Yoshiaki Ohkuma

ANALYSIS OF THE ROLE OF TFIIE IN BASAL TRANSCRIPTION AND TFIIH-MEDIATED CARBOXY-TERMINAL DOMAIN PHOSPHORYLATION THROUGH STRUCTURE-FUNCTION STUDIES OF TFIIE-ALPHA

The general transcription factor TFIIE recruits TFIIH at a late stage of transcription initiation complex formation and markedly stimulates TFIIH-dependent phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II. To study this function of TFIIE in more detail, systematic deletion mutations were introduced into the large subunit of TFIIE (TFIIE-alpha) and were analyzed with regard to their effects on TFIIH-dependent CTD phosphorylation, TFIIE-dependent basal and enhancer-dependent transcription, and interactions of TFIIE-alpha with both TFIIE-beta and TFIIH. The amino (N)-terminal half of TFIIE-alpha, which possesses several putative structural motifs, was sufficient for the phosphorylation and transcription activities and for TFIIE-beta interactions, whereas a site effecting both strong interactions with TFIIH and large stimulatory effects on transcription and CTD phosphorylation was localized to an acidic region near the carboxy (C) terminus. The fact that these activities appear to be tightly linked supports the idea that TFIIE interacts physically and functionally with TFIIH and that CTD phosphorylation is essential for transcription under normal conditions. The present results suggest that TFIIE, via its effect on TFIIH, may act as a checkpoint for formation of a preinitiation complex.

Analysis of structure-function relationships of yeast TATA box binding factor TFIID

A systematic series of N-terminal, C-terminal, and internal deletion mutants of S. cerevisiae TFIID were expressed in vitro and tested for TATA box binding and basal level transcription activities using, respectively, DNA mobility shift and in vitro transcription assays. The domains responsible for these activities were colocalized to a surprisingly large region containing C-terminal residues 63-240. This region was noted previously to contain potentially interesting structural motifs (central basic core, direct repeats, and sigma factor homology) and, more recently, to be highly conserved among TFIID from different species. Deletion mutant cotranslation studies revealed that TFIID binds DNA as a monomer. The implications of these results for TFIID structure and function are discussed.

The carboxy-terminal domain of the XPC protein plays a crucial role in nucleotide excision repair through interactions with transcription factor IIH.

The xeroderma pigmentosum group C (XPC) protein specifically involved in genome-wide damage recognition for nucleotide excision repair (NER) was purified as a tight complex with HR23B, one of the two mammalian homologs of RAD23 in budding yeast. This XPC-HR23B complex exhibits strong binding affinity for single-stranded DNA, as well as preferential binding to various types of damaged DNA. To examine the structure-function relationship of XPC, a series of truncated mutant proteins were generated and assayed for various binding activities. The two domains participating in binding to HR23B and damaged DNA, respectively, were mapped within the carboxy-terminal half of XPC, which also contains an evolutionary conserved amino acid sequence homologous to the yeast RAD4 protein. We established that the carboxy-terminal 125 amino acids are dispensable for both HR23B and damaged DNA binding, while interactions with transcription factor IIH (TFIIH) are significantly impaired by truncation of this domain. Furthermore, deletion of the extreme carboxy-terminal domain totally abolished XPC activity in the cell-free NER reaction. These results suggest that following initial damage recognition, the carboxy terminus of XPC may be essential for the recruitment of TFIIH, and that most truncation mutations identified in XP-C patients result in non-functional proteins.

Mechanism of Promoter Melting by the Xeroderma Pigmentosum Complementation Group B Helicase of Transcription Factor IIH Revealed by Protein-DNA Photo-Cross-Linking

ABSTRACT The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions −9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions −5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.

MCAF mediates MBD1-dependent transcriptional repression

ABSTRACT DNA methylation is involved in a variety of genome functions, including gene control and chromatin dynamics. MBD1 is a transcriptional regulator through the cooperation of a methyl-CpG binding domain, cysteine-rich CXXC domains, and a transcriptional repression domain. A yeast two-hybrid screen was performed to investigate the role of MBD1 in methylation-based transcriptional repression. We report a mediator, MBD1-containing chromatin-associated factor (MCAF), that interacts with the transcriptional repression domain of MBD1. MCAF harbors two conserved domains that allow it to interact with MBD1 and enhancer-like transactivator Sp1. MCAF possesses a coactivator-like activity, and it seems to facilitate Sp1-mediated transcription. In contrast, the MBD1-MCAF complex blocks transcription through affecting Sp1 on methylated promoter regions. These data provide a mechanistic basis for direct inhibition of gene expression via methylation-dependent and histone deacetylation-resistant processes.

Structure of the central core domain of TFIIEβ with a novel double-stranded DNA-binding surface

Human general transcription factor TFIIE consists of two subunits, TFIIEα and TFIIEβ. Recently, TFIIEβ has been found to bind to the region where the promoter starts to open to be single‐stranded upon transcription initiation by RNA polymerase II. Here, the central core domain of human TFIIEβ (TFIIEβc) has been identified by a limited proteolysis. This solution structure has been determined by NMR. It consists of three helices with a β hairpin at the C–terminus, resembling the winged helix proteins. However, TFIIEβc shows a novel double‐stranded DNA‐binding activity where the DNA‐binding surface locates on the opposite side to the previously reported winged helix motif by forming a positively charged furrow. A model will be proposed that TFIIE stabilizes the preinitiation complex by binding not only to the general transcription factors together with RNA polymerase II but also to the promoter DNA, where double‐stranded DNA starts to open to be single‐stranded upon activation of the preinitiation complex.

