Figure 3

Simultaneous treatment of cells to increase histone acetylation and decrease DNA methylation. Rhabdomyosarcoma cells were treated with the indicated amounts of AzaC in the absence (lanes 1–5) or presence (+TSA; lanes 6–10) of 500 nM TSA. RNA was extracted and separated on an agarose gel before transferring to a nylon membrane and hybridizing with the indicated probes. (A) H19 transcription is reactivated by AzaC in a dose-dependent fashion with higher doses resulting in greater reactivation (lanes 1–5). Combining the AzaC treatment with 500 nM TSA however results in lower levels of reactivation of the gene (compare lanes 6–10 with lanes 1–5). (B) The same membrane stripped and rehybridized with a probe for IGF2. Levels of IGF2 show a slight decrease as H19 transcription increases in the cells treated with AzaC alone (lanes 1–5). H19 and IGF2 compete for common enhancer elements, which would explain this effect. The effect is diminished in cells treated with both TSA and AzaC (lanes 6–10), which also show less H19 expression. (C) 28S rRNA loading control for the Northern.