The Smad transcriptional corepressor TGIF recruits mSin3.

The homeodomain protein TG-interacting factor (TGIF) represses transcription by histone deacetylase-dependent and -independent means. Heterozygous mutations in human TGIF result in holoprosencephaly, a severe genetic disorder affecting craniofacial development, suggesting that TGIF is critical for normal development. After transforming growth factorbeta (TGFbeta) stimulation, Smad proteins enter the nucleus and form transcriptional activation complexes or interact with TGIF, which functions as a corepressor. The relative levels of Smad corepressors and coactivators present within the cell may determine the outcome of a TGFbeta response. We show that TGIF interacts directly with the paired amphipathic alpha-helix 2 domain of the mSin3 corepressor, and TGIF recruits mSin3 to a TGFbeta-activated Smad complex. The mSin3 interaction domain of TGIF has been shown to be essential for repression of a TGFbeta transcriptional response. Thus, TGIF represents a targeting component of the mSin3 corepressor complex.

SAP30, a component of the mSin3 corepressor complex involved in N-CoR-mediated repression by specific transcription factors.

The transcriptional corepressor mSin3 is found in a large multiprotein complex containing the histone deacetylases HDAC1 and HDAC2, in addition to at least five tightly associated polypeptides. We have cloned and characterized a novel component of the mSin3 complex, SAP30, SAP30 binds to mSin3 and is capable of mediating transcriptional repression via histone deacetylases. SAP30 also binds the N-CoR corepressor and is required for N-CoR-mediated repression by antagonist-bound estrogen receptor and the homeodomain protein Rpx, as well as N-CoR suppression of transactivation by the POU domain protein Pit-1. However, SAP30 is not required for N-CoR-mediated repression by unliganded retinoic acid receptor or thyroid hormone receptor, suggesting that SAP30 is involved in the functional recruitment of the mSin3-histone deacetylase complex to a specific subset of N-CoR corepressor complexes.

Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.

Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development and in repression of viral genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression.

Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity.

Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression.

Transcriptional repression by Mad-Max heterodimers requires interaction of Mad with the corepressors mSin3A/B. Sin3p, the S. cerevisiae homolog of mSin3, functions in the same pathway as Rpd3p, a protein related to two recently identified mammalian histone deacetylases, HDAC1 and HDAC2. Here, we demonstrate that mSin3A and HDAC1/2 are associated in vivo. HDAC2 binding requires a conserved region of mSin3A capable of mediating transcriptional repression. In addition, Mad1 forms a complex with mSin3 and HDAC2 that contains histone deacetylase activity. Trichostatin A, an inhibitor of histone deacetylases, abolishes Mad repression. We propose that Mad-Max functions by recruiting the mSin3-HDAC corepressor complex that deacetylates nucleosomal histones, producing alterations in chromatin structure that block transcription.

Mad proteins contain a dominant transcription repression domain.

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.

Human T cell leukemia virus type I Tax and phorbol 12-myristate 13-acetate induce expression of the A20 zinc finger protein by distinct mechanisms involving nuclear factor kappa B.

A20 was originally identified as a primary tumor necrosis factor alpha (TNF)-responsive gene, which encodes a 790-amino acid zinc finger protein. A20 is expressed in a wide variety of cell lines, including fibroblasts, in which A20 expression protects cells from TNF cytotoxicity. An analysis of A20 expression in lymphocytic and monocytic cells lines revealed that A20 protein expression correlates with lymphocyte activation and monocyte differentiation. A20 expression was also induced in Jurkat T cells expressing the human T cell leukemia virus type I Tax protein. Transient transfection studies demonstrated that stimulation of A20 transcription by TNF, phorbol 12-myristate 13-acetate (PMA), and Tax was mediated by two kappa B elements within the A20 promoter. Accordingly, DNA electrophoretic mobility shift assays confirmed inducible binding of nuclear factor kappa B (NF-kappa B) to a promoter fragment containing both A20 kappa B elements. Analysis of individual A20 kappa B sites revealed that both kappa B sites were required for TNF or PMA activation of the A20 promoter; however, Tax activation required only one kappa B site. Overexpression of NF-kappa B subunits activated the wild type A20 promoter, but did not activate mutated forms containing single kappa B sites. Thus, Tax activation of A20 transcription occurs through a mechanism distinct from PMA and TNF, possibly due to differential activation of NF-kappa B complexes or transcriptional cofactors.

Thrombospondin 1 and thrombospondin 2 are expressed as both homo- and heterotrimers.

There exist two distinct thrombospondin molecules (designated TSP1 and TSP2) which are encoded by separate genes. TSP1 is a trimeric cell surface and extracellular matrix molecule. Sequence comparison reveals that the 2 cysteines involved in interchain disulfide linkage and trimer assembly in TSP1 are conserved in TSP2 (Laherty, C. D., O'Rourke, K., Wolf, F. W., Katz, R., Seldin, M. F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 3274-3281). Swiss 3T3 fibroblasts express both TSP1 and TSP2, and, therefore, an important question is whether TSP in such cells is expressed as homotrimers or as heterotrimers. We find that Swiss 3T3 cells and epithelial cells transfected with TSP expression vectors express both homo- and heterotrimeric forms of TSP. In addition, homotrimeric TSP2 has a lower affinity for heparin than homotrimeric TSP1. Thus, the heparin affinity of TSP can be modulated by the expression of TSP as homo- or heterotrimers.

The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B.

A20 is an inducible zinc finger protein that confers resistance to tumor necrosis factor alpha cytotoxicity. A survey of various cell lines revealed that A20 was constitutively expressed in Epstein-Barr virus (EBV)-immortalized B-cells. Transfection experiments demonstrated that the EBV latent membrane protein LMP1 induced A20 expression. LMP1 is a transforming protein of EBV that has dramatic effects on cell growth, activation, and survival. An integral membrane phosphoprotein, LMP1 bears no homology to other recognized membrane signaling molecules, and its signal transduction pathway is not known. However, studies using the A20 promoter demonstrated that LMP1 transcriptionally activates the A20 gene through cis-acting kappa B sites. In addition, electrophoretic mobility shift assays confirmed LMP1-inducible binding of an NF-kappa B-like factor to kappa B sequences within the A20 promoter. This is the first report implicating NF-kappa B in signaling by LMP1, a fundamentally important viral transforming protein.

Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements.

A20 was first identified as a tumor necrosis factor (TNF) primary response transcript encoding a 790-amino acid protein with a unique zinc finger motif. Recently, A20 was shown to protect cells from TNF-induced cytotoxicity in a variety of cell lines. Nuclear run-on studies previously established that TNF activates A20 at the transcriptional level. To further characterize the mechanism by which TNF activates the A20 gene, we have cloned the A20 5'-flanking sequences and identified TNF-responsive elements within the promoter. The transcription initiation site was mapped by both primer extension and S1 nuclease protection experiments to a position 4.2 kilobases (kb) upstream of the initiator methionine; the first and second exon were separated by a 3.9-kb intron. Sequences upstream of the transcription start site were 76% GC-rich and contained six Sp1 binding sites and a TATA-like sequence at -29 but lacked a consensus CCAAT site. Transfection of Jurkat T-cells with an array of A20 promoter CAT constructs showed that two kappa B elements residing at -54 and -66 were required for induction by TNF. Supporting this notion, DNA electrophoretic mobility shift assays using nuclear extracts from unstimulated and TNF-stimulated Jurkat cells demonstrated kappa B-specific binding of a TNF-activated factor to an end-labeled probe containing the two A20 kappa B sequences. Finally, evidence obtained from cotransfection experiments showed that A20 negatively regulated its own expression.

Characterization of mouse thrombospondin 2 sequence and expression during cell growth and development.

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose expression has been associated with a variety of cellular processes including growth and embryogenesis. The recent discovery of the existence of a second mouse TSP gene necessitates careful examination of the discrete biochemical and functional properties associated with each molecule. In this report, the primary structures of human TSP, mouse TSP1 (mTSP1), mouse TSP2 (mTSP2), and chicken TSP are compared; and the expression of mTSP1 and mTSP2 during embryogenesis and growth factor-mediated cell proliferation is examined. The cloning and sequencing of the entire coding regions of mTSP1 and mTSP2 revealed considerable conservation of residues critical for TSP structure and function; these data suggest that TSP2 is capable of trimer formation and many of the same cell-surface and ligand interactions that mediate TSP function. Comparison of the various TSP sequences also allowed the assignment based on sequence homology of previously reported human TSP as TSP1 and chicken TSP as TSP2. mTSP2, like mTSP1, was shown to be a primary response gene when quiescent Swiss 3T3 cells were stimulated with serum, platelet-derived growth factor BB, basic fibroblast growth factor, or interleukin-1 beta. Interestingly, TSP1 and TSP2 exhibited markedly different tissue- and stage-specific patterns of mRNA expression during mouse embryogenesis, implying that the two TSP molecules possess discrete functional properties important for development. Additionally, the TSP genes (Thbs1 and Thbs2) were mapped to single loci on mouse chromosomes 2 and 17, respectively.

Characterization of the promoter region of the human thrombospondin gene. DNA sequences within the first intron increase transcription.

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose synthesis and secretion by mesenchymal cells is regulated at the level of gene transcription by platelet-derived growth factor. To examine the transcriptional regulation of the TSP gene at the molecular level, a genomic clone containing the human TSP promoter and flanking sequence was isolated and characterized. A 3.8-kilobase pair (kb) DNA fragment containing the first three exons, the first two introns, and 2.2 kb of 5'-flanking region was sequenced, and the site of transcription initiation was determined by both primer extension and S1 nuclease mapping. Consensus sequences for several potential regulatory elements were found in the 5'-flanking sequence, including a TATA box consensus sequence, TTTAAAA, located 24 base pairs upstream from the transcription start site. A chimeric gene was constructed containing the first intron, the first exon, and 2.0 kb of 5'-flanking sequence of the TSP gene fused to the promoterless gene for chloramphenicol acetyltransferase. When transfected into COS-1 or NIH3T3 cells this gene construct was transcribed, indicating the presence of a functional promoter in the TSP sequence. Transient transfection studies using deletion mutants of this TSP-chloramphenicol acetyltransferase construct were performed to locate cis-acting regulatory sequences. The deletion of flanking sequence 5' to position -234 had little or no effect on transcriptional activity, whereas deletion of 5'-flanking sequence extending further in the 3' direction resulted in the gradual loss of transcriptional activity. The removal of the first intron resulted in a 4-fold decrease in transcript levels, indicating the presence of a cis-acting positive element(s) in the first intron of the human TSP gene. This element(s) was further localized to the region between position +576 and position +727.