Novel adenomatous polyposis coli gene promoter is located 40 kb upstream of the initiating methionine.

The product of the oncosuppressor adenomatous polyposis coli (APC) gene is involved in cell cycle arrest and apoptosis and its loss of function is associated with the development of colorectal carcinogenesis. Its transcriptional regulation seems rather complex and has not been completely elucidated up to now. In an attempt to identify the transcription start sites for the mouse Apc gene we have detected a novel transcript in mouse embryonic stem (ES) cells and colon tissue. This transcript contains an untranslated exon, whose flanking sequences exhibited strong promoter activity in transient transfection experiments. These results suggest that we have identified a novel promoter for the mouse Apc gene, localized about 40 kb upstream of the initiating methionine, which drives expression of the unique Apc transcript type detected in undifferentiated totipotent ES cells. Transcripts bearing the novel exon combined either with exon 1 or with exon 2 were detected in all mouse tissues tested.

Somatic activation of beta-catenin bypasses pre-TCR signaling and TCR selection in thymocyte development.

Mutation or ablation of T cell factor 1 and lymphocyte enhancer factor 1 indicated involvement of the Wnt pathway in thymocyte development. The central effector of the Wnt pathway is beta-catenin, which undergoes stabilization upon binding of Wnt ligands to frizzled receptors. We report here that conditional stabilization of beta-catenin in immature thymocytes resulted in the generation of single positive T cells that lacked the alpha beta TCR and developed in the absence of pre-TCR signaling and TCR selection. Although active beta-catenin induced differentiation in the absence of TCRs, its action was associated with reduced proliferation and survival when compared to developmental changes induced by the pre-TCR or the alpha beta TCR.

Constitutive pre-TCR signaling promotes differentiation through Ca2+ mobilization and activation of NF-kappaB and NFAT.

Pre-T cell antigen receptor (pre-TCR) signaling plays a crucial role in the development of immature T cells. Although certain aspects of proximal pre-TCR signaling have been studied, the intermediate signal transducers and the distal transcription modulators have been poorly characterized. We report here a correlation between pre-TCR signaling and a biphasic rise in the cytosolic Ca2+ concentration. In addition, we show that constitutive pre-TCR signaling is associated with an increased rate of Ca2+ influx through store-operated plasma membrane Ca2+ channels. We show also that the biphasic nature of the observed pre-TCR-induced rise in cytosolic Ca2+ differentially modulates the activities of the transcription factors NF-kappaB and NFAT in developing T cells.

The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.

EblacZ tumor dormancy in bone marrow and lymph nodes: active control of proliferating tumor cells by CD8+ immune T cells.

A well-defined lacZ gene tagged DBA/2 lymphoma (EblacZ) was used to examine the role of host immune responses in controlling tumor dissemination and persistence, as well as metastasis. In s.c. and intra-ear pinna-inoculated mice, low numbers of EblacZ cells homed to the bone marrow and lymph nodes. The frequency of bone marrow-residing tumor cells did not change with the growth of primary tumor or with multiple inoculations of tumor cells. The bone marrow-residing tumor cells expressed the proliferation-associated Ki67 antigen and expanded upon CD8+ depletion. In contrast, inoculation of nu/nu or severe combined immunodeficiency mice or of immune-suppressed DBA/2 mice led to the rapid outgrowth of EblacZ cells in the bone marrow and their metastasis to other organs. Transfer of bone marrow from EblacZ immunized MHC congenic or syngeneic DBA/2 donors, but not from naive donors, protected s.c.-inoculated DBA/2 mice. Protection was abrogated by in vitro depletion of CD8+ T cells prior to transfer of bone marrow. These experiments show that bone marrow and lymph nodes are privileged sites where potentially lethal tumor cells are controlled in a dormant state by the immune system. Metastasis may be a consequence of the breakdown of this immune control.

The extended packaging sequence of MoMLV contains a constitutive mRNA nuclear export function.

