TFII-I-mediated polymerase pausing antagonizes GLI2 induction by TGFß.

The modulation of GLI2, an oncogenic transcription factor commonly upregulated in cancer, is in many cases not due to genetic defects, suggesting dysregulation through alternative mechanisms. The identity of these molecular events remains for the most part unknown. Here, we identified TFII-I as a novel repressor of GLI2 expression. Mapping experiments suggest that the INR region of the GLI2 promoter is necessary for GLI2 repression. ChIP studies showed that TFII-I binds to this INR. TFII-I knockdown decreased the binding of NELF-A, a component of the promoter-proximal pausing complex at this site, and enriched phosphorylated RNAPII serine 2 in the GLI2 gene body. Immunoprecipitation studies demonstrate TFII-I interaction with SPT5, another pausing complex component. TFII-I overexpression antagonized GLI2 induction by TGFß, a known activator of GLI2 in cancer cells. TGFß reduced endogenous TFII-I binding to the INR and increased RNAPII SerP2 in the gene body. We demonstrate that this regulatory mechanism is not exclusive of GLI2. TGFß-induced genes CCR7, TGFß1 and EGR3 showed similar decreased TFII-I and NELF-A INR binding and increased RNAPII SerP2 in the gene body post-TGFß treatment. Together these results identify TFII-I as a novel repressor of a subset of TGFß-responsive genes through the regulation of RNAPII pausing.

The matrix protein of vesicular stomatitis virus inhibits host-directed transcription of target genes via interaction with the TFIIH subunit p8.

In response to viral infection, the host innate antiviral response is elicited to limit viral replication. Many viruses have evolved various strategies to circumvent the host antiviral response. It has been reported that matrix (M) protein of vesicular stomatitis virus (VSV) can inhibit host gene expression to evade the host innate immune response. However, the molecular mechanism remains unclear. Here, we demonstrated that VSV M protein inhibited transcription of a reporter gene transfected into BSR-T7/5 cells. To further investigate the underlying mechanism, a yeast two-hybrid screen was performed to search for host proteins that interact with the M protein. The subunit of transcription/repair factor TFIIH, p8, was identified as an M binding partner, and the interaction was validated with a GST pull-down assay and laser confocal microscopy. Through a mutagenesis analysis, we found that the p8-M interaction was impaired when I96, E156, R159 and R160 residues on M were replaced with Ala. These mutants reduced the inhibitory effect on transcription of the reporter gene. Furthermore, the transcription inhibition mediated by M was impaired when co-expressed with p8. These results indicate that the p8-M interaction plays an important role in inhibiting transcription of host genes.

A Common Polymorphism in a Williams Syndrome Gene Predicts Amygdala Reactivity and Extraversion in Healthy Adults.

BACKGROUND: Williams syndrome (WS), a genetic disorder resulting from hemizygous microdeletion of chromosome 7q11.23, has emerged as a model for identifying the genetic architecture of socioemotional behavior. Common polymorphisms in GTF2I, which is found within the WS microdeletion, have been associated with reduced social anxiety in the general population. Identifying neural phenotypes affected by these polymorphisms would help advance our understanding not only of this specific genetic association but also of the broader neurogenetic mechanisms of variability in socioemotional behavior. METHODS: Through an ongoing parent protocol, the Duke Neurogenetics Study, we measured threat-related amygdala reactivity to fearful and angry facial expressions using functional magnetic resonance imaging, assessed trait personality using the Revised NEO Personality Inventory, and imputed GTF2I rs13227433 from saliva-derived DNA using custom Illumina arrays. Participants included 808 non-Hispanic Caucasian, African American, and Asian university students. RESULTS: The GTF2I rs13227433 AA genotype, previously associated with lower social anxiety, predicted decreased threat-related amygdala reactivity. An indirect effect of GTF2I genotype on the warmth facet of extraversion was mediated by decreased threat-related amygdala reactivity in women but not men. CONCLUSIONS: A common polymorphism in the WS gene GTF2I associated with reduced social anxiety predicts decreased threat-related amygdala reactivity, which mediates an association between genotype and increased warmth in women. These results are consistent with reduced threat-related amygdala reactivity in WS and suggest that common variation in GTF2I contributes to broader variability in socioemotional brain function and behavior, with implications for understanding the neurogenetic bases of WS as well as social anxiety.

Intracisternal Gtf2i Gene Therapy Ameliorates Deficits in Cognition and Synaptic Plasticity of a Mouse Model of Williams-Beuren Syndrome.

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by a heterozygous deletion of 26-28 genes at chromosome band 7q11.23. Haploinsufficiency at GTF2I has been shown to play a major role in the neurobehavioral phenotype. By characterizing the neuronal architecture in four animal models with intragenic, partial, and complete deletions of the WBS critical interval (ΔGtf2i(+/-), ΔGtf2i( -/-), PD, and CD), we clarify the involvement of Gtf2i in neurocognitive features. All mutant mice showed hypersociability, impaired motor learning and coordination, and altered anxiety-like behavior. Dendritic length was decreased in the CA1 of ΔGtf2i(+/-), ΔGtf2i ( -/-), and CD mice. Spine density was reduced, and spines were shorter in ΔGtf2i ( -/-), PD, and CD mice. Overexpression of Pik3r1 and downregulation of Bdnf were observed in ΔGtf2i(+/-), PD, and CD mice. Intracisternal Gtf2i-gene therapy in CD mice using adeno-associated virus resulted in increased mGtf2i expression and normalization of Bdnf levels, along with beneficial effects in motor coordination, sociability, and anxiety, despite no significant changes in neuronal architecture. Our findings further indicate that Gtf2i haploinsufficiency plays an important role in the neurodevelopmental and cognitive abnormalities of WBS and that it is possible to rescue part of this neurocognitive phenotype by restoring Gtf2i expression levels in specific brain areas.

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation.

In eukaryotes, RNA polymerase (RNAP) III transcribes hundreds of genes for tRNAs and 5S rRNA, among others, which share similar promoters and stable transcription initiation complexes (TIC), which support rapid RNAP III recycling. In contrast, RNAP II transcribes a large number of genes with highly variable promoters and interacting factors, which exert fine regulatory control over TIC lability and modifications of RNAP II at different transitional points in the transcription cycle. We review data that illustrate a relatively smooth continuity of RNAP III initiation-elongation-termination and reinitiation toward its function to produce high levels of tRNAs and other RNAs that support growth and development.

Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair.

BACKGROUND: In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair. METHODS: Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay. RESULTS: Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency. CONCLUSION: These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.

TFII-I regulates target genes in the PI-3K and TGF-ß signaling pathways through a novel DNA binding motif.

General transcription factor (TFII-I) is a multi-functional protein involved in the transcriptional regulation of critical developmental genes, encoded by the GTF2I gene located on chromosome 7q11.23. Haploinsufficiency at GTF2I has been shown to play a major role in the neurodevelopmental features of Williams-Beuren syndrome (WBS). Identification of genes regulated by TFII-I is thus critical to detect molecular determinants of WBS as well as to identify potential new targets for specific pharmacological interventions, which are currently absent. We performed a microarray screening for transcriptional targets of TFII-I in cortex and embryonic cells from Gtf2i mutant and wild-type mice. Candidate genes with altered expression were verified using real-time PCR. A novel motif shared by deregulated genes was found and chromatin immunoprecipitation assays in embryonic fibroblasts were used to document in vitro TFII-I binding to this motif in the promoter regions of deregulated genes. Interestingly, the PI3K and TGFß signaling pathways were over-represented among TFII-I-modulated genes. In this study we have found a highly conserved DNA element, common to a set of genes regulated by TFII-I, and identified and validated novel in vivo neuronal targets of this protein affecting the PI3K and TGFß signaling pathways. Overall, our data further contribute to unravel the complexity and variability of the different genetic programs orchestrated by TFII-I.

Conservation between the RNA polymerase I, II, and III transcription initiation machineries.

Recent studies of the three eukaryotic transcription machineries revealed that all initiation complexes share a conserved core. This core consists of the RNA polymerase (I, II, or III), the TATA box-binding protein (TBP), and transcription factors TFIIB, TFIIE, and TFIIF (for Pol II) or proteins structurally and functionally related to parts of these factors (for Pol I and Pol III). The conserved core initiation complex stabilizes the open DNA promoter complex and directs initial RNA synthesis. The periphery of the core initiation complex is decorated by additional polymerase-specific factors that account for functional differences in promoter recognition and opening, and gene class-specific regulation. This review outlines the similarities and differences between these important molecular machines.

Transcriptional regulation by Pol II(G) involving mediator and competitive interactions of Gdown1 and TFIIF with Pol II.

Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (noninitiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.

PI3K/Akt-dependent functions of TFII-I transcription factors in mouse embryonic stem cells.

Activation of PI3K/Akt signaling is sufficient to maintain the pluripotency of mouse embryonic stem cells (mESC) and results in down-regulation of Gtf2i and Gtf2ird1 encoding TFII-I family transcription factors. To investigate how these genes might be involved in the process of embryonic stem cell differentiation, we performed expression microarray profiling of mESC upon inhibition of PI3K by LY294002. This analysis revealed significant alterations in expression of genes for specific subsets of chromatin-modifying enzymes. Surprisingly, genome-wide promoter ChIP-chip mapping indicated that the majority of differently expressed genes could be direct targets of TFII-I regulation. The data support the hypothesis that upregulation of TFII-I factors leads to activation of a specific group of developmental genes during mESC differentiation.

The TFIIH subunit Tfb3 regulates cullin neddylation.

Cullin proteins are scaffolds for the assembly of multisubunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING domain of Hrt1 nor required the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 and the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING domain protein Tfb3 controls activation of a subset of cullins.

Multifunctional transcription factor TFII-I is an activator of BRCA1 function.

BACKGROUND: The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams-Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date. METHODS: A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated. RESULTS: We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.

Role for transcription factor TFII-I in the suppression of SSeCKS/Gravin/Akap12 transcription by Src.

The SSeCKS/Gravin/AKAP12 gene, encoding a kinase scaffolding protein with metastasis-suppressing activity, is transcriptionally downregulated in Src-transformed cells through the recruitment of HDAC1 to a Src-responsive proximal promoter site charged with Sp1, Sp3 and USF1. However, the ectopic expression of these proteins formed a suppressive complex in Src-transformed but not in parental NIH3T3 cells, suggesting the involvement of additional repressor factors. Transcription factor II-I (TFII-I) [general transcription factor 2i (Gtf2i)] was identified by mass spectrometry as being associated with the SSeCKS promoter complex in NIH3T3/Src cells, and moreover, the Src-induced tyrosine phosphorylation of TFII-I significantly increased its binding to the SSeCKS proximal promoter. siRNA-mediated knockdown of TFII-I or the expression of TFII-I(Y248/249F) caused the derepression of SSeCKS in NIH3T3/Src cells. Taken with previous data showing that the tyrosine phosphorylation of TFII-I facilitates its nuclear translocation, these data suggest that Src-family kinase-mediated phosphorylation converts a portion of TFII-I into a transcriptional repressor.

Essential role of the N-terminal region of TFII-I in viability and behavior.

BACKGROUND: GTF2I codes for a general intrinsic transcription factor and calcium channel regulator TFII-I, with high and ubiquitous expression, and a strong candidate for involvement in the morphological and neuro-developmental anomalies of the Williams-Beuren syndrome (WBS). WBS is a genetic disorder due to a recurring deletion of about 1,55-1,83 Mb containing 25-28 genes in chromosome band 7q11.23 including GTF2I. Completed homozygous loss of either the Gtf2i or Gtf2ird1 function in mice provided additional evidence for the involvement of both genes in the craniofacial and cognitive phenotype. Unfortunately nothing is now about the behavioral characterization of heterozygous mice. METHODS: By gene targeting we have generated a mutant mice with a deletion of the first 140 amino-acids of TFII-I. mRNA and protein expression analysis were used to document the effect of the study deletion. We performed behavioral characterization of heterozygous mutant mice to document in vivo implications of TFII-I in the cognitive profile of WBS patients. RESULTS: Homozygous and heterozygous mutant mice exhibit craniofacial alterations, most clearly represented in homozygous condition. Behavioral test demonstrate that heterozygous mutant mice exhibit some neurobehavioral alterations and hyperacusis or odynacusis that could be associated with specific features of WBS phenotype. Homozygous mutant mice present highly compromised embryonic viability and fertility. Regarding cellular model, we documented a retarded growth in heterozygous MEFs respect to homozygous or wild-type MEFs. CONCLUSION: Our data confirm that, although additive effects of haploinsufficiency at several genes may contribute to the full craniofacial or neurocognitive features of WBS, correct expression of GTF2I is one of the main players. In addition, these findings show that the deletion of the fist 140 amino-acids of TFII-I altered it correct function leading to a clear phenotype, at both levels, at the cellular model and at the in vivo model.

Plasmodium falciparum: Preinitiation complex occupancy of active and inactive promoters during erythrocytic stage.

Over 80% of Plasmodium falciparum genes are developmentally regulated during the parasite's life cycle with most genes expressed in a "just in time" fashion. However, the molecular mechanisms of gene regulation are still poorly understood. Analysis of P. falciparum genome shows that the parasite appears to encode relatively few transcription factors homologous to those in other eukaryotes. We used Chromatin immunoprecipitation (ChIP) to study interaction of PfTBP and PfTFIIE with stage specific Plasmodium promoters. Our results indicate that PfTBP and PfTFIIE are bound to their cognate sequence in active and inactive erythrocytic-expressed promoters. In addition, TF occupancy appears to extend beyond the promoter regions, since PfTBP interaction with the coding and 3' end regions was also detected. No PfTBP or PfTFIIE interaction was detected on csp and pfs25 genes which are not active during the erythrocytic asexual stage. Furthermore, PfTBP and PfTFIIE binding did not appear to correlate with histone 3 and/or 4 acetylation, suggesting that histone acetylation may not be a prerequisite for PfTBP or PfTFIIE promoter interaction. Based on our observations we concluded that the PfTBP/PfTFIIE-containing preinitiation complex (PIC) would be preassembled on promoters of all erythrocytic-expressed genes in a fashion independent of histone acetylation, providing support for the "poised" model. Contrary to the classical model of eukaryotic gene regulation, PIC interaction with Plasmodium promoters occurred independent of transcriptional activity and to the notion that chromatin acetylation leads to PIC assembly.

The complex of TFII-I, PARP1, and SFPQ proteins regulates the DYX1C1 gene implicated in neuronal migration and dyslexia.

DYX1C1 was first identified as a candidate gene for dyslexia susceptibility, and its role in controlling neuronal migration during embryogenesis and effect on learning in rodents have been verified. In contrast, genetic association studies have been ambiguous in replicating its effects on dyslexia. To better understand the regulation of DYX1C1 and the possible functional role of genetic variation in the promoter of DYX1C1, we selected three single-nucleotide polymorphisms (SNPs) with predicted functional consequences or suggested associations to dyslexia for detailed study. Electrophoretic mobility shift assays suggested the allele-specific binding of the transcription factors TFII-I (to rs3743205) and Sp1 (to rs16787 and rs12899331) that could be verified by competition assays. In addition, we purified a complex of protein factors binding to the previously suggested dyslexia-related SNP, -3G/A (rs3743205). Three proteins, TFII-I, PARP1, and SFPQ, were unambiguously identified by mass spectrometry and protein sequencing. Two SNPs, rs16787 and rs3743205, showed significant allelic differences in luciferase assays. Our results show that TFII-I, PARP1, and SFPQ proteins, each previously implicated in gene regulation, form a complex controlling transcription of DYX1C1. Furthermore, allelic differences in the promoter or 5' untranslated region of DYX1C1 may affect factor binding and thus regulation of the gene.

Functional characterization of the native NH2-terminal transactivation domain of the human androgen receptor: binding kinetics for interactions with TFIIF and SRC-1a.

The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.

Signal-induced functions of the transcription factor TFII-I.

We have learned a great deal over the last several years about the molecular mechanisms that govern cell growth, cell division and cell death. Normal cells pass through cell cycle (growth) and divide in response to mitogenic signals that are transduced through their cognate cell surface receptors to the nucleus. Despite the fact that cellular growth and division are mechanistically distinct steps, they are usually coordinately regulated, which is critical for normal cellular proliferation. The precise mechanistic basis for this coordinated regulation is unclear. TFII-I is a unique, signal-induced multifunctional transcription factor that is activated upon a variety of signaling pathways and appears to participate in distinct phases of cell growth. For instance, TFII-I is required for growth factor-induced transcriptional activation of the c-fos gene, which is essential for cell cycle entry. Two alternatively spliced isoforms of TFII-I exhibit opposing but necessary functions for mitogen-induced transcriptional activation of c-fos. Besides transcriptional activation of the c-fos proto-oncogene and eventual entry into cell cycle, TFII-I also appears to have a role in later phases of the cell cycle and cell division. Here we discuss how a multitude of signaling inputs target TFII-I isoforms, which may exert their functions in distinct phases of the cell cycle and play a key role in the coordinated regulation of cellular proliferation.

Induction of chromosomally integrated HIV-1 LTR requires RBF-2 (USF/TFII-I) and Ras/MAPK signaling.

The HIV-1 LTR is regulated by multiple signaling pathways responsive to T cell activation. In this study, we have examined the contribution of the MAPK, calcineurin-NFAT and TNFalpha-NF-kappaB pathways on induction of chromosomally integrated HIV-1 LTR reporter genes. We find that induction by T-cell receptor (CD3) cross-linking and PMA is completely dependent upon a binding site for RBF-2 (USF1/2-TFII-I), known as RBEIII at -120. The MAPK pathway is essential for induction of the wild type LTR by these treatments, as the MEK inhibitors PD98059 and U0126 block induction by both PMA treatment and CD3 cross-linking. Stimulation of cells with ionomycin on its own has no effect on the integrated LTR, indicating that calcineurin-NFAT is incapable of causing induction in the absence of additional signals, but stimulation with both PMA and ionomycin produces a synergistic response. In contrast, stimulation of NF-kappaB by treatment with TNFalpha causes induction of both the wild type and RBEIII mutant LTRs, an effect that is independent of MAPK signaling. USF1, USF2 and TFII-I from unstimulated cells are capable of binding RBEIII in vitro, and furthermore can be observed on the LTR in vivo by chromatin imunoprecipitation from untreated cells. DNA binding activity of USF1/2 is marginally stimulated by PMA/ ionomycin treatment, and all three factors appear to remain associated with the LTR throughout the course of induction. These results implicate major roles for the MAPK pathway and RBF-2 (USF1/2-TFII-I) in coordinating events necessary for transition of latent integrated HIV-1 to active transcription in response to T cell signaling.