Modified niche optima and breadths explain the historical contingency of bacterial community responses to eutrophication in coastal sediments.

Previous studies have shown that the response of bacterial communities to disturbances depends on their environmental history. Historically fluctuating habitats host communities that respond better to disturbance than communities of historically stable habitats. However, the exact ecological mechanism that drives this dependency remains unknown. Here, we experimentally demonstrate that modifications of niche optima and niche breadths of the community members are driving this dependency of bacterial responses to past environmental conditions. First, we develop a novel, simple method to calculate the niche optima and breadths of bacterial taxa regarding single environmental gradients. Then, we test this method on sediment bacterial communities of three habitats, one historically stable and less loaded and two historically more variable and more loaded habitats in terms of historical chlorophyll-α water concentration, that we subject to hypoxia via organic matter addition ex situ. We find that communities containing bacterial taxa differently adapted to hypoxia show different structural and functional responses, depending on the sediment's environmental history. Specifically, in the historically less fluctuating and loaded sediments where we find more taxa poorly adapted to hypoxic conditions, communities change a lot over time and organic matter is not degraded efficiently. The opposite is true for the historically more fluctuating and loaded sediments where we find more taxa well adapted to hypoxia. Based on the community responses observed here, we also propose an alternative calculation of community resistance that takes into account how rapidly the communities respond to disturbances and not just the initial and final states of the community.

Functional consequences of HLA-DQ8 homozygosity versus heterozygosity for islet autoimmunity in type 1 diabetes.

Human leukocyte antigen (HLA) class II haplotypes are established risk factors in type 1 diabetes (T1D). The heterozygous DQ2/8 genotype confers the highest risk, whereas the DQ6/8 genotype is protective. We hypothesized that DQ2/8 trans-molecules composed of α and ß chains from DQ2 and DQ8 express unique ß-cell epitopes, whereas DQ6 may interfere with peptide binding to DQ8. Here we show that a single insulin epitope (InsB13-21) within the T1D prototype antigenic InsB6-22 peptide can bind to both cis- and trans-dimers, although these molecules display different peptide binding patterns. DQ6 binds a distinct insulin epitope (InsB6-14). The phenotype of DQ8-restricted T cells from a T1D patient changed from proinflammatory to anti-inflammatory in the presence of DQ6. Our data provide new insights into both susceptible and protective mechanism of DQ, where protecting HLA molecules bind autoantigens in a different (competing) binding register leading to 'epitope stealing', thereby inducing a regulatory, rather than a pathogenic immune response.

Immortalized keratinocytes derived from patients with epidermolytic ichthyosis reproduce the disease phenotype: a useful in vitro model for testing new treatments.

BACKGROUND: Epidermolytic ichthyosis (EI) is a skin fragility disorder caused by mutations in genes encoding suprabasal keratins 1 and 10. While the aetiology of EI is known, model systems are needed for pathophysiological studies and development of novel therapies. OBJECTIVES: To generate immortalized keratinocyte lines from patients with EI for studies of EI cell pathology and the effects of chemical chaperones as putative therapies. METHODS: We derived keratinocytes from three patients with EI and one healthy control and established immortalized keratinocytes using human papillomavirus 16-E6/E7. Growth and differentiation characteristics, ability to regenerate organotypic epidermis, keratin expression, formation of cytoskeletal aggregates, and responses to heat shock and chemical chaperones were assessed. RESULTS: The cell lines EH11 (K1_p.Val176_Lys197del), EH21 (K10_p.156Arg>Gly), EH31 (K10_p.Leu161_Asp162del) and NKc21 (wild-type) currently exceed 160 population doublings and differentiate when exposed to calcium. At resting state, keratin aggregates were detected in 9% of calcium-differentiated EH31 cells, but not in any other cell line. Heat stress further increased this proportion to 30% and also induced aggregates in 3% of EH11 cultures. Treatment with trimethylamine N-oxide and 4-phenylbutyrate (4-PBA) reduced the fraction of aggregate-containing cells and affected the mRNA expression of keratins 1 and 10 while 4-PBA also modified heat shock protein 70 (HSP70) expression. Furthermore, in situ proximity ligation assay suggested a colocalization between HSP70 and keratins 1 and 10. Reconstituted epidermis from EI cells cornified but EH21 and EH31 cells produced suprabasal cytolysis, closely resembling the in vivo phenotype. CONCLUSIONS: These immortalized cell lines represent a useful model for studying EI biology and novel therapies.

