David Wotton

Transcriptional control by the TGF‐β/Smad signaling system

The deployment of a cells genetic program in a multicellular organism must be tightly controlled for the sake of the organism as a whole. Over the past 20 years the transforming growth factor‐β (TGF‐β) family of secretory polypeptides has emerged as a major source of signals exerting this type of control. This family includes various forms of TGF‐β, the bone morphogenetic proteins (BMPs), the Nodals, the Activins, the anti‐Mullerian hormone, and many other structurally related factors in vertebrates, insects and nematodes (Massague, 1998). Produced by diverse cell types, these factors regulate cell migration, adhesion, multiplication, differentiation and death throughout the life span of the organism. Many of these responses result from changes in the expression of key target genes. Hence, transcriptional control by the TGF‐β family has become a subject of intense investigation in recent years. The present knowledge of these mechanisms is reviewed here. One basic concept concerning the role of the TGF‐β family as hormonally active agents warrants mention at the outset. Unlike classical hormones, whose actions are few and concrete, the members of the TGF‐β family have many different effects depending on the type and state of the cell. For example, in the same healing wound TGF‐β may stimulate or inhibit cell proliferation depending on whether the target is a fibroblast or a keratinocyte (Ashcroft et al ., 1999); in mammary epithelial cells TGF‐β will cause growth arrest or metastatic behavior depending on the level of oncogenic Ras activity present in the cell (Oft et al ., 1996); and human BMP4 and its Drosophila ortholog, DPP, can signal dorsalization in the fly (Padgett et al ., 1993) yet bone formation in a vertebrate (Sampath et al ., 1993). TGF‐β family members are multifunctional hormones, the nature of their effects depending on what has been called ‘the cellular context’. It was plausible …

A Smad transcriptional corepressor.

Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell.

The polycomb protein Pc2 is a SUMO E3

Polycomb group (PcG) proteins form large multimeric complexes (PcG bodies) which are involved in the stable repression of gene expression. The human PcG protein, Pc2, has been shown to recruit the transcriptional corepressor, CtBP, to PcG bodies. We show that CtBP is sumoylated at a single lysine. In vitro, CtBP sumoylation minimally requires the SUMO E1 and E2 (Ubc9) and SUMO-1. However, Pc2 dramatically enhances CtBP sumoylation. In vivo, this is likely due to the ability of Pc2 to recruit both CtBP and Ubc9 to PcG bodies, thereby bringing together substrate and E2, and stimulating the transfer of SUMO to CtBP. These results demonstrate that Pc2 is a SUMO E3, and suggest that PcG bodies may be sumoylation centers.

Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination

Holoprosencephaly (HPE) is the most common structural defect of the developing forebrain in humans (1 in 250 conceptuses, 1 in 16,000 live-born infants). HPE is aetiologically heterogeneous, with both environmental and genetic causes. So far, three human HPE genes are known: SHH at chromosome region 7q36 (ref. 6); ZIC2 at 13q32 (ref. 7); and SIX3 at 2p21 (ref. 8). In animal models, genes in the Nodal signalling pathway, such as those mutated in the zebrafish mutants cyclops (refs 9,10), squint (ref. 11) and one-eyed pinhead (oep; ref. 12), cause HPE. Mice heterozygous for null alleles of both Nodal and Smad2 have cyclopia. Here we describe the involvement of the TG-interacting factor (TGIF), a homeodomain protein, in human HPE. We mapped TGIF to the HPE minimal critical region in 18p11.3. Heterozygous mutations in individuals with HPE affect the transcriptional repression domain of TGIF, the DNA-binding domain or the domain that interacts with SMAD2. (The latter is an effector in the signalling pathway of the neural axis developmental factor NODAL, a member of the transforming growth factor-β (TGF-β) family.) Several of these mutations cause a loss of TGIF function. Thus, TGIF links the NODAL signalling pathway to the bifurcation of the human forebrain and the establishment of ventral midline structures.

Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4

Smad2 and Smad4 are related tumour-suppressor proteins, which, when stimulated by the growth factor TGF-β, form a complex to inhibit growth. The effector function of Smad2 and Smad4 is located in the conserved carboxy-terminal domain (C domain) of these proteins and is inhibited by the presence of their amino-terminal domains (N domain). This inhibitory function of the N domain is shown here to involve an interaction with the C domain that prevents the association of Smad2 with Smad4. This inhibitory function is increased in tumour-derived forms of Smad2 and 4 that carry a missense mutation in a conserved N domain arginine residue. The mutant N domains have an increased affinity for their respective C domains, inhibit the Smad2–Smad4 interaction, and prevent TGFβ-induced Smad2–Smad4 association and signalling. Whereas mutations in the C domain disrupt the effector function of the Smad proteins, N-domain arginine mutations inhibit SMAD signalling through a gain of autoinhibitory function. Gain of autoinhibitory function is a new mechanism for inactivating tumour suppressors.

Cooperative binding of Ets-1 and core binding factor to DNA.

Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription.

Multiple Modes of Repression by the Smad Transcriptional Corepressor TGIF

TGIF is a DNA-binding homeodomain protein that has been demonstrated to play a role in transforming growth factor β-regulated transcription and implicated in the control of retinoid-responsive transcription. We investigated the intrinsic transcriptional activity of TGIF fused to a heterologous DNA-binding domain. Our results demonstrate that TGIF is a transcriptional repressor able to repress transcription from several different promoters. Repression by TGIF is insensitive to the distance at which it is bound from the promoter. Moreover, the wild type TGIF effectively represses transcription when bound to its cognate DNA-binding site via its homeodomain. Deletion analysis revealed the presence of at least two separable repression domains within TGIF. Repression by one of these is dependent on the activity of histone deacetylases (HDACs), whereas the other appears not to require HDAC activity. Finally, we demonstrate that TGIF interacts with HDACs via its carboxyl-terminal repression domain. Together, these results suggest that TGIF is a multifunctional transcriptional repressor, which acts in part by recruiting HDAC activity.

Epidermal growth factor signaling via Ras controls the Smad transcriptional co‐repressor TGIF

Smad transcription factors mediate the actions of transforming growth factor‐β (TGF‐β) cytokines during development and tissue homeostasis. TGF‐β receptor‐activated Smad2 regulates gene expression by associating with transcriptional co‐activators or co‐repressors. The Smad co‐repressor TGIF competes with the co‐activator p300 for Smad2 association, such that TGIF abundance helps determine the outcome of a TGF‐β response. Small alterations in the physiological levels of TGIF can have profound effects on human development, as shown by the devastating brain and craniofacial developmental defects in heterozygotes carrying a hypomorphic TGIF mutant allele. Here we show that TGIF levels modulate sensitivity to TGF‐β‐mediated growth inhibition, that TGIF is a short‐lived protein and that epidermal growth factor (EGF) signaling via the Ras–Mek pathway causes the phosphorylation of TGIF at two Erk MAP kinase sites, leading to TGIF stabilization and favoring the formation of Smad2–TGIF co‐repressor complexes in response to TGF‐β. These results identify the first mechanism for regulating TGIF levels and suggest a potential link for Smad and Ras pathway convergence at the transcriptional level.

The structure of the human CD2 gene and its expression in transgenic mice.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5′ flanking sequences, 15 kb containing the genes five exons and 9 kb of 3′ flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.

The Interaction of the Carboxyl Terminus-binding Protein with the Smad Corepressor TGIF Is Disrupted by a Holoprosencephaly Mutation in TGIF

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-β-activated Smads, thereby repressing genes normally activated by TGF-β. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIFresults in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. We demonstrate that TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-β-activated gene responses by TGIF is dependent on interaction with CtBP, and we show that TGIF is able to recruit CtBP to a TGF-β-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF.