Abstract: Exactly twenty years ago TFII-I was discovered as a biochemical entity that was able to bind to and function via a core promoter element called the Initiator (Inr). Since then several different properties of this signal-induced multifunctional factor were discovered. Here I update these ever expanding functions of TFII-I--focusing primarily on the last ten years since the first review appeared in this journal.
Abstract: Primary response genes (PRGs) are a set of genes that are induced in response to both cell-extrinsic and cell-intrinsic signals and do not require de novo protein synthesis for their expression. These "first responders" in the waves of transcription of signal-responsive genes play pivotal roles in a wide range of biological responses, including neuronal survival and plasticity, cardiac stress response, innate and adaptive immune responses, glucose metabolism, and oncogeneic transformation. Here we bring together recent advances and our current understanding of the signal-induced transcriptional and epigenetic regulation of PRGs.
Abstract: Transcriptional regulation of eukaryotic protein-coding genes requires the participation of site-specific transcription factors that bind distal regulatory elements, as well as factors that, together with RNA polymerase II, form the basal transcription machinery at the core promoter. Gene regulation requires proper communication between promoters and enhancers, often over great distances. Therefore, it is important to understand the potentially inter-related transcription factor interactions at both of these elements. How this is achieved on tissue-specific genes, such as the immunoglobulin heavy chain (IgH) in B cells remains unclear. Here, we review known interactions at the Igh variable region (V(H)) promoters and present our perspective on promoter-enhancer interactions that are likely important for Ig gene regulation in B cells.
Abstract: Abstract Williams-Beuren Syndrome (WBS), an autosomal dominant genetic disorder, is characterized by a unique cognitive profile and craniofacial defects. WBS results from a microdeletion at the chromosomal location 7q11.23 that encompasses the genes encoding the members of TFII-I family of transcription factors. Given that the haploinsuficiency for TFII-I is causative to the craniofacial phenotype in humans, we set out to analyze the effect of post-transcriptional silencing of TFII-I during BMP-2-driven osteoblast differentiation in the C2C12 cell line. Our results show that TFII-I plays an inhibitory role in regulating genes that are essential in osteogenesis and intersects with the bone specific transcription factor Runx2 and the retinoblastoma protein, pRb. Identification of pathways regulated by TFII-I family transcription factors may begin to shed light on the molecular determinants of WBS.
Abstract: GTF2I and GTF2IRD1 encoding the multifunctional transcription factors TFII-I and BEN are clustered at the 7q11.23 region hemizygously deleted in Williams-Beuren syndrome (WBS), a complex multisystemic neurodevelopmental disorder. Although the biochemical properties of TFII-I family transcription factors have been studied in depth, little is known about the specialized contributions of these factors in pathways required for proper embryonic development. Here, we show that homozygous loss of either Gtf2ird1 or Gtf2i function results in multiple phenotypic manifestations, including embryonic lethality; brain hemorrhage; and vasculogenic, craniofacial, and neural tube defects in mice. Further analyses suggest that embryonic lethality may be attributable to defects in yolk sac vasculogenesis and angiogenesis. Microarray data indicate that the Gtf2ird1 homozygous phenotype is mainly caused by an impairment of the genes involved in the TGFbetaRII/Alk1/Smad5 signal transduction pathway. The effect of Gtf2i inactivation on this pathway is less prominent, but downregulation of the endothelial growth factor receptor-2 gene, resulting in the deterioration of vascular signaling, most likely exacerbates the severity of the Gtf2i mutant phenotype. A subset of Gtf2ird1 and Gtf2i heterozygotes displayed microcephaly, retarded growth, and skeletal and craniofacial defects, therefore showing that haploinsufficiency of TFII-I proteins causes various developmental anomalies that are often associated with WBS.
Abstract: TCR signaling leads to the activation of kinases such as inducible tyrosine kinase (Itk), a key regulatory protein in T-lymphocyte activation and function. The homolog of Itk in B cells is Bruton's tyrosine kinase, previously shown to bind and phosphorylate the transcription factor TFII-I. TFII-I plays major roles in transcription and signaling. Our purpose herein was twofold: first, to identify some of the molecular determinants involved in TFII-I activation downstream of receptor crosslinking in T cells and second, to uncover the existence of Itk-TFII-I signaling in T lymphocytes. We report for the first time that TFII-I is tyrosine phosphorylated upon TCR, TCR/CD43, and TCR/CD28 co-receptor engagement in human and/or murine T cells. We show that Itk physically interacts with TFII-I and potentiates TFII-I-driven c-fos transcription. We demonstrate that TFII-I is phosphorylated upon co-expression of WT, but not kinase-dead, or kinase-dead/R29C mutant Itk, suggesting these residues are important for TFII-I phosphorylation, presumably via an Itk-dependent mechanism. Structural analysis of TFII-I-Itk interactions revealed that the first 90 residues of TFII-I are dispensable for Itk binding. Mutations within Itk's kinase, pleckstrin-homology, and proline-rich regions did not abolish TFII-I-Itk binding. Our results provide an initial step in understanding the biological role of Itk-TFII-I signaling in T-cell function.