The types II and III transforming growth factor-beta receptors form homo-oligomers.

Affinity-labeling experiments have detected hetero-oligomers of the types I, II, and III transforming growth factor beta (TGF-beta) receptors which mediate intracellular signaling by TGF-beta, but the oligomeric state of the individual receptor types remains unknown. Here we use two types of experiments to show that a major portion of the receptor types II and III forms homo-oligomers both in the absence and presence of TGF-beta. Both experiments used COS-7 cells co-transfected with combinations of these receptors carrying different epitope tags at their extracellular termini. In immunoprecipitation experiments, radiolabeled TGF-beta was bound and cross-linked to cells co-expressing two differently tagged type II receptors. Sequential immunoprecipitations using anti-epitope monoclonal antibodies showed that type II TGF-beta receptors form homo-oligomers. In cells co-expressing epitope-tagged types II and III receptors, a low level of co-precipitation of the ligand-labeled receptors was observed, indicating that some hetero-oligomers of the types II and III receptors exist in the presence of ligand. Antibody-mediated cross-linking studies based on double-labeling immunofluorescence explored co-patching of the receptors at the cell surface on live cells. In cells co-expressing two differently tagged type II receptors or two differently tagged type III receptors, forcing one receptor into micropatches by IgG induced co-patching of the receptor carrying the other tag, labeled by noncross-linking monovalent Fab'. These studies showed that homo-oligomers of the types II and III receptors exist on the cell surface in the absence or presence of TGF-beta 1 or -beta 2. In cells co-expressing types II and III receptors, the amount of heterocomplexes at the cell surface was too low to be detected in the immunofluorescence co-patching experiments, confirming that hetero-oligomers of the types II and III receptors are minor and probably transient species.

Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner.

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-beta (TGF-beta) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-beta type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-beta1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-beta1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-beta1. 4) Importin-beta1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-beta receptor leads to enhanced interaction with importin-beta1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.

Operational criteria for selecting a cDNA microarray data normalization algorithm.

Microarray technology allows gene expression profiling at a global level. Many algorithms for the normalization of raw microarray data have been proposed, but no attempt has yet been made to propose operationally verifiable criteria for their comparative evaluation, which is necessary for the selection of the most appropriate method for a given dataset. This study develops a set of operational criteria for assessing the impact of various normalization algorithms in terms of accuracy (bias), precision (variance) and over-fitting (information reduction). The use of these criteria is illustrated by applying the three most widely used algorithms (global median normalization, spiked-in based normalization and lowess) on a specifically designed, multiply-controlled dataset.

Functional consequences of tumorigenic missense mutations in the amino-terminal domain of Smad4.

Smads, the intracellular effectors of transforming growth factor-beta (TGF-beta) family members, are somatically mutated at high frequency in particular types of human cancers. Certain of these mutations affect the Smad amino-terminal domain, which, in the case of Smad3 and Smad4, binds DNA. We investigated the functional consequences of four missense mutations in the Smad4 amino-terminal domain found in human tumors. The mutant proteins were found to have impaired abilities to bind DNA although they were fully capable of forming complexes with Smad3. All four Smad4 mutants showed decreased protein stability compared to wild-type Smad4. Two of the Smad4 mutants (G65V and P130S) were translocated to the nucleus and were capable of transactivating a Smad-dependent promoter in a ligand-dependent manner. In contrast, the L43S and R100T mutants were not translocated efficiently to the nucleus and consequently resulted in severely defective transcriptional responses to TGF-beta. Moreover, we demonstrate here the critical importance of two basic residues in the beta-hairpin loop of Smad3 or Smad4 for DNA binding, consistent with predictions from the Smad3 crystal structure. In addition, our results reveal that in the TGF-beta-induced heteromeric signaling complex, loss of DNA binding of Smad4 can be compensated by Smad3, however, both Smad3 and Smad4 are needed for efficient DNA binding and signaling. In conclusion, mutations in the amino-terminal domain of Smad4, that are found in cancer, show loss of multiple functional properties which may contribute to tumorigenesis.

Expression and characterization of Cys374 mutated human beta-actin in two different mammalian cell lines: impaired microfilament organization and stability.

