Stefan G.E. Roberts

TFIIB and the regulation of transcription by RNA polymerase II

Accurate transcription of a gene by RNA polymerase II requires the assembly of a group of general transcription factors at the promoter. The general transcription factor TFIIB plays a central role in preinitiation complex assembly, providing a bridge between promoter-bound TFIID and RNA polymerase II. TFIIB makes extensive contact with the core promoter via two independent DNA-recognition modules. In addition to interacting with other general transcription factors, TFIIB directly modulates the catalytic center of RNA polymerase II in the transcription complex. Moreover, TFIIB has been proposed as a target of transcriptional activator proteins that act to stimulate preinitiation complex assembly. In this review, we will discuss our current understanding of these activities of TFIIB.

The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling.

The cycle of eukaryotic transcription, from initiation to elongation and termination is regulated at multiple steps. Coordinated action of regulatory factors keeps in check the transcriptional competence of RNA polymerase II (RNAPII) at different stages. Productive transcription requires the escape of the paused RNAPII from the promoter and transition to rapid elongation of the transcript. Numerous studies have identified diverse mechanisms of initiating transcription by overriding inhibitory signals at the gene promoter. The general theme that has emerged is that the balance between positive and negative regulatory factors determines the overall rate of transcription. Recently transcription termination has emerged as an important area of transcriptional regulation that is coupled with the efficient recycling of RNAPII. The factors associated with transcription termination can also mediate gene looping and thereby determine the efficiency of re-initiation. This review highlights these regulatory steps, the key modulators involved in transcription dynamics, and the emerging tools to analyze them.

Phosphorylation of TFIIB Links Transcription Initiation and Termination

Summary The general transcription factor TFIIB plays a central role in preinitiation complex (PIC) assembly and the recruitment of RNA polymerase II (RNA pol II) to the promoter [1]. Recent studies have revealed that TFIIB engages in contact with the transcription termination region and also with complexes that are involved in 3′ end processing and/or termination [2–9]. Here we report that TFIIB can be phosphorylated within the N terminus at serine 65 in vivo and that the phosphorylated form of TFIIB is present within (PICs). Surprisingly, TFIIB serine 65 phosphorylation is required after the phosphorylation of serine 5 of RNA pol II C-terminal domain (CTD) has occurred, but before productive transcription initiation begins. We show that phosphorylation of TFIIB at serine 65 regulates the interaction between TFIIB and the CstF-64 component of the CstF 3′ cleavage and polyadenylation complex. This directs the recruitment of CstF (cleavage stimulatory factor) to the terminator and also the recruitment of the CstF and CPSF (cleavage and polyadenylation specific factor) complexes to the promoter. Our results reveal that phosphorylation of TFIIB is a critical event in transcription that links the gene promoter and terminator and triggers initiation by RNA pol II.

Dynamic interaction between WT1 and BASP1 in transcriptional regulation during differentiation

The Wilms’ tumour suppressor protein WT1 plays a central role in the development of the kidney and also other organs. WT1 can act as a transcription factor with highly context-specific activator and repressor functions. We previously identified Brain Acid Soluble Protein 1 (BASP1) as a transcriptional cosuppressor that can block the transcriptional activation function of WT1. WT1 and BASP1 are co-expressed during nephrogenesis and both proteins ultimately become restricted to the podocyte cells of the adult kidney. Here, we have analysed the WT1/BASP1 complex in a podocyte precursor cell line that can be induced to differentiate. Chromatin immunoprecipitation revealed that WT1 and BASP1 occupy the promoters of the Bak, c-myc and podocalyxin genes in podocyte precursor cells. During differentiation-dependent upregulation of podocalyxin expression BASP1 occupancy of the podocalyxin promoter is reduced compared to that of WT1. In contrast, the repressive WT1/BASP1 occupancy of the c-myc and Bak promoters is maintained and these genes are downregulated during the differentiation process. We provide evidence that the regulation of BASP1 promoter occupancy involves the sumoylation of BASP1. Our results reveal a dynamic cooperation between WT1 and BASP1 in the regulation of gene expression during differentiation.

