Sp1 and SRE 1 sites of the LDL receptor promoter when cells were treated with SP600125. By monitoring the protection from DMS methylation, we observed a significant and reproducible decrease on both strands in the occupancy of the Sp1 site, which has been shown to be involved in controlling basal LDL receptor expression. The possibility that reduced occupancy of the Sp1 site by SP600125 is due to reductions in the expression, DNA binding, or transactivation potential of Sp1 was also examined. No significant differences were observed between untreated and SP600125 treated HepG2 cells, suggesting that lower accessibility of Sp1, and not changes in expression, DNA binding, or transactivation, is responsible for the compromised interaction of Sp1 at the LDL receptor promoter.