Previous studies have demonstrated that addition of glutathione at the penultimate Cys374 residue of actin results in filaments with diminished mechanical stability. In the present work substitutions introducing a negatively charged (Asp and Glu) or a neutral (Ala) amino acid at position 374 of the human beta-actin and tagged at the N-terminus with the flag epitope were studied by transient transfections into Ishikawa human endometrial and opossum kidney cells. Immunofluorescence revealed that microfilaments which incorporated negatively charged mutants were partially to severely disorganized when compared to the almost well-formed actin-Ala374 filaments or the wild type actin filaments. Furthermore, microfilaments containing either negatively charged mutant were more sensitive to the destabilizing action of cytochalasin B. In addition, Triton fractionation resealed a considerable reduction of flag-actin content in the Triton insoluble fraction for cells expressing Asp374 or Glu374 mutant compared to wild type actin. These results demonstrate that negatively charged amino acid residues at the exposed C-terminal tail strongly affect actin microfilament organization and dynamics in vivo.

Focal vitreomacular traction: a prospective study of the evolution to macular hole: the mathematical approach.

UNLABELLED: PURPORSE: To determine the associated balance of forces of the vitreofoveal interface in focal vitreomacular traction evolving to full-thickness macular hole (FTMH) and to link/explain the observed changes in the context of mathematical and physics models. PATIENTS AND METHODS: This is a multicenter, prospective, and observational case series conducted at the Vitreoretinal Department of three different referral centers. Fellow eyes of patients with unilateral idiopathic FTMH were included. Eighty-nine patients were included in the analysis. The fellow normal eye of the study patients was imaged with spectral-domain optical coherence tomography. The main outcome measure was the optical-coherence-tomography-defined characteristics of the vitreofoveal interface and their analysis with mathematical and physics models at the end of follow-up period. RESULTS: Of the included 89 patients (66 women and 23 men; mean age±SD, 68.5 years±9.8), 10 (11.2%) developed FTMH at the fellow eye at the end of the follow-up period. We observed two types of vitreofoveal attachment. A V-shaped (cord-like) configuration and a U-shaped configuration. The eyes with the V-shaped attachment demonstrated initial structural changes in the outer foveal layers and the eyes with the U-shaped attachment showed inner morphological changes. CONCLUSION: We hypothesize that the type (V- or U-shaped) of the vitreofoveal attachment may affect the type and location of the initial structural change leading to the formation of FTMH from the stage of the focal vitreomacular traction.

Snail depletes the tumorigenic potential of glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive brain malignancy characterized by high heterogeneity and invasiveness. It is increasingly accepted that the refractory feature of GBM to current therapies stems from the existence of few tumorigenic cells that sustain tumor growth and spreading, the so-called glioma-initiating cells (GICs). Previous studies showed that cytokines of the bone morphogenetic protein (BMP) family induce differentiation of the GICs, and thus act as tumor suppressors. Molecular pathways that explain this behavior of BMP cytokines remain largely elusive. Here, we show that BMP signaling induces Smad-dependent expression of the transcriptional regulator Snail in a rapid and sustained manner. Consistent with its already established promigratory function in other cell types, we report that Snail silencing decreases GBM cell migration. Consequently, overexpression of Snail increases GBM invasiveness in a mouse xenograft model. Surprisingly, we found that Snail depletes the GBM capacity to form gliomaspheres in vitro and to grow tumors in vivo, both of which are important features shared by GICs. Thus Snail, acting downstream of BMP signaling, dissociates the invasive capacity of GBM cells from their tumorigenic potential.

Differential regulation of the two RhoA-specific GEF isoforms Net1/Net1A by TGF-ß and miR-24: role in epithelial-to-mesenchymal transition.

In the present study we analyzed the regulation of the two isoforms of the RhoA-specific guanine nucleotide exchange factor Net1 by transforming growth factor-ß (TGF-ß) in keratinocytes. We report that short-term TGF-ß treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-ß-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-ß. Interestingly, long-term TGF-ß treatment resulted in Net1 mRNA downregulation and Net1A protein degradation by the proteasome. Furthermore, we identified the microRNA miR-24 as a novel post-transcriptional regulator of Net1A expression. Silencing of Net1A resulted in disruption of E-cadherin- and zonula occludens-1 (ZO-1)-mediated junctions, as well as expression of the transcriptional repressor of E-cadherin, Slug and the mesenchymal markers N-cadherin, plasminogen activator inhibitor-1 (PAI-1) and fibronectin, indicating that late TGF-ß-induced downregulation of Net1A is involved in epithelial-to-mesenchymal transition (EMT). Finally, miR-24 was found to be implicated in the regulation of the EMT program in response to TGF-ß and was shown to be directly involved in the TGF-ß-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short- and long-term TGF-ß-mediated regulation of EMT.