The present report shows that incorporation of defined sequences from the Moloney murine leukaemia virus (MoMLV) into Rex dependent expression vectors based on the human T-cell leukaemia virus (HTLV-1) allows Rex independent gene expression. Deletion mutagenesis of the MoMLV derived sequences allowed this function to be localised to a 312 nt length sequence overlapping the MoMLV gag p15/p12 open reading frame. This 'extended packaging sequence' has been reported to markedly increase the titre of in vitro packaged retroviral vectors. Using fluorescent in situ hybridisation combined with confocal microscopy we show that the 312 nt element can replace Rex mediated nuclear export and expression of transcripts containing HTLV-1 cis acting repressive elements. Our observations are consistent with the extended packaging sequence of MoMLV exerting a constitutive mRNA nuclear export function.

The mouse filensin gene: structure and evolutionary relation to other intermediate filament genes.

Filensin and phakinin are two lens-specific members of the intermediate filament (IF) superfamily of proteins. They coassemble to form a beaded submembraneous filamentous network, the beaded filaments (BFs). The low sequence homology and differences in assembly compared to other IF proteins do not allow their classification in any of the five IF subgroups. The organization of the phakinin gene exon/intron boundaries provides evidence that this partner may be sharing a common origin with type I cytokeratin genes. Here we report the molecular cloning, sequence and characterization of the mouse filensin gene. The filensin gene consists of 8 exons and 7 introns, with 6 introns interrupting its rod domain in a highly conserved manner characteristic of type III IF genes, like vimentin, desmin, or peripherin. Of the two tail domain exons the one adjacent to the rod domain, compares to exon 7 of the non-neuronal cytoplasmic IF gene of helix aspersa and to the lamin region bridging the end of the rod domain to the nuclear localization signal. Altogether, these observations indicate that the lens beaded filaments form an independent class of IF.

The activation domain of a hormone inducible HTLV-1 Rex protein determines colocalization with the nuclear pore.

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Rex is an essential regulatory protein that acts at the posttranscriptional level to promote expression of unspliced and singly spliced genes of the virus. Rex functions have been attributed to at least three separate domains of the protein determining nuclear/nucleolar accumulation and RNA binding (overlapping), multimerization, and nuclear export of Rex-responsive RNA. The steady-state intracellular localization of functional Rex molecules is mainly nucleolar. Fusions of wild-type Rex and the ligand binding domain of human estrogen receptor (ER) produced conditional molecules (ERRex and ERalaRex), which remained cytoplasmic in the absence of hormone and in response to hormone colocalized with the nuclear pore complex (NPC). These molecules induced in a hormone-dependent manner the expression of a Rex reporter plasmid and of the HTLV-1 Env protein and fusion of Env expressing cells. In contrast, activation domain mutants (ERRex delta and ERRexGly) translocated from the cytoplasm and acquired a diffuse nuclear localization. These mutants did not associate with the NPC and failed to show any of the expected Rex functions. Rex functions were perturbed by inactivating the RNA binding domain (mutant ERM2) or the oligomerization domain (mutant ERM7). However, these two mutant fusion proteins exhibited a hormone-dependent NPC colocalization. These observations provide in vivo evidence that intranuclear translocation of intact Rex to the NPC is dependent exclusively on a functional activation domain and is not influenced by binding to the target RNA.

To bead or not to bead? Lens-specific intermediate filaments revisited.

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to 'mainstream' IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

Contributions of the structural domains of filensin in polymer formation and filament distribution.

Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.

Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells.

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host-specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.

The beaded intermediate filaments and their potential functions in eye lens.

The elongated fiber cells of the eye lens contain a unique cytoskeletal system, the beaded chain filaments (BFs). The BFs had been morphologically identified more than two decades ago, but the precise identity of their subunit molecules remained unknown. Recently, use of recombinant DNA approaches, refined morphological and immunochemical studies and experiments with mutant mice have allowed the molecular dissection of these structures and provided clues about their potential functions. The BFs represent a highly specialized network of intermediate filaments (IFs) juxtaposed to the plasma membrane. They are obligate heteropolymers composed of two lens-specific polypeptides, filensin and phakinin. In this review we discuss the properties, molecular interactions and in situ arrangement of these two proteins, and comment on their potential roles during lens development.

The 47-kD lens-specific protein phakinin is a tailless intermediate filament protein and an assembly partner of filensin.