Mediator Complex Recruits Epigenetic Regulators via Its Two Cyclin-dependent Kinase Subunits to Repress Transcription of Immune Response Genes

Background: Two CDK subunits of the Mediator complex play pivotal roles in transcription by a mechanism that has not yet been elucidated. Results: The histone arginine methyltransferase PRMT5 is a Mediator CDK-interacting protein. Conclusion: Mediator-associated PRMT5 symmetrically dimethylates histone H4 arginine 3, and this might cause transcriptional repression. Significance: This work enables further exploration of Mediator functions in transcriptional repression. The Mediator complex (Mediator) plays pivotal roles in activating transcription by RNA polymerase II, but relatively little is known about its roles in repression. Here, we identified the histone arginine methyltransferase PRMT5 and WD repeat protein 77/methylosome protein 50 (WDR77/MEP50) as Mediator cyclin-dependent kinase (CDK)-interacting proteins and studied the roles of PRMT5 in the transcriptional regulation of CCAAT enhancer-binding protein (C/EBP) β target genes. First, we purified CDK8- and CDK19-containing complexes from HeLa nuclear extracts and subjected these purified complexes to mass spectrometric analyses. These experiments revealed that two Mediator CDKs, CDK8 and CDK19, individually interact with PRMT5 and WDR77, and their interactions with PRMT5 cause transcriptional repression of C/EBPβ target genes by regulating symmetric dimethylation of histone H4 arginine 3 (H4R3me2s) in the promoter regions of those genes. Furthermore, the recruitment of the DNA methyltransferase DNMT3A correlated with H4R3 dimethylation potentially leading to DNA methylation at the promoter proximal region and tight inhibition of preinitiation complex formation. In vertebrates, C/EBPβ regulates many genes involved in immune responses and cell differentiation. These findings shed light on the molecular mechanisms of the repressive roles of Mediator CDKs in transcription of C/EBPβ target genes and might provide clues that enable future studies of the functional associations between Mediators and epigenetic regulation.

Analysis of the role of TFIIE in transcriptional regulation through structure-function studies of the TFIIEbeta subunit.

The general transcription factor TFIIE plays important roles at two distinct but sequential steps in transcription as follows: preinitiation complex formation and activation (open complex formation), and the transition from initiation to elongation. The large subunit of human TFIIE (TFIIEα) binds to and facilitates the enzymatic functions of TFIIH, but TFIIE also functions independently from TFIIH. To determine functional roles of the small subunit of human TFIIE (TFIIEβ), deletion mutations were systematically introduced into putative structural motifs and characteristic sequences. Here we show that all of these structures that lie within the central 227-amino acid region of TFIIEβ are necessary and sufficient for both basal and activated transcription. We further demonstrate that two C-terminal basic regions are essential for physical interaction with both TFIIEα and single-stranded DNA, as well as with other transcription factors including theDrosophila transcriptional regulator Krüppel. In addition, we analyzed the effects of the TFIIEβ deletion mutations on TFIIH-dependent phosphorylation of the C-terminal domain of RNA polymerase II and on wild type TFIIEβ-driven basal transcription. Both responsible regions also mapped within the essential 227-amino acid region. Our results suggest that TFIIE engages in communication with both transcription factors and promoter DNA via the TFIIEβ subunit.

Structural insight into the TFIIE-TFIIH interaction: TFIIE and p53 share the binding region on TFIIH.

RNA polymerase II and general transcription factors (GTFs) assemble on a promoter to form a transcription preinitiation complex (PIC). Among the GTFs, TFIIE recruits TFIIH to complete the PIC formation and regulates enzymatic activities of TFIIH. However, the mode of binding between TFIIE and TFIIH is poorly understood. Here, we demonstrate the specific binding of the C‐terminal acidic domain (AC‐D) of the human TFIIEα subunit to the pleckstrin homology domain (PH‐D) of the human TFIIH p62 subunit and describe the solution structures of the free and PH‐D‐bound forms of AC‐D. Although the flexible N‐terminal acidic tail from AC‐D wraps around PH‐D, the core domain of AC‐D also interacts with PH‐D. AC‐D employs an entirely novel binding mode, which differs from the amphipathic helix method used by many transcriptional activators. So the binding surface between PH‐D and AC‐D is much broader than the specific binding surface between PH‐D and the p53 acidic fragments. From our in vitro studies, we demonstrate that this interaction could be a switch to replace p53 with TFIIE on TFIIH in transcription.

Binding site-dependent direct activation and repression of in vitro transcription by Drosophila homeodomain proteins

Fushi tarazu and engrailed are two of the genes required for proper segmentation of the Drosophila embryo. Their protein products Fushi tarazu and Engrailed (Ftz and En) each contain a homeodomain and have been shown to act as transcriptional regulators in transient expression experiments in a Drosophila cell culture system. We used an in vitro transcription system to test whether the effects of Ftz and En on transcription were direct or indirect. Purified Ftz directly activates in vitro transcription by binding to homeodomain binding sites inserted upstream of the TATA box of the Drosophila hsp70 promoter. Equimolar amounts of purified En repress this activation by competition with Ftz for binding to these sites. These results indicate that Ftz and En act directly as transcription factors and suggest that such homeodomain proteins regulate development by combinatorial transcriptional control.