Sustained TGF beta exposure suppresses Smad and non-Smad signalling in mammary epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis.

To better understand the dual, tumour-suppressive and tumour-promoting function of transforming growth factor-beta (TGFbeta), we analysed mammary epithelial NMuMG cells in response to short and long-term TGFbeta exposure. NMuMG cells became proliferation-arrested and apoptotic after exposure to TGFbeta for 2-5 days, whereas surviving cells underwent epithelial-mesenchymal transition (EMT). After chronic TGFbeta exposure (2-3 weeks), however, NMuMG cells became resistant to proliferation arrest and apoptosis, showing sustained EMT instead (TD cells). EMT was fully reversed by a pharmacologic TGFbeta-receptor-I kinase inhibitor or withdrawal of TGFbeta for 6-12 days. Interestingly, both cell cycle arresting/proapoptotic (Smads, p38 kinase) and antiapoptotic, proliferation and EMT-promoting signalling pathways (PI3K-PKB/Akt, ERK) were co-suppressed to low, but significant levels. Except for PI3K-Akt, TGFbeta-dependent downregulation of these signalling pathways in transdifferentiated (TD) cells was fully reversed upon TGFbeta withdrawal, together with partial re-induction of proliferation arrest and apoptosis. Co-injection of non-tumorigenic NMuMG cells with tumour-forming CHO cells oversecreting exogenous TGFbeta1 (CHO-TGFbeta1) allowed outgrowth of epithelioid cells in CHO-TGFbeta1 cell-induced tumours. These epithelial islands enhanced CHO-TGFbeta1 tumour cell proliferation, possibly due to chemokines (for example, JE/MCP-1) secreted by NMuMG/TD cells. We conclude that suppression of antiproliferative, proapoptotic TGFbeta signalling in TD cells may permit TGFbeta-dependent proliferation, survival and EMT-enhancing signalling pathways to act at low levels. Thus, TGFbeta may modulate its own signalling to facilitate switching from tumour suppression to tumour progression.

Regulation of actin organisation by TGF-beta in H-ras-transformed fibroblasts.

The actin cytoskeleton undergoes architectural changes during the processes of cell transformation and tumourigenesis. Transforming growth factors beta arrest cell cycle progression, regulate differentiation and modulate the onset of oncogenesis and tumourigenesis. Here, we investigated the direct role of transforming growth factor beta-1 in altering the transformed phenotype and regulating the actin organisation of oncogenic fibroblasts that constitutively or inducibly express the H-ras oncogene. Following transforming growth factor beta-1 treatment, these transformed fibroblasts undergo a dramatic morphological alteration that includes a discrete reorganisation of their actin cytoskeleton and focal adhesions. Quantitative biochemical analysis demonstrated that transforming growth factor beta-1 potently induced polymerisation of globular to filamentous actin, thus corroborating the morphological analysis. The effect of transforming growth factor beta-1 on the cytoskeleton correlates with the ability of this cytokine to suppress anchorage-independent growth of the transformed fibroblasts. Furthermore, transforming growth factor beta-1 upregulates considerably the levels of the RhoB small GTPase and less the RhoA levels. Finally, The beta GTPase inhibitor, C3 exotransferase, blocks the ability of TGF-beta1 to induce cytoskeletal reorganisation. These findings indicate that transforming growth factor beta can regulate cell morphology and growth in a concerted manner possibly via mechanisms that control the actin cytoskeleton.

Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members.

The cell cycle inhibitor p21/WAF1/Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. We report herein that the p21 gene is constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter showed that it contains a distal (positions -2,300/-210) and a proximal (positions -124 to -61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important role in the transduction of extracellular signals such as transforming growth factor beta, activin, etc. Constitutive expression of p21 was severely reduced by a C-terminally truncated form of Smad4 that was shown previously to block signaling through Smads. Smad3/4 and to a much lesser extent Smad2/4 caused high levels of transcriptional activation of the p21 promoter. Transactivation was compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, we show that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells.

TGF-beta receptors: structure and function.

