POTENTIATION OF RNA POLYMERASE-II TRANSCRIPTION BY GAL4-VP16 DURING BUT NOT AFTER DNA-REPLICATION AND CHROMATIN ASSEMBLY

Purified, reconstituted chromatin templates containing regular, physio logical nucleosome spacing were transcribed in vitro by RNA polymerase Il along with the Gal4-VP16 activator. When Gal4-VP16 was prebound to DNA before reconstitution of either H1-deficient or H1-containing chr omatin, the resulting templates were transcribed with a similar effici ency. Under such conditions, we observed long-range (1000 bp) activati on of transcription in vitro with H1-containing chromatin, but not nak ed DNA templates. When Gal4-VP16 was added to preassembled chromatin, the H1-deficient chromatin was transcriptionally active, whereas the H 1-containing chromatin, which possessed properties similar to native c hromatin, was transcriptionally inert. We then mimicked DNA replicatio n and chromatin assembly at a replication fork and found that Gal4-VP1 6 could potentiate transcription during, but not after, replication an d assembly of histone H1-containing chromatin. These experiments provi de biochemical data that support a DNA replication-dependent mechanism for reconfiguration of chromatin structure and activation of transcri ption by Gal4-VP16.