TFIIB co-localizes and interacts with α-tubulin during oocyte meiosis in the mouse and depletion of TFIIB causes arrest of subsequent embryo development.

TFIIB (transcription factor IIB) is a transcription factor that provides a bridge between promoter-bound TFIID and RNA polymerase II, and it is a target of various transcriptional activator proteins that stimulate the pre-initiation complex assembly. The localization and/or attachment matrix of TFIIB in the cytoplast is not well understood. This study focuses on the function of TFIIB and its interrelationship with α-tubulins in a mouse model. During oocyte maturation TFIIB distributes throughout the entire nucleus of the germinal vesicle (GV). After progression to GV breakdown (GVBD), TFIIB and α-tubulin co-localize and accumulate in the vicinity of the condensed chromosomes. During the MII stage, the TFIIB signals are more concentrated at the equatorial plate and the kinetochores. Colcemid treatment of oocytes disrupts the microtubule (MT) system, although the TFIIB signals are still present with the altered MT state. Injection of oocytes with TFIIB antibodies and siRNAs causes abnormal spindle formation and irregular chromosome alignment. These findings suggest that TFIIB dissociates from the condensed chromatids and then tightly binds to microtubules from GVBD to the MII phase. The assembly and disassembly of TFIIB may very well be associated with and driven by microtubules. TFIIB maintains its contact with the α-tubulins and its co-localization forms a unique distribution pattern. Depletion of Tf2b in oocytes results in a significant decrease in TFIIB expression, although polar body extrusion does not appear to be affected. Knockdown of Tf2b dramatically affects subsequent embryo development with more than 85% of the embryos arrested at the 2-cell stage. These arrested embryos still maintain apparently normal morphology for at least 96h without any obvious degeneration. Analysis of the effects of TFIIB in somatic cells by co-transfection of BiFC plasmids pHA-Tf2b and pFlag-Tuba1α further confirms a direct interaction between TFIIB and α-tubulins.

Transcription initiation factor IIB involves in Schwann cell differentiation after rat sciatic nerve crush.

Transcription Initiation Factor IIB (TFIIB), as a general transcription factor, plays an essential role in preinitiation complex assembly and transcription initiation by recruiting RNA polymerase II to the promoter. However, its distribution and function in peripheral system lesion and repair were still unknown. Here, we investigated the spatiotemporal expression of TFIIB in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that TFIIB was expressed in normal sciatic nerve. It gradually increased, reached a peak at the seventh day after crush, and then returned to the normal level at 4 weeks. We observed that TFIIB expressed mainly increased in Schwann cells and co-localized with Oct-6. In vitro, we induced Schwann cell differentiation with cyclic adenosine monophosphate (cAMP) and found that TFIIB expression was increased in the differentiated process. TFIIB-specific siRNA inhibited cAMP-induced Schwann cell morphological change and the expression of P0. Collectively, we hypothesized peripheral nerve crush-induced upregulation of TFIIB in the sciatic nerve was associated with Schwann cell differentiation.

Phosphorylation of TFIIB links transcription initiation and termination.

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. 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. 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.

TFIIB aptamers inhibit transcription by perturbing PIC formation at distinct stages.

Transcription in eukaryotes is a multistep process involving the assembly and disassembly of numerous inter- and intramolecular interactions between transcription factors and nucleic acids. The roles of each of these interactions and the regions responsible for them have been identified and studied primarily by the use of mutants, which destroy the inherent properties of the interacting surface. A less intrusive but potentially effective way to study the interactions as well as the surfaces responsible for them is the use of RNA aptamers that bind to the interacting factors. Here, we report the isolation and characterization of high-affinity RNA aptamers that bind to the yeast general transcription factor TFIIB. These aptamers fall into two classes that interfere with TFIIB's interactions with either TBP or RNA polymerase II, both of which are crucial for transcription in yeast. We demonstrate the high affinity and specificity of these reagents, their effect on transcription and preinitiation complex formation and discuss their potential use to address mechanistic questions in vitro as well as in vivo.

Global distribution of negative cofactor 2 subunit-alpha on human promoters.

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters in vitro. Its association with TBP prevents the binding of TFIIB and leads to inhibition of preinitiation complex formation. Here, we investigate the association of NC2 subunit-alpha with human RNA polymerase II promoter regions by using gene-specific ChIP and genome-wide promoter ChIPchip analyses. We find NC2alpha associated with a large number of human promoters, where it peaks close to the core regions. NC2 occupancy in vivo positively correlates with mRNA levels, which perhaps reflects its capacity to stabilize TBP on promoter regions. In single gene analyses, we confirm core promoter binding and in addition map the NC2 complex to enhancer proximal regions. High-occupancy histone genes display a stable NC2/TFIIB ratio during the cell cycle, which otherwise varies markedly from one gene to another. The latter is at least in part explained by an observed negative correlation of NC2 occupancy with the presence of the TFIIB recognition element in core promoter regions. Our data establish the genome-wide basis for general and gene-specific functions of NC2 in mammalian cells.

The unique IR2 protein of equine herpesvirus 1 negatively regulates viral gene expression.

The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dose-dependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transient-transfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.