The transforming growth factor beta (TGF-beta) superfamily of dimeric polypeptide growth factors regulate cell growth and differentiation and play important roles in embryonic development and the immune response. The molecular cloning of three types of high affinity cell-surface receptors (type I, type II and type III) for these growth factors provide insights into their poorly understood mechanisms of action. Here we focus on the structure of these receptors and on the complex interactions which occur between different receptors that are necessary for ligand-induced signal transduction. The type I and type II receptors are both related transmembrane serine/threonine kinase receptors which may hetero-oligomerize to transduce proper signals. The type III receptor modulates the binding of ligands to the signaling complex of type I and type II receptors. These findings suggest that serine/threonine phosphorylation and heteromeric receptor interactions are important elements of TGF-beta induced signaling.

Effects of the open reading frames in the Rous sarcoma virus leader RNA on translation.

Three short open reading frames (ORFs) reside in the 5' leader of Rous sarcoma virus (RSV) and are conserved in all avian sarcoma-leukosis retroviruses. Both extensions of the lengths of the ORFs and alterations in their initiation codons affect viral replication and gene expression. To determine whether the effects on viral replication were due to translational regulation mediated by the ORFs, we examined translation following mutation of the initiation and termination codons of each of the three ORFs. We found that the ORFs marginally enhanced downstream gene expression. Moreover, repression of downstream gene translation was proportional to the lengths of the elongated ORFs and depended on the initiation contexts of the AUG codons. Although the ORFs play a major role in viral activities, their effects on translation were relatively minor. Rather, the ORFs may affect the fate of unspliced avian retroviral RNA in chronically infected cells by participating in the sorting of viral RNA for either translation or encapsidation into virions.

A mutation in the short 5'-proximal open reading frame on Rous sarcoma virus RNA alters virus production.

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production.

Modelling of the MHC II allele I-A(g7) of NOD mouse: pH-dependent changes in specificity at pockets 9 and 6 explain several of the unique properties of this molecule.

AIMS/HYPOTHESIS: We modelled the three-dimensional structure of I-A(g7), the chief genetic component of diabetes in non-obese diabetic mice, to understand the unusual properties of this molecule. METHODS: Modelling was done, in complex with established antigenic peptides, based on the structure of I-A(k). RESULTS: The selectivity of the I-A(g7) molecule changes greatly at pockets 9 and 6 but hardly at all at pockets 1, 4 and 7, between endosomal pH (5.0) and extracellular pH (7.0), in agreement with previous results. This selectivity is attributed to the unique combination of beta9His, beta56His and beta57Ser. The positive charges in and around pocket 9 at pH 5, favour binding by negatively charged residues. At pH 7 however, the uncharged alpha68, beta9 and beta56 histidines favour the accommodation of the bulky residues lysine, arginine, phenylalanine and tyrosine at pocket 9. The combination of beta9His and alpha66Glu is responsible for the pH-dependent selectivity at pocket 6. Furthermore, the lack of repulsion between beta56His and alpha76Arg at pH 7 leads to a more stable ternary complex. CONCLUSION/INTERPRETATION: These results reconcile previous conflicts over the peptide binding ability of I-A(g7) and its motif. They furthermore provide possible explanations for the short lifetime of cell-surface I-A(g7) complexes in vivo, the higher threshold of thymic negative selection and inherent self-reactivity shown by immunocytes in these mice and the protection from diabetes afforded to them by several transgenically expressed mouse class II alleles. This contributes to an understanding of the pathogenesis of Type I (insulin-dependent) diabetes mellitus in this animal.

c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Sp1.

The cell cycle inhibitor protein p21(WAF1/Cip1) (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation. We have recently reported that the transforming growth factor-beta pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Sp1. In the present study we show that the Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2. By using gel electrophoretic mobility shift assays we show that this region does not contain a binding site for c-Jun. In accordance with the DNA binding data, c-Jun was unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coexpression of c-Jun and Sp1 in these cells resulted in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene was transactivated by overexpressed c-Jun in HepG2 cells. The above data propose functional cooperation between c-Jun and Sp1. Physical interactions between the two factors were demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Jun. The region of c-Jun mediating interaction with Sp1 was mapped within the basic region leucine zipper domain. In vivo, functional interactions between c-Jun and Sp1 were demonstrated using a GAL4-based transactivation assay. Overexpressed c-Jun transactivated a chimeric promoter consisting of five tandem GAL4-binding sites only when coexpressed with GAL4-Sp1-(83-778) fusion proteins in HepG2 cells. By utilizing the same assay, we found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) was sufficient for c-Jun-induced transactivation of the p21 promoter. In conclusion, our data support a mechanism of superactivation of Sp1 by c-Jun, which is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters.