S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression

Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phas e of the cell cycle. Thus treatment of cells with an inhibitor of translati on, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cy cle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase- dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and s how that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, wh ich is again S-phase-dependent. The modest effect of CX in inducing the act ivity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observ e transcriptional activation. We therefore suggest that the uncoupled DNA r eplication and histone synthesis that occur after CX treatment induces a ge neral modification of chromatin structure, and propose that this general di sorganization of chromatin structure is responsible for a widespread activa tion of RNA polymerase II-mediated gene transcription.