Human Müllerian-inhibiting substance promoter contains a functional TFII-I-binding initiator.

Müllerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (-6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I.

Regulation of nuclear localization and transcriptional activity of TFII-I by Bruton's tyrosine kinase.

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.

Regulation of TFII-I activity by phosphorylation.

The transcription factor TFII-I binds to distinct promoter sequences including an initiator element in several eukaryotic genes. Here we demonstrate that TFII-I is phosphorylated in vivo at serine/threonine and tyrosine residues in the absence of any apparent extracellular signals. This "basal" phosphorylation of TFII-I is not required and does not affect its specific DNA binding, but is critical for its in vitro transcriptional properties via the Vbeta promoter. To better assess the functional role of phosphorylation in regulating TFII-I activity, we focused on tyrosine phosphorylation of TFII-I. Ectopically expressed recombinant TFII-I, like its native counterpart, exhibits tyrosine phosphorylation in the absence of distinct extracellular signals. More important, mutation of a potential consensus tyrosine phosphorylation site in TFII-I leads to severe reduction in its basal transcriptional activation of the Vbeta promoter in vivo. Taken together, these data suggest that tyrosine phosphorylation of TFII-I is important for its initiator-dependent transcriptional activity.

TFII-I regulates Vbeta promoter activity through an initiator element.

In our effort to understand the transcriptional regulation of naturally occurring TATA-less but initiator (Inr)-containing genes, we have employed the murine T-cell receptor Vbeta 5.2 promoter as a model. Here we show by transient-transfection assays that the Inr binding transcription factor TFII-I is required for efficient expression of the Vbeta promoter in vivo. Mutations in the Inr element that reduced binding of TFII-I also abolished the Vbeta promoter activity by ectopic TFII-I. We further biochemically identified a protease-resistant N-terminal DNA binding fragment of TFII-I, p70. When ectopically expressed, recombinant p70 bound to the Vbeta Inr element with a specificity similar to that of wild-type TFII-I. More importantly, p70, which lacks independent activation functions, behaved as a dominant negative mutant that inhibited Inr-specific function of wild-type TFII-I. However, the activation functions of p70 were restored when fused to the heterologous activation domain of the yeast activator protein GAL4. Taken together, these data suggest that TFII-I functions in vivo require an intact Inr element and that the Inr-specific transcriptional functions of TFII-I are solely dictated by its N-terminal DNA binding domain and do not require its own C-terminal activation domain.

A multifunctional DNA-binding protein that promotes the formation of serum response factor/homeodomain complexes: identity to TFII-I.

The human homeodomain protein Phox1 interacts functionally with serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. Here, we report the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. We show that this protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. Furthermore, SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPIN exhibits modest affinity for a characterized initiator sequence in vitro. We propose that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery.

Methods for studying the biochemical properties of an Inr element binding protein: TFII-I.

Transcription initiation in eukaryotic mRNA coding genes is brought about by a host of general transcription factors, which assemble into a functional preinitiation complex (PIC) at the core promoter region, and gene-specific factors, which exert their effects on the rate and/or stability of the PIC. The core promoter region consists of a well-characterized TATA box and/or a less well-characterized pyrimidine-rich initiator element (Inr). While the biochemical mechanisms of TATA-mediated transcription initiation are extensively studied and known to be directed by the TATA binding protein, the mechanisms via the Inr element are poorly understood, as several factors have been shown to bind to an Inr. Here, we describe the biochemical properties of an Inr binding protein, TFII-I, employing the naturally occurring TATA-less but Inr-containing promoter derived from the T-cell receptor beta chain gene (V beta).

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.

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 (TATA+Inr-) promoters are different from the transcriptional requirements for the TATA-less, Inr-containing (TATA-Inr+) promoters. Although both types of promoters require the transcription initiation factor (TFIID) in addition to other common initiation factors, a TATA-Inr+ promoter requires accessory components. Here we have employed in vitro analyses to address the transcription factor requirements for a TATA-Inr+ promoter. We demonstrate that in addition to TFIID, a naturally occurring TATA-Inr+ promoter requires TFII-I, an Inr element-dependent transcription factor. Consistent with its Inr element-dependent activities, TFII-I is dispensable for a TATA+Inr- 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 TATA+Inr- and TATA-Inr+ promoters.