Raul Urrutia

Sp1- and Krüppel-like transcription factors

SummarySp1-like proteins and Krüppel-like factors (KLFs) are highly related zinc-finger proteins that are important components of the eukaryotic cellular transcriptional machinery. By regulating the expression of a large number of genes that have GC-rich promoters, Sp1-like/KLF transcription regulators may take part in virtually all facets of cellular function, including cell proliferation, apoptosis, differentiation, and neoplastic transformation. Individual members of the Sp1-like/KLF family can function as activators or repressors depending on which promoter they bind and the coregulators with which they interact. A long-standing research aim has been to define the mechanisms by which Sp1-like factors and KLFs regulate gene expression and cellular function in a cell- and promoter-specific manner. Most members of this family have been identified in mammals, with at least 21 Sp1-like/KLF proteins encoded in the human genome, and members are also found in frogs, worms and flies. Sp1-like/KLF proteins have highly conserved carboxy-terminal zinc-finger domains that function in DNA binding. The amino terminus, containing the transcription activation domain, can vary significantly between family members.

KRAB-containing zinc-finger repressor proteins

SummaryThe largest family of zinc-finger transcription factors comprises those containing the Krüppel-associated box (or KRAB domain), which are present only in tetrapod vertebrates. Many genes encoding KRAB-containing proteins are arranged in clusters in the human genome, with one cluster close to chromosome 9ql3 and others in centromeric and telomeric regions of other chromosomes, but other genes occur individually throughout the genome. The KRAB domain, which is found in the amino-terminal region of the proteins, behaves as a transcriptional repressor domain by binding to corepressor proteins, whereas the C2H2 zinc-finger motifs bind DNA. The functions currently proposed for members of the KRAB-containing protein family include transcriptional repression of RNA polymerase I, II, and III promoters and binding and splicing of RNA. Members of the family are involved in maintenance of the nucleolus, cell differentiation, cell proliferation, apoptosis, and neoplastic transformation.

Glycogen Synthase Kinase-3β Participates in Nuclear Factor κB–Mediated Gene Transcription and Cell Survival in Pancreatic Cancer Cells

Recent studies using glycogen synthase kinase-3beta (GSK-3beta)-deficient mouse embryonic fibroblasts suggest that GSK-3beta positively regulates nuclear factor kappaB (NFkappaB)-mediated gene transcription. Because NFkappaB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3beta in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3beta and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3beta by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3beta influences NFkappaB-mediated gene transcription at a point distal to the Ikappa kinase complex, as only ectopic expression of the NFkappaB subunits p65/p50, but not an Ikappa kinase beta constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3beta inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3beta in cancer cell survival and proliferation and suggest GSK-3beta as a potential therapeutic target in the treatment of pancreatic cancer.

Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells.

Members of the TGFbeta family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGFbeta peptides is necessary for a better understanding of normal morphogenesis as well as carcinogenesis of the pancreas. In this study, we have characterized the expression and function in exocrine pancreatic epithelial cells of the TGFbeta-inducible early gene (TIEG), a Krüppel-like zinc finger transcription factor encoding gene previously isolated from mesodermally derived osteoblastic cells. We demonstrate that this gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we show that the expression of TIEG is regulated by TGFbeta1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG in the TGFbeta-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG in linking TGFbeta-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.

Molecular Cloning and Characterization of TIEG2Reveals a New Subfamily of Transforming Growth Factor-β-inducible Sp1-like Zinc Finger-encoding Genes Involved in the Regulation of Cell Growth

Sp1-like zinc finger transcription factors are involved in the regulation of cell growth and differentiation. Recent evidence demonstrating that mammalian cells express novel, yet uncharacterized, Sp1-like proteins has stimulated a search for new members of this family. We and others have recently reported that the transforming growth factor (TGF)-β-regulated gene TIEGencodes a new Sp1-like protein that inhibits cell growth in cultured cells. Here we report the identification, nuclear localization, DNA binding activity, transcriptional repression activity, and growth inhibitory effects of TIEG2, a novel TGF-β-inducible gene related to TIEG. TIEG2 is ubiquitously expressed in human tissues, with an enrichment in pancreas and muscle. TIEG2 shares 91% homology with TIEG1 within the zinc finger region and 44% homology within the N terminus. Biochemical characterization reveals that TIEG2 is a nuclear protein, which, as predicted from the primary structure, specifically binds to an Sp1-like DNA sequence in vitroand can repress a promoter containing Sp1-like binding sites in transfected Chinese hamster ovary epithelial cells. Furthermore, functional studies using [3H]thymidine uptake and MTS (3-(4,3-dimethyltiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays demonstrate that the overexpression of TIEG2 in Chinese hamster ovary cells inhibits cell proliferation. Thus, TIEG2, together with TIEG1, defines a new subfamily of TGF-β-inducible Sp1-like proteins involved in the regulation of cell growth.

Association of distinct α2 adrenoceptor and serotonin transporter polymorphisms with constipation and somatic symptoms in functional gastrointestinal disorders

Background: The role of genetics in the phenotypic manifestations of irritable bowel syndrome (IBS) is unclear. Our aims were: (1) to compare the prevalence of polymorphisms of alpha 2 (α2) adrenoceptors, norepinephrine transporter, and serotonin transporter protein (soluble carrier protein member 4 (SLC6A4)) promoter in patients with lower functional gastrointestinal disorders (FGID) and in healthy controls; and (2) to test associations of these genetic variations with symptoms of IBS and high somatic symptom scores. Methods: Validated bowel and somatic symptom questionnaires characterised the phenotype: 90 with IBS constipation (IBS-C), 128 IBS diarrhoea, 38 IBS alternating bowel function, and 20 chronic abdominal pain. Logistic regression analyses assessed associations of different polymorphisms for α2 adrenoceptor and SLC6A4 with IBS or chronic abdominal pain phenotypes and high somatic score. Results: Two distinct polymorphisms independently appeared to be associated with the phenotype IBS-C: α2C Del 322–325 (odds ratio (OR) 2.48 (95% confidence interval (CI) 0.98, 6.28); p = 0.05) and α2A −1291 (C→G) (OR 1.66 (95% CI 0.94, 2.92); p = 0.08) relative to wild-type. Overall, the α2C Del 322–325 polymorphism (alone or combined with other polymorphisms) was also significantly associated with a high somatic symptom score (OR 2.2 (95% CI 1.06, 4.64); p = 0.03). Combinations of polymorphisms were also associated with high somatic scores. Conclusion: Functionally distinct α2A and α2C adrenoceptor and serotonin transporter polymorphisms are associated with constipation and high somatic symptoms in patients with lower functional gastrointestinal disorders, although the strength of the genetic contribution to the phenotype is unclear.

A Conserved α-Helical Motif Mediates the Interaction of Sp1-Like Transcriptional Repressors with the Corepressor mSin3A

ABSTRACT Sp1-like proteins are defined by three highly homologous C2H2 zinc finger motifs that bind GC-rich sequences found in the promoters of a large number of genes essential for mammalian cell homeostasis. Here we report that TIEG2, a transforming growth factor β-inducible Sp1-like protein with antiproliferative functions, represses transcription through recruitment of the mSin3A-histone deacetylase complex. The interaction of TIEG2 with mSin3A is mediated by an alpha-helical repression motif (α-HRM) located within the repression domain (R1) of TIEG2. This α-HRM specifically associates with the second paired amphipathic helix (PAH2) domain of mSin3A. Mutations in the TIEG2 α-HRM domain that disrupt its helical structure abolish its ability to both bind mSin3A and repress transcription. Interestingly, the α-HRM is conserved in both the TIEG (TIEG1 and TIEG2) and BTEB (BTEB1, BTEB3, and BTEB4) subfamilies of Sp1-like proteins. The α-HRM from these proteins also mediates direct interaction with mSin3A and represses transcription. Surprisingly, we found that the α-HRM of the Sp1-like proteins characterized here exhibits structural and functional resemblance to the Sin3A-interacting domain previously described for the basic helix-loop-helix protein Mad1. Thus, our study defines a mechanism of transcriptional repression via the interactions of the α-HRM with the Sin3-histone deacetylase complex that is utilized by at least five Sp1-like transcriptional factors. More importantly, we demonstrate that a helical repression motif which mediates Sin3 interaction is not an exclusive structural and functional characteristic of the Mad1 subfamily but rather has a wider functional impact on transcriptional repression than previously demonstrated.

The family feud: turning off Sp1 by Sp1-like KLF proteins.

Sp1 is one of the best characterized transcriptional activators. The biological importance of Sp1 is underscored by the fact that several hundreds of genes are thought to be regulated by this protein. However, during the last 5 years, a more extended family of Sp1-like transcription factors has been identified and characterized by the presence of a conserved DNA-binding domain comprising three Krüppel-like zinc fingers. Each distinct family member differs in its ability to regulate transcription, and, as a consequence, to influence cellular processes. Specific activation and repression domains located within the N-terminal regions of these proteins are responsible for these differences by facilitating interactions with various co-activators and co-repressors. The present review primarily focuses on discussing the structural, biochemical and biological functions of the repressor members of this family of transcription factors. The existence of these transcriptional repressors provides a tightly regulated mechanism for silencing a large number of genes that are already known to be activated by Sp1.

Sp1 and its likes: biochemical and functional predictions for a growing family of zinc finger transcription factors.

Abstract: The discovery and functional characterization of Sp1 as a GC‐rich binding zinc finger protein provided a useful paradigm for understanding mechanisms mediating transcriptional activation in eukaryotic cells. This early paradigm suggested that promoters carrying GC‐rich sequences are activated by Sp1 through its interaction with proteins from the basal transcriptional machinery to upregulate gene expression. Since the time of this seminal work, studies from several laboratories have led to the discovery of many Sp1‐like transcription factors containing highly homologous DNA binding motifs that bind to similar sequences. Consequently, this knowledge poses many important questions regarding whether these related proteins have similar or antagonistic biochemical and functional properties to Sp1. The goal of this article is to use available database information and recent experimental evidence to describe the current repertoire of Sp1‐like zinc finger transcription factors in mammalian cells. Furthermore, we discuss structural and functional studies that reveal that these proteins may share a role in morphogenetic pathways. Altogether, this information is aimed at better understanding how this growing family of transcription factors work to regulate gene expression and morphogenesis.

Sin3: master scaffold and transcriptional corepressor.

Sin3 was isolated over two decades ago as a negative regulator of transcription in budding yeast. Subsequent research has established the protein as a master transcriptional scaffold and corepressor capable of transcriptional silencing via associated histone deacetylases (HDACs). The core Sin3-HDAC complex interacts with a wide variety of repressors and corepressors, providing flexibility and expanded specificity in modulating chromatin structure and transcription. As a result, the Sin3/HDAC complex is involved in an array of biological and cellular processes, including cell cycle progression, genomic stability, embryonic development, and homeostasis. Abnormal recruitment of this complex or alteration of its enzymatic activity has been implicated in neoplastic transformation.