Ananda L. Roy

Cloning of an Inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1

The transcription factor TFII‐I has been shown to bind independently to two distinct promoter elements, a pyrimidine‐rich initiator (Inr) and a recognition site (E‐box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII‐I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E‐box elements. The primary structure of TFII‐I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix–loop/span–helix homology. These unique structural features suggest that TFII‐I may have the capacity for multiple protein–protein and, potentially, multiple protein–DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII‐I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E‐box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions.

Core promoters and transcriptional control

Many biological processes are controlled, spatially and temporally, at the level of transcription. Thus, understanding the mechanisms of transcriptional regulation of gene expression is critical in deciphering the molecular modes of differentiation and development of a eukaryotic cell. Transcriptional control is mediated largely through interactions of regulatory transcription factors with their cognate enhancer elements. The regulatory signals generated at enhancer elements are communicated to the general transcription machinery formed at the core promoter elements of all genes. Recent observations suggest that the general transcription machinery can also generate regulatory signals independent of enhancer-generated interactions. Thus, the transcriptional regulation of gene expression, both in time and in space, may result from appropriate interfacing of independent signals generated at the core promoter and at the enhancer.

Regulation of Nuclear Localization and Transcriptional Activity of TFII-I by Bruton’s Tyrosine Kinase

ABSTRACT Bruton’s tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function.

TFII-I Enhances Activation of the c-fos Promoter through Interactions with Upstream Elements

ABSTRACT The transcription factor TFII-I was initially isolated as a factor that can bind to initiator elements in core promoters. Recent evidence suggests that TFII-I may also have a role in signal transduction. We have found that overexpression of TFII-I can enhance the response of the wild-type c-fos promoter to a variety of stimuli. This effect depends on the c-fosc-sis–platelet-derived growth factor-inducible factor binding element (SIE) and serum response element (SRE). There is no effect of cotransfected TFII-I on the TATA box containing the c-fos basal promoter. Three TFII-I binding sites can be found in c-fos promoter. Two of these overlap the c-fos SIE and SRE, and another is located just upstream of the TATA box. Mutations that distinguish between serum response factor (SRF), STAT, and TFII-I binding to the c-fos SIE and SRE suggest that the binding of TFII-I to these elements is important for c-fos induction in conjunction with the SRF and STAT transcription factors. Moreover, TFII-I can form in vivo protein-protein complexes with the c-fos upstream activators SRF, STAT1, and STAT3. These results suggest that TFII-I may mediate the functional interdependence of the c-fos SIE and SRE elements. In addition, the ras pathway is required for TFII-I to exert its effects on the c-fos promoter, and growth factor stimulation enhances tyrosine phosphorylation of TFII-I. These results indicate that TFII-I is involved in signal transduction as well as transcriptional activation of the c-fospromoter.

Biochemistry and biology of the inducible multifunctional transcription factor TFII-I

An animal cell has the capability to respond to a variety of external signals through cell surface receptors. The response is usually manifested in terms of altered gene expression in the nucleus. Thus, in modern molecular and cell biology, it has become important to understand how the communication between extracellular signals and nuclear gene transcription is achieved. Originally discovered as a basal factor required for initiator-dependent transcription in vitro, recent evidence suggests that TFII-I is also an inducible multifunctional transcription factor that is activated in response to a variety of extracellular signals and translocates to the nucleus to turn on signal-induced genes. Here I review the biochemical and biological properties of TFII-I and related proteins in nuclear gene transcription, signal transduction and genetic disorders.

Identification of TFII-I as the Endoplasmic Reticulum Stress Response Element Binding Factor ERSF: Its Autoregulation by Stress and Interaction with ATF6

ABSTRACT When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca2+ store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N9)CCACG. Within a subset of mammalian ERSEs, N9 represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca2+ store, the level of TFII-I transcript was elevated in Tg-stressed cells, correlating with an increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant TFII-I isoforms bind directly to the ERSEs of grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the consensus tyrosine phosphorylation site of TFII-I and the GGC sequence motif of the ERSE. We further discovered that TFII-I is an interactive protein partner of ATF6 and that optimal stimulation of ERSE by ATF6 requires TFII-I.

Physical and functional interactions of histone deacetylase 3 with TFII-I family proteins and PIASxβ

TFII-I family proteins are characterized structurally by the presence of multiple reiterated I-repeats, each containing a putative helix–loop–helix domain. Functionally, they behave as multifunctional transcription factors that are activated by a variety of extracellular signals. In studying their subcellular localization, we noticed that these transcription factors frequently reside in subnuclear domains/dots. Because nuclear dots are believed often to harbor components of histone deacetylase enzymes (HDACs), we investigated whether TFII-I family proteins colocalize and interact with HDACs. Here, we show that TFII-I and its related member hMusTRD1/BEN physically and functionally interact with HDAC3. The TFII-I family proteins and HDAC3 also show nearly identical expression patterns in early mouse development. Consistent with our earlier observation that TFII-I family proteins also interact with PIASxβ, a member of the E3 ligase family involved in the small ubiquitin-like modifier (SUMO) pathway, we show further that PIASxβ physically and functionally interacts with HDAC3 and relieves the transcriptional repression exerted by HDAC3 upon TFII-I-mediated gene activation. These results suggest a complex interplay between two posttranslational pathways—histone modification and SUMOylation—brokered in part by TFII-I family proteins.

Essential functions of the Williams-Beuren syndrome-associated TFII-I genes in embryonic development

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 TGFβRII/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.

TFII is required for transcription of the naturally TATA-less but initiator-containing Vbeta promoter.

The proximal or core promoter of a typical eukaryotic protein coding gene comprises distinct elements, TATA and/or initiator (Inr). The existence of TATA or Inr at the core promoter suggests that the mechanism of transcription initiation mediated by these two genetic elements may be different. Accordingly, it has been demonstrated that the transcriptional requirements for the TATA-containing, Inr-less (TATAInr) promoters are different from the transcriptional requirements for the TATA-less, Inr-containing (TATAInr) promoters. Although both types of promoters require the transcription initiation factor (TFIID) in addition to other common initiation factors, a TATAInr promoter requires accessory component(s). Here we have employed in vitro analyses to address the transcription factor requirements for a TATAInr promoter. We demonstrate that in addition to TFIID, a naturally occurring TATAInr promoter requires TFII-I, an Inr element-dependent transcription factor. Consistent with its Inr element-dependent activities, TFII-I is dispensable for a TATAInr promoter. Furthermore, we demonstrate that both TFII-I and TFIID activities in nuclear extracts are temperature-sensitive. However, TFII-I is heat-inactivated at temperatures lower than that required to inactivate TFIID. Therefore, differential heat treatment of nuclear extracts provides an assay to discriminate between transcriptional requirements at TATAInr and TATAInr promoters.

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.—Tapia‐Páez, I., Tammimies, K., Massinen, S., Roy. A. L., Kere, J. The complex of TFII‐I, PARP1, and SFPQ proteins regulates the DYX1C1 gene implicated in neuronal migration and dyslexia. FASEB J. 22, 3001–3009 (2008)