Different Functional Modes of p300 in Activation of RNA Polymerase III Transcription from Chromatin Templates

Abstract

Transcriptional coactivators that regulate the activity of human RNA polymerase III (Pol III) in the context of chromatin have not been reported. Here, we describe a completely defined in vitro system for transcription of a human tRNA gene assembled into a chromatin template. Transcriptional activation and histone acetylation in this system depend on recruitment of p300 by general initiation factor TFIIIC, thus providing a new paradigm for recruitment of histone-modifying coactivators. Beyond its role as a chromatin-modifying factor, p300 displays an acetyltransferase-independent function at the level of preinitiation complex assembly. Thus, direct interaction of p300 with TFIIIC stabilizes binding of TFIIIC to core promoter elements and results in enhanced transcriptional activity on histone-free templates. Additional studies show that p300 is recruited to the promoters of actively transcribed tRNA and U6 snRNA genes in vivo. These studies identify TFIIIC as a recruitment factor for p300 and thus may have important implications for the emerging concept that tRNA genes or TFIIIC binding sites act as chromatin barriers to prohibit spreading of silenced heterochromatin domains.

ACKNOWLEDGMENTS

We thank Zhengxin Wang and Martin Teichmann for Pol III reagents, Annika E. Wallberg for chromatin assembly in initial pilot experiments, Michael Carey and Joshua C. Black for advice with immobilized-template assays, James T. Kadonaga for ACF and p300 MutAT2 baculovirus expression vectors, Yoshihiro Nakatani for p300-HAT domain expression vector and PCAF baculovirus expression vector, and the Kazusa DNA Research Institute for TFNR cDNA.

C.M. was supported by a fellowship from the Deutsche Forschungsgemeinschaft. This work was supported by grants from the National Institutes of Health to R.G.R.