In fact, the degree of p42 44MAPK activation determines the extent of induction . In contrast, stress activated p38MAPK negatively AZD8931 regulates LDL receptor expression. Recently, we observed that phorbol ester dependent LDL receptor induction was associated with an increase in local histone H3 Ser10 phosphorylation independent of p42 44MAPK in HepG2 cells. However, information is lacking regarding precisely how this modification integrates with the transcriptional machinery responsible for regulating LDL receptor induction. Furthermore, while the majority of histone H3 Ser10 phosphorylation studies have focused on the mechanisms of gene activation, the results obtained have been limited in their application to the transcriptional initiation process.
During our investigation of the role of c Jun N terminal kinase in cytokine induced LDL receptor transcription, we made an observation that treating human hepatoma HepG2 cells with SP600125 {anthrapyrazol 6 one} alone blocked global histone H3 Ser10 phosphorylation. In the present study, we expanded on this early observation and directly evaluated the influence CHIR-99021 of global histone H3 Ser10 dephosphorylation on localized histone H3 Ser10 phosphorylation and the basal expression of genes involved in cholesterol homeostasis. We show that SP600125 strongly and specifically inhibited global histone H3 Ser10 phosphorylation, possibly independent of the p46 54JNK pathway. Moreover, SP600125 reduced the basal endogenous levels of LDL receptor expression without affecting the expression of other genes involved in cholesterol homeostasis.
Lastly, using the techniques of chromatin immunoprecipitation, a direct correlation was observed between the levels of histone H3 Ser10 dephosphorylation, the loss of Sp1 site occupancy, and LDL receptor transcription. These results suggest a model whereby histone H3 Ser10 phosphorylation regulates the accessibility and activity of basal transcriptional machinery for specific genes and enable us to discuss models by which repression may be mediated. Our findings reveal important differences in the precise ways by which the LDL receptor and other genes involved in cholesterol homeostasis are regulated, and they suggest that the existence of subtle differences in the nature of histone H3 Ser10 phosphorylation may be responsible for an efficient and gene specific transcriptional control. Thus, the LDL receptor represents one of the few known natural model systems with which to study the effects of histone H3 Ser10 phosphorylation in vivo.
Global hypophosphorylation of histone H3 Ser10 by SP600125. Initial studies were performed to determine the effect of SP600125 on different histone H3 modifications. HepG2 cells were incubated with 10 M SP600125 for periods of up to 6 h, and cell extracts were probed with a panel of modification specific antibodies. SP600125 dramatically reduced global histone H3 Ser10 phosphorylation without affecting expression. The reduction in phosphorylation started at 10 min, with near maximal reduction occurring after 20 min of treatment with SP600125, whereas global acetylation of lysine 14 remained largely unaffected. To determine whether SP600125 exhibited dose dependent effects on histone H3 Ser10 phosphorylation, cells were also treated with increasing doses of SP600125 for 1 h.