Project

RNA polymerase III transcription

Goal: This project integrates the several Pol III-related sub-projects into a comprehensive view.

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Martin Teichmann
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Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases’ recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell’s developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.
Martin Teichmann
added 2 research items
Resolving RNA polymerase structures at the atomic level has revolutionized our understanding of transcription. Three articles now published in Nature Structural & Molecular Biology and Nature Communications decipher unique properties of human RNA polymerase III and propose built-in modules within the enzyme that mediate transcriptional activation, repression and antirepression.
A Correction to this paper has been published: https://doi.org/10.1038/s41594-021-00574-w.
Martin Teichmann
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Martin Teichmann
added 2 research items
How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.
Martin Teichmann
added 2 research items
In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.
In Eukaryotes, tRNAs, 5S RNA and U6 RNA are transcribed by RNA polymerase (Pol) III. Human Pol III is composed of 17 subunits. Three specific Pol III subunits form a stable ternary subcomplex (RPC62-RPC39-RPC32α/β) being involved in pre-initiation complex formation. No paralogues for subunits of this subcomplex subunits have been found in Pols I or II, but hRPC62 was shown to be structurally related to the general Pol II transcription factor hTFIIEα. Here we show that these structural homologies extend to functional similarities. hRPC62 as well as hTFIIEα possess intrinsic ATP-dependent 3'-5' DNA unwinding activity. The ATPase activities of both proteins are stimulated by single-stranded DNA. Moreover, the eWH domain of hTFIIEα can replace the first eWH (eWH1) domain of hRPC62 in ATPase and DNA unwinding assays. Our results identify intrinsic enzymatic activities in hRPC62 and hTFIIEα.
Martin Teichmann
added 43 research items
Human transcription factor IIIA (htFIIIA), specifically required for transcription of the gene for 5S ribosomal RNA has been characterized with respect to some of its physical, immunological and functional properties. TFIIIA from HeLa cells, which selectively binds 5S RNA, is a monomer of approximately 35 kDa with a Stokes' radius of approximately 2.65 nm and a sedimentation coefficient of approximately 2.8 S. These values indicate that the human protein is of rather globular shape and hence diverges not only in molecular mass but also in most of the molecular properties from its highly asymmetric counterpart in Xenopus laevis oocytes. By raising specific polyclonal antibodies against hTFIIIA it was shown in Western immunoblots that there was no cross-reaction between anti-hTFIIIA antibodies and the amphibian protein. Conversely, monoclonal antibodies against three domains of X. laevis TFIIIA antibodies and the amphibian protein. Conversely, monoclonal antibodies against three domains of X. laevis TFIIIA did not cross-react with the human transcription factor. The polyclonal antisera raised against hTFIIIA specifically neutralized binding of the human transcription factor to 5S DNA and abolished in vitro transcription of 5S RNA but these antibodies were unable to inhibit 5S RNA synthesis in cellular extracts from Xenopus, Drosophila or yeast cells. Finally, the species variation of TFIIIA could be substantiated by electrophoretic mobility shift assays revealing preferential binding of hTFIIIA to the homologous 5S RNA gene.
TFIIIC in yeast and humans is required for transcription of tRNA and 5 S RNA genes by RNA polymerase III. In the yeast Saccharomyces cerevisiae, TFIIIC is composed of six subunits, five of which are conserved in humans. We report the identification, molecular cloning, and characterization of the sixth subunit of human TFIIIC, TFIIIC35, which is related to the smallest subunit of yeast TFIIIC. Human TFIIIC35 does not contain the phosphoglycerate mutase domain of its yeast counterpart, and these two proteins display only limited homology within a 34-amino acid domain. Homologs of the sixth TFIIIC subunit are also identified in other eukaryotes, and their phylogenic evolution is analyzed. Affinity-purified human TFIIIC from an epitope-tagged TFIIIC35 cell line is active in binding to and in transcription of the VA1 gene in vitro. Furthermore, TFIIIC35 specifically interacts with the human TFIIIC subunits TFIIIC63 and, to a lesser extent, TFIIIC90 in vitro. Finally, we determined a limited region in the smallest subunit of yeast TFIIIC that is sufficient for interacting with the yeast TFIIIC subunit ScTfc1 (orthologous to TFIIIC63) and found it to be adjacent to and overlap the 34-amino acid domain that is conserved from yeast to humans.
The 17-subunit human RNA polymerase III (hPol III) transcribes small, untranslated RNA genes that are involved in the regulation of transcription, splicing and translation. hPol III subunits hRPC62, hRPC39 and hRPC32 form a stable ternary subcomplex required for promoter-specific transcription initiation by hPol III. Here, we report the crystal structure of hRPC62. This subunit folds as a four-tandem extended winged helix (eWH) protein that is structurally related to the transcription factor TFIIEα N terminus. Through biochemical analyses, we mapped the protein-protein interactions of hRPC62, hRPC32 and hRPC39. In addition, we demonstrated that hRPC62 and hRPC39 bind single-stranded and duplex DNA, respectively, in a sequence-independent manner. Overall, we shed light on structural similarities between the hPol III–specific subunit hRPC62 and TFIIEα and propose specific functions for hRPC39 and hRPC62 in transcription initiation by hPol III.
Martin Teichmann
added a project goal
This project integrates the several Pol III-related sub-projects into a comprehensive view.