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.It is well established that assembly of regulatory DNA sequences into nucleosomes represses transcription by restricting access of the transcriptional machinery to DNA (reviewed in reference 28). Thus, overcoming nucleosomal repression through alteration of chromatin structure is essential for gene activation. Eukaryotic cells contain at least two general classes of chromatin-modifying factors, the ATP-dependent chromatin-remodeling complexes (43) and histone-modifying enzymes that effect histone acetylation, methylation, phosphorylation, and ubiquitination (63). Whereas the function and mechanism of action of both types of factors have been extensively studied on genes transcribed by RNA polymerase II (Pol II), much less is known about chromatin-modifying factors involved in the regulation of Pol III-dependent genes.Pol III synthesizes small structural RNAs such as 5S RNA, tRNA, adenovirus VA1 RNA, and U6 RNA. Accurate and specific transcription of DNA templates by Pol III requires the assistance of the general initiation transcription factors TFIIIB and TFIIIC and, in some cases, various gene-specific factors such as the 5S gene-specific factor TFIIIA (for review, see references 15 and 53). In the case of tRNA and VA1 genes (subclass 2 genes), gene-internal core promoter elements (A and B boxes) are recognized directly by TFIIIC, which in turn directs the sequential binding of TFIIIB and Pol III. Human TFIIIC has been chromatographically separated into two distinct activities, TFIIIC1 and TFIIIC2 (74). TFIIIC2 is a stable complex of six subunits (220, 110, 102, 90,63, and 35 kDa) that binds directly to the B box (10a, 30, 75). TFIIIC1 displays no strong DNA-binding activity on its own but stabilizes TFIIIC2 binding to A and B boxes (66, 74). Other factors derived from TFIIIC preparations, such as coactivator PC4, also stabilize TFIIIC bind...