Abstract: The post-embryonic cells, in a non-proliferating quiescent state (G(0)), require mitogenic signaling to drive them into cell cycle entry (G(1)). However, cell cycle events become largely independent of external signaling once cells begin DNA synthesis in S phase. Given these two phases of cell cycle are mechanistically distinct, it is unclear whether there could be coordinated transcriptional regulation between these phases. The signal induced multifunctional transcription factor TFII-I, upon growth factor signaling, undergoes tyrosine phosphorylation, which is essential for its transcriptional activation function and corresponding G(0)-G(1) transition. Here we show that silencing of TFII-I has unexpected defects in S-phase. The TFII-I KD cells exhibit significant delay entering into and executing S-phase progression and entry into G(2)/M phase but do not exhibit any significant mitotic defects as evidenced by post-mitotic G(1) entry and frequency of binucleation. Microarray analysis, coupled with functional validation, reveals cyclin D1 and PKC-beta as major downstream targets of TFII-I. Cyclin D1 is induced in G(1) and is necessary for G(1)/S transition. PKC-beta also activates cyclin D1 via NFkappaB. These observations suggest a transcriptional network during early phases of cell cycle mediated by TFII-I. Finally, we show that Cdk1 phosphorylates TFII-I at the G(2)/M boundary, which likely leads to its displacement from the condensed chromatin during prophase to pro-metaphase transition. Taken together, TFII-I appears to have distinct roles in distinct phases of the mammalian cell cycle.
Abstract: The ubiquitously expressed TFII-I family of multifunctional transcription factors is involved in gene regulation as well as signaling. Despite the fact that they share significant sequence homology, these factors exhibit varied and distinct functions. The lack of knowledge about its binding sites and physiological target genes makes it more difficult to assign a definitive function for the TFII-I-related protein, BEN. We set out to determine its optimal binding site with the notion of predicting its physiological target genes. Here we report the identification of an optimal binding sequence for BEN by SELEX (systematic evolution of ligands by exponential enrichment) and confirm the relevance of this sequence by functional assays. We further performed a data base search to assign genes that have this consensus site(s) and validate several candidate genes by quantitative PCR upon stable silencing of BEN and by chromatin immunoprecipitation assay upon stable expression of BEN. Given that haploinsufficiency in BEN is causative to Williams-Beuren syndrome, these results may further lead to the identification of a set of physiologically relevant target genes for BEN and may help identify molecular determinants of Williams-Beuren syndrome.
Abstract: 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.
Abstract: 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.
Abstract: The multifunctional transcription factor TFII-I physically and functionally interacts with Bruton's tyrosine kinase in murine B cells. However, the downstream functions of TFII-I in B cells are unknown. Toward achieving this goal, we established stable posttranscriptional silencing of TFII-I in WEHI-231 immature murine B cells, which undergoes growth arrest and apoptosis either upon anti-IgM or TGF-beta signaling. In this study, we show that TFII-I promotes growth arrest of cells in a signal-dependent manner. Unlike control cells, B cells exhibiting loss of TFII-I function fail to undergo arrest upon signaling due to up-regulation of c-Myc expression and concomitant down-regulation of both p21 and p27. Loss of TFII-I is also associated with simultaneous increase in nuclear c-rel and decrease in p50 homodimer binding. Thus, besides controlling c-myc transcription, TFII-I controls B cell proliferation by regulating both nuclear translocation of c-rel and DNA-binding activity of p50 NF-kappaB.
Abstract: The promyelocytic leukemia (PML) gene codes for a tumor suppressor protein that is associated with distinct subnuclear macromolecular structures called the PML bodies. The PML gene is frequently involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL). The translocation results in a fusion gene product, PML-RARalpha, in which the PML gene fuses to the retinoic acid receptor alpha (RARalpha) gene. PML-RARalpha has been shown to promote transcriptional repression of genes involved in myeloid terminal differentiation and to disrupt the architecture of PML bodies, a phenotype reversed by treatment with all trans retinoic acid (ATRA). However, there are several alternatively spliced isoforms of PML-RARalpha. Here, we addressed the differences between the short and the long isoforms of PML-RARalpha (L and S) since both are associated with APL. We demonstrate that PML-RARalphaL, but not PML-RARalphaS, can directly promote cell growth by transcriptionally activating the pro-proliferative gene, c-fos, in response to mitogenic stimulation. The activity of the PML-RARalphaL is completely sensitive to ATRA. We further show that this activation is not via direct recruitment of the protein to the c-fos promoter but indirectly by altering the chromosomal environment of the c-fos gene, thereby rendering it more accessible to the signal induced transcriptional activators. Our results suggest that in addition to antagonizing the PML-tumor suppressor or the PML-pro-apoptotic activity, PML-RARalpha proteins can also directly promote cell growth by activating c-fos.
Abstract: Multifunctional transcription factor TFII-I has two spliced isoforms (Delta and beta) in murine fibroblasts. Here we show that these isoforms have distinct subcellular localization and mutually exclusive transcription functions in the context of growth factor signaling. In the absence of signaling, TFII-Ibeta is nuclear and recruited to the c-fos promoter in vivo. But upon growth factor stimulation, the promoter recruitment is abolished and it is exported out of the nucleus. Moreover, isoform-specific silencing of TFII-Ibeta results in transcriptional activation of the c-fos gene. In contrast, TFII-IDelta is largely cytoplasmic in the resting state but translocates to the nucleus upon growth factor signaling, undergoes signal-induced recruitment to the same site on the c-fos promoter, and activates the gene. Importantly, activated TFII-IDelta interacts with Erk1/2 (MAPK) kinase in the cell cytoplasm and imports the Erk1/2 to the nucleus, thereby transducing growth factor signaling. Our results identify a unique growth factor signaling pathway controlled by opposing activities of two TFII-I spliced isoforms.
Abstract: Bright/ARID3a/Dril1, a member of the ARID family of transcription factors, is expressed in a highly regulated fashion in B lymphocytes, where it enhances immunoglobulin transcription three- to sixfold. Recent publications from our lab indicated that functional, but not kinase-inactive, Bruton's tyrosine kinase (Btk) is critical for Bright activity in an in vitro model system, yet Bright itself is not appreciably tyrosine phosphorylated. These data suggested that a third protein, and Btk substrate, must contribute to Bright-enhanced immunoglobulin transcription. The ubiquitously expressed transcription factor TFII-I was identified as a substrate for Btk several years ago. In this work, we show that TFII-I directly interacts with human Bright through amino acids in Bright's protein interaction domain and that specific tyrosine residues of TFII-I are essential for Bright-induced activity of an immunoglobulin reporter gene. Moreover, inhibition of TFII-I function in a B-cell line resulted in decreased heavy-chain transcript levels. These data suggest that Bright functions as a three-component protein complex in the immunoglobulin locus and tie together previous data indicating important roles for Btk and TFII-I in B lymphocytes.
Abstract: In response to extracellular ligands, surface receptor tyrosine kinases and G-protein-coupled receptors activate isoforms of phospholipase C (PLC) and initiate calcium signaling. PLC can activate expression of surface transient receptor potential channels (TRPC) such as TRPC3, which modulate calcium entry through the plasma membrane. A recent paper shows that competitive binding of cytoplasmic TFII-I, a transcription factor, to PLC-gamma results in inhibition of TRPC3-mediated agonist-induced Ca(2+) entry. These results establish a novel cytoplasmic function for TFII-I.
Abstract: Reproduction, or the faithful passage of genetic information from one cell to its progeny, is central to life. To achieve reproduction faithfully cells have developed the cell cycle during which error-free replication of DNA, followed by division of the nucleus and partitioning of the cytoplasm yields two daughter cells. Prior to committing to reproduction, cells must sense mitogen levels in their environment and transduce those signals to the nucleus through a series of biochemical steps, resulting in spatial and/or temporal activation of genes that will drive cell cycle progression past the restriction point and initiate DNA replication. One way external signals are transmitted to the nucleus is via mitogen inducible transcription factors that shuttle between the cytoplasm and nucleus. Here we introduce a newly discovered pathway by which TFII-I, a growth signal induced transcription factor, operates in the prerestriction point and mitogen dependent phase of the cell cycle. We also discuss a potential role for TFII-I in DNA repair and how it might be involved in signal dependent DNA repair versus cell cycle.
Abstract: FF domains are poorly understood protein motifs found in all eukaryotes but in a very small number of proteins. They typically occur in tandem arrays and appear predominantly in splicing and transcription factors. Curiously, they are also present in the p190 family of cytoplasmic Rho GTPase activating proteins (GAPs). We identified the serum-responsive transcriptional regulator TFII-I as a specific interactor with the p190 RhoGAP FF domains. p190 sequesters TFII-I in the cytoplasm via the FF domains, but upon PDGF receptor-mediated phosphorylation of an FF domain, TFII-I is released from p190 and translocates to the nucleus where it can activate transcription of serum-inducible genes including c-fos. These findings reveal a pathway by which mitogens promote gene transcription and indicate a role for FF domains in phosphorylation-mediated signal transduction.
Abstract: Goosecoid (Gsc) is a homeodomain-containing transcription factor present in a wide variety of vertebrate species and known to regulate formation and patterning of embryos. Here we show that in embryonic carcinoma P19 cells, the transcription factor TFII-I forms a complex with Smad2 upon transforming growth factor beta (TGFbeta)/activin stimulation, is recruited to the distal element (DE) of the Gsc promoter, and activates Gsc transcription. Downregulation of endogenous TFII-I by small inhibitory RNA in P19 cells abolishes the TGFbeta-mediated induction of Gsc. Similarly, Xenopus embryos with endogenous TFII-I expression downregulated by injection of TFII-I-specific antisense oligonucleotides exhibit decreased Gsc expression. Unlike TFII-I, the related factor BEN (binding factor for early enhancer) is constitutively recruited to the distal element in the absence of TGFbeta/activin signaling and is replaced by the TFII-I/Smad2 complex upon TGFbeta/activin stimulation. Overexpression of BEN in P19 cells represses the TGFbeta-mediated transcriptional activation of Gsc. These results suggest a model in which TFII-I family proteins have opposing effects in the regulation of the Gsc gene in response to a TGFbeta/activin signal.
Abstract: TFII-I is a signal-induced multi-functional transcription factor that has recently been implicated as a regulatory component of the endoplasmic reticulum (ER) stress response. TFII-I acts through ER stress-induced binding to the ER stress element, which is highly conserved in promoters of ER stress-inducible genes such as Grp78/BiP. Interestingly, its tyrosine phosphorylation sites are required for its activation of the Grp78 promoter. Toward understanding the link between TFII-I, the tyrosine kinase signaling pathway, and Grp78 activation, we discovered that Tg stress induces a dramatic increase of TFII-I phosphorylation at Tyr248 and localization of this form of TFII-I to the nucleus. Chromatin immunoprecipitation analysis further reveals enhanced TFII-I binding to the Grp78 promoter in vivo upon ER stress. Previously, we reported that genistein, a general inhibitor of tyrosine kinase, could suppress ER stress induction of Grp78 by inhibiting complex formation on the ER stress element; however, the mechanism is not known. Consistent with TFII-I being a target of genistein suppression, we observed that genistein could suppress Tg stress-induced phosphorylation of TFII-I. We further demonstrate that c-Src, which is one of kinases identified to mediate phosphorylation of TFII-I at Tyr248, is activated by Tg stress and is able to stimulate the Grp78 promoter activity. Lastly, using stable cell lines with suppressed TFII-I levels, we show that TFII-I is required for optimal induction of Grp78 by ER stress. Our studies provide a molecular link that connects the c-Src tyrosine kinase transduction pathway to ER stress-induced transcriptional activation of Grp78 mediated by TFII-I.
Abstract: The multifunctional transcription factor TFII-I is tyrosine phosphorylated in response to extracellular growth signals and transcriptionally activates growth-promoting genes. However, whether activation of TFII-I also directly affects the cell cycle profile is unknown. Here we show that under normal growth conditions, TFII-I is recruited to the cyclin D1 promoter and transcriptionally activates this gene. Most strikingly, upon cell cycle arrest resulting from genotoxic stress and p53 activation, TFII-I is ubiquitinated and targeted for proteasomal degradation in a p53- and ATM (ataxia telangiectasia mutated)-dependent manner. Consistent with a direct role of TFII-I in cell cycle regulation and cellular proliferation, stable and ectopic expression of wild-type TFII-I increases cyclin D1 levels, resulting in accelerated entry to and exit from S phase, and overcomes p53-mediated cell cycle arrest, despite radiation. We further show that the transcriptional regulation of cyclin D1 and cell cycle control by TFII-I are dependent on its tyrosine phosphorylation at positions 248 and 611, sites required for its growth signal-mediated transcriptional activity. Taken together, our data define TFII-I as a growth signal-dependent transcriptional activator that is critical for cell cycle control and proliferation and further reveal that genotoxic stress-induced degradation of TFII-I results in cell cycle arrest.