Core Promoter-Dependent TFIIB Conformation and a Role for TFIIB Conformation in Transcription Start Site Selection

ABSTRACT The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

Core promoter elements recognized by transcription factor IIB

The general transcription factor TFIIB (transcription factor IIB) plays a critical role in the assembly of the RNA polymerase II pre-initiation complex. TFIIB can make sequence-specific DNA contacts both upstream and downstream of the TATA box. This has led to the definition of two core promoter BREs (TFIIB-recognition elements), one upstream [BRE(u) (upstream BRE)] and one downstream of TATA box [BRE(d) (downstream BRE)]. TFIIB-BRE(u) and TFIIB-BRE(d) contacts are mediated by two independent DNA-recognition motifs within the core domain of TFIIB. Both the BRE(u) and the BRE(d) modulate the transcriptional potency of a promoter. However, the net effect of the BREs on promoter activity is dependent on the specific blend of elements present within a core promoter.

Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II

The general transcription factor TFIIB has a central role in the assembly of the preinitiation complex at the promoter, providing a platform for the entry of RNA polymerase II/TFIIF. We used an RNA interference (RNAi)‐based system in which TFIIB expression is ablated in vivo and replaced with a TFIIB derivative that contains a silent mutation and is refractory to the RNAi. Using this approach, we found that transcriptionally defective TFIIB amino‐terminal mutants showed distinct effects on the basis of their ability to compete with wild‐type TFIIB in vivo. Moreover, analysis of the TFIIB mutant derivatives by chromatin immunoprecipitation showed that promoter occupancy by TFIIB is dependent on the association with RNA polymerase II. Together, our results support a mode of preinitiation complex assembly in which TFIIB/RNA polymerase II recruitment to the promoter occurs in vivo.

TFIIB Recognition Elements Control the TFIIA-NC2 Axis in Transcriptional Regulation

ABSTRACT TFIIB recognizes DNA sequence-specific motifs that can flank the TATA elements of the promoters of protein-encoding genes. The TFIIB recognition elements (BREu and BREd) can have positive or negative effects on transcription in a promoter context-dependent manner. Here we show that the BREs direct the selective recruitment of TFIIA and NC2 to the promoter. We find that TFIIA preferentially associates with BRE-containing promoters while NC2 is recruited to promoters that lack consensus BREs. The functional relevance of the BRE-dependent recruitment of TFIIA and NC2 was determined by small interfering RNA-mediated knockdown of TFIIA and NC2, both of which elicited BRE-dependent effects on transcription. Our results confirm the established functional reciprocity of TFIIA and NC2. However, our findings show that TFIIA assembly at BRE-containing promoters results in reduced transcriptional activity, while NC2 acts as a positive factor at promoters that lack functional BREs. Taken together, our results provide a basis for the selective recruitment of TFIIA and NC2 to the promoter and give new insights into the functional relationship between core promoter elements and general transcription factor activity.

TFIIB dephosphorylation links transcription inhibition with the p53-dependent DNA damage response

The general transcription factor II B (TFIIB) plays a central role in both the assembly of the transcription complex at gene promoters and also in the events that lead to transcription initiation. TFIIB is phosphorylated at serine-65 at the promoters of several endogenous genes, and this modification is required to drive the formation of gene promoter–3′ processing site contacts through the cleavage stimulation factor 3′ (CstF 3′)-processing complex. Here we demonstrate that TFIIB phosphorylation is dispensable for the transcription of genes activated by the p53 tumor suppressor. We find that the kinase activity of TFIIH is critical for the phosphorylation of TFIIB serine-65, but it is also dispensable for the transcriptional activation of p53-target genes. Moreover, we demonstrate that p53 directly interacts with CstF independent of TFIIB phosphorylation, providing an alternative route to the recruitment of 3′-processing complexes to the gene promoter. Finally, we show that DNA damage leads to a reduction in the level of phospho-ser65 TFIIB that leaves the p53 transcriptional response intact, but attenuates transcription at other genes. Our data reveal a mode of phospho-TFIIB-independent transcriptional regulation that prioritizes the transcription of p53-target genes during cellular stress.

Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB.

The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2 binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2 binding reduces backbone mobility in the b-turn (Arg28-Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2-dependent structural changes. These structural effects of Zn2 binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II.