In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed filensin. Filensin does not self-assemble into regular IFs but is known to associate with another 47-kD lens-specific protein which has been suggested to represent its assembly partner. To address this possibility, we cloned and sequenced the cDNA coding for the bovine 47-kD protein which we have termed phakinin (from the greek phi alpha kappa omicron sigma = phakos = lens). The predicted sequence comprises 406 amino acids and shows significant similarity (31.3% identity over 358 residues) to type I cytokeratins. Phakinin possesses a 95-residue, non-helical domain (head) and a 311 amino acid long alpha-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakinin lacks a COOH-terminal tail domain and it therefore represents the second known example of a naturally tailless IF protein. Confocal microscopy on frozen lens sections reveals that phakinin colocalizes with filensin and is distributed along the periphery of the lens fiber cells. Quantitative immunoblotting with whole lens fiber cell preparations and fractions of washed lens membranes suggest that the natural stoichiometry of phakinin to filensin is approximately 3:1. Under in vitro conditions, phakinin self-assembles into metastable filamentous structures which tend to aggregate into thick bundles. However, mixing of phakinin and filensin at an optimal ratio of 3:1 yields stable 10-nm filaments which have a smooth surface and are ultrastructurally indistinguishable from "mainstream" IFs. Immunolabeling with specific antibodies shows that these filaments represent phakinin/filensin heteropolymers. Despite its homology to the cytokeratins, phakinin does not coassemble with acidic (type I), or basic (type II) cytokeratins. From these data we conclude that filensin and phakinin are obligate heteropolymers which constitute a new membrane-associated, lens-specific filament system related to, but distinct from the known classes of IFs.

Bovine filensin possesses primary and secondary structure similarity to intermediate filament proteins.

The cDNA coding for calf filensin, a membrane-associated protein of the lens fiber cells, has been cloned and sequenced. The predicted 755-amino acid-long open reading frame shows primary and secondary structure similarity to intermediate filament (IF) proteins. Filensin can be divided into an NH2-terminal domain (head) of 38 amino acids, a middle domain (rod) of 279 amino acids, and a COOH-terminal domain (tail) of 438 amino acids. The head domain contains a di-arginine/aromatic amino acid motif which is also found in the head domains of various intermediate filament proteins and includes a potential protein kinase A phosphorylation site. By multiple alignment to all known IF protein sequences, the filensin rod, which is the shortest among IF proteins, can be subdivided into three subdomains (coils 1a, 1b, and 2). A 29 amino acid truncation in the coil 2 region accounts for the smaller size of this domain. The filensin tail contains 6 1/2 tandem repeats which match analogous motifs of mammalian neurofilament M and H proteins. We suggest that filensin is a novel IF protein which does not conform to any of the previously described classes. Purified filensin fails to form regular filaments in vitro (Merdes, A., M. Brunkener, H. Horstmann, and S. D. Georgatos. 1991. J. Cell Biol. 115:397-410), probably due to the missing segment in the coil 2 region. Participation of filensin in a filamentous network in vivo may be facilitated by an assembly partner.

Mapping of the gene TCF2 coding for the transcription factor LFB3 to human chromosome 17 by polymerase chain reaction.

A human clone corresponding to the gene for the DNA-binding factor LFB3, a protein highly homologous to the liver-specific transcription factor LFB1, has been isolated and partially sequenced. This gene is designated TCF2. Oligonucleotide primers have been designed for LFB3 and used to amplify specifically the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction. By this means, the human LFB3 gene has been mapped to the long arm of chromosome 17, between the centromere and the APL breakpoint.

Amino-terminal domain of NF1 binds to DNA as a dimer and activates adenovirus DNA replication.

NF1 is a DNA-binding protein involved in initiation of adenovirus DNA replication as well as in modulating the rate of transcription initiation of genes containing the sequence TGGCA. We show here that recombinant NF1 expressed via vaccinia virus is transported into the nucleus and binds to its cognate sequences with the same specificity as NF1 purified from HeLa cells. Furthermore, the recombinant NF1 forms oligomers in solution and binds as a dimer to palindromic as well as half-site sequences. NF1 expressed via vaccinia virus stimulates the initiation of adenovirus replication in vitro. The N-terminal 240 amino acids of the protein are sufficient for full DNA-binding activity as well as stimulation of adenovirus replication. By analysis of several NF1 mutants translated in vitro, we also define the minimal DNA-binding domain and localize the region responsible for DNA binding on the N-terminal and for oligomerization on the C-terminal side of this domain.

Transcription of the promoter of the rat NF-1 gene depends on the integrity of an Sp1 recognition site.

The transcription start site and promoter of the rat gene coding for the transcription factor NF-1 have been identified. The NF-1 promoter was fused to the chloramphenicol acetyltransferase-coding sequence, and the resulting plasmid was transcriptionally active in the HepG2 cell line. Footprinting and gel retardation analysis indicated that the transcription factor Sp1 binds to the NF-1 promoter. Mutants in the Sp1-binding site displayed a strong reduction in transcriptional activity.

Purification of a NF1-like DNA-binding protein from rat liver and cloning of the corresponding cDNA.

NF1-like proteins play a role in transcription of liver-specific genes. A DNA-binding protein, recognizing half of the canonical NF1 binding site (TGGCA) present on the human albumin and retinol-binding protein genes, has been purified from rat liver. Several peptides deriving from a tryptic digest of the purified protein were sequenced and the sequence was used to synthesize specific oligonucleotides. Two overlapping cDNA clones were obtained from a rat-liver cDNA library; their sequence reveals an open reading frame coding for 505 amino acids, including all the peptides sequenced from the purified protein. The DNA-binding domain, most likely located within the first 250 amino acids, is highly homologous to the sequence of CTF/NF1 purified from HeLa cells. Northern analysis reveals several mRNA species present in different combinations in various rat tissues.

Gene reactivation: a tool for the isolation of mammalian DNA methylation mutants.

We report the isolation and characterization of a mammalian strain (tsm) that has a temperature-sensitive mutation in DNA methylation. The isolation procedure was based on the observation that treatment of a CHO TK- MT- cell line with demethylating agents introduces up to 46% demethylation, resulting in phenotypic reversion and transcriptional activation of the thymidine kinase (TK) and metallothionein (MT) genes at frequencies ranging from 1% to 59%. Seven thousand individual colonies from an EMS-mutagenized CHO TK- MT- population were screened for spontaneous reversion to TK+ phenotype after treatment at 39 degrees C. Successful isolates were subsequently examined for MT+ reversion. A single clone (tsm) was obtained that showed temperature-dependent reactivation of both TK and MT genes at frequencies of 7.2 X 10(-4) and 6 X 10(-4), respectively. The tsm cells were viable at 39 degrees C and showed no increased mutation frequency. Reactivation correlated with transcriptional activation of the respective genes, whereas backreversion to the TK- phenotype was associated with transcriptional inactivation. TK- backrevertants were reactivable again with demethylating agents. Although demethylation in tsm cells was not detectable by HPLC, Southern blot analysis revealed that reactivants, irrespective of their mode of generation, showed specific demethylation of both TK and MT genes. Also, after about 150 cell generations after treatment, reactivants from both temperature-induced tsm and cells exposed to demethylating agents gained 60% and 23%, respectively, in 5-methylcytosine (5mC). It is proposed that the phenotype of tsm cells is due to a mutation involved in the regulation of DNA methylation. The further characterization of this and other mammalian mutants should help to clarify the physiological role of DNA methylation, as well as its regulation.

Beta-globin gene promoter generates 5' truncated transcripts in the embryonic/fetal erythroid environment.

We report here the localisation of sequences responsible for the faulty expression of human beta-globin gene in Putko and K562 cells. Complete beta-globin gene introduced into these cells produces transcripts with abnormal 5' ends, while cotransfected mouse H2 gene is expressed correctly. Using hybrid constructs of these two genes we demonstrate that aberrant activity is conferred by sequences 5' of the beta-globin gene. Thus beta-globin promoter attached to the H2 coding sequence produces H2 transcripts with truncated 5' ends. By introducing a series of deletions in the beta-globin promoter we restrict these sequences to the -77/+28 base pair region spanning the CAAT element to the translation initiation site. These results are consistent with the lack of recognition of the beta-globin gene major cap site in Putko and K562 cells. We suggest that inactivity of the adult globin gene in the embryonic/fetal environment is at least in part conferred by sequences within the beta-globin gene promoter.