Genome-wide profiling reveals ~1500 Pol III-associated loci in K562 cells. In general, the expected 400–500 Pol III-transcribed genes encoding tRNAs and ncRNAs show very high levels of all three Pol III factors. In contrast, ~1000 Pol III-associated loci have not been previously described, and the vast majority of these (90%) are located near SINEs. The SINE-associated loci show much lower levels of Pol III factors as compared to tRNA genes, although these levels are clearly above the background. The transcriptional products of and biological functions of these Pol III-associated loci near SINEs are unknown. The prevalence of SINEs near these Pol III-associated loci might reflect DNA sequences resembling B or A blocks in a subset of SINEs.
Most, but not all, tRNA genes are occupied by the complete Pol III transcription machinery, and Pol III association at expressed vs. non-expressed genes differs by a factor of 100. Strikingly, the genomic regions in the vicinity of expressed and non-expressed Pol III genes are different. Expressed Pol III genes are located close to regions that have histone modifications characteristic of functional Pol II promoters and Pol II itself. In contrast, this distinctive chromatin signature is absent from non-expressed Pol III genes. These observations suggest that Pol III factors bind preferentially to genomic regions with a histone modification pattern generated by a functional Pol II promoter. We think it unlikely that non-expressed Pol III genes have defective TFIIIC recognition sites, because they generally possess high-quality B box sequences. Conversely, TFIIIC associates with fewer than 2% of the B box motifs in the human genome, indicating that the presence of a B box alone is insufficient for binding in vivo. In addition, the fact that ETC loci are located near Pol II promoters and associated histone modification strongly argues that TFIIIC association depends on Pol II-generated chromatin regions.
The characteristic pattern of histone modifications at Pol II promoters depends on Pol II preinitiation complexes but not on extensive elongation, because Pol II is often paused just downstream of many promoters in a manner that precludes any appreciable transcription26,27
. This suggests that TFIIIC binding to regions near functional Pol II promoters is largely, and perhaps completely, independent of Pol II transcriptional activity (i.e. mRNA synthesis). In accord with this suggestion, treatment of human cells with alpha-amanitin, an inhibitor that blocks Pol II transcription after preinitiation complex formation, has limited effects on Pol III transcription17
. For these reasons, we speculate that TFIIIC binding is strongly enhanced by the chromatin structure generated by nearby functional Pol II promoters. In principle, TFIIIC binding might be increased by promoter accessibility due to histone acetylation reducing histone-DNA contacts, direct interactions with modified histones via an effector domain(s) in a TFIIIC subunit or associated protein, or a histone variant (e.g. H2AZ) near Pol II promoter regions.
Accessibility to the recognition factor TFIIIC is not, however, the only determinant of Pol III association in human cells. In S. cerevisiae, TFIIIC levels are highly correlated with Pol III occupancy, and recruitment of the complete Pol III machinery exhibits a one-to-one correspondence with TFIIIC binding. In contrast, the human TFIIIC/Pol III ratio is considerably more variable than it is in yeast, and a wide range of TFIIIC levels may recruit a given amount of polymerase. The presence of a Brf2 mechanism for Pol III recruitment in human cells is one obvious alternative pathway, allowing transcription with little or no TFIIIC. At the opposite extreme are the ETC loci, which recruit little or no polymerase despite having high occupancy by TFIIIC. Even at standard type 2 Pol III genes, the ratio of TFIIIC to polymerase varies over a wide range, in contrast to the TFIIIB/Pol III ratio, which is relatively consistent.
The different patterns of histone marks near ETC
-type and transcriptionally active TFIIIC sites suggest that TFIIIC’s ability to recruit TFIIIB to type 2 genes might depend on a particular histone modification pattern. Indeed, Myc-dependent activation of Pol III transcription is associated with targeted histone acetylation and increased association of TFIIIB28
. In a similar vein, transcription of type 3 genes is influenced by the CHD8 chromatin modifying protein29
. Interestingly, TFIIIC either possesses histone acetylase activity30,31
and/or recruits the p300 histone acetylase32
, such that it could actively participate in generating a chromatin state appropriate for TFIIIB recruitment. Association of the initiator TFIIIB is an important rate-limiting step in Pol III gene expression in humans, because the excellent correlation of TFIIIB and polymerase levels suggests that Pol III occupancy follows linearly from TFIIIB recruitment. In this regard, TFIIIB is a target of regulation Maf133
, and Myc28
The presence of TFIIIC at many loci without TFIIIB or Pol III, much like similar loci in yeast, suggests a role for human TFIIIC beyond its function in Pol III transcription. TFIIIC bound to the intergenic region of closely spaced, divergently transcribed Pol II genes might be important for the regulation and separate expression of the two genes. In yeast, TFIIIC-bound loci function as heterochromatin barriers and insulators10,21,22
and also participate in higher-order chromosome organization10,37
. The correlation of TFIIIC peaks with CTCF is noteworthy and suggests the possible involvement of human TFIIIC in chromosome organization.