Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes

ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with α-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.

An interaction between DNA ligase I and proliferating cell nuclear antigen: Implications for Okazaki fragment synthesis and joining

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 Å, enabling direct visualization of α-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i.e., insertion of peptides, ≈10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 Å resolution. Hepatitis B virus capsids are icosahedral particles, ≈300 Å in diameter, made up of T-shaped dimers (subunit Mr, 16–21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and −7 (in the residual propeptide) in the “e-antigen” form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

Derepression of human embryonic ζ-globin promoter by a locus-control region sequence

A multiple protein–DNA complex formed at a human α-globin locus-specific regulatory element, HS-40, confers appropriate developmental expression pattern on human embryonic ζ-globin promoter activity in humans and transgenic mice. We show here that introduction of a 1-bp mutation in an NF-E2/AP1 sequence motif converts HS-40 into an erythroid-specific locus-control region. Cis-linkage with this locus-control region, in contrast to the wild-type HS-40, allows erythroid lineage-specific derepression of the silenced human ζ-globin promoter in fetal and adult transgenic mice. Furthermore, ζ-globin promoter activities in adult mice increase in proportion to the number of integrated DNA fragments even at 19 copies/genome. The mutant HS-40 in conjunction with human ζ-globin promoter thus can be used to direct position-independent and copy number-dependent expression of transgenes in adult erythroid cells. The data also supports a model in which competitive DNA binding of different members of the NF-E2/AP1 transcription factor family modulates the developmental stage specificity of an erythroid enhancer. Feasibility to reswitch on embryonic/fetal globin genes through the manipulation of nuclear factor binding at a single regulatory DNA motif is discussed.

Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein–protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10–20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Sequence of the FRA3B common fragile region: Implications for the mechanism of FHIT deletion

The hypothesis that chromosomal fragile sites may be “weak links” that result in hot spots for cancer-specific chromosome rearrangements was supported by the discovery that numerous cancer cell homozygous deletions and a familial translocation map within the FHIT gene, which encompasses the common fragile site, FRA3B. Sequence analysis of 276 kb of the FRA3B/FHIT locus and 22 associated cancer cell deletion endpoints shows that this locus is a frequent target of homologous recombination between long interspersed nuclear element sequences resulting in FHIT gene internal deletions, probably as a result of carcinogen-induced damage at FRA3B fragile sites.

Sequences within the Cytoplasmic Domain of Gp180/Carboxypeptidase D Mediate Localization to the Trans-Golgi Network

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12–43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.

Direct Visualization of the Human Estrogen Receptor α Reveals a Role for Ligand in the Nuclear Distribution of the Receptor

The human estrogen receptor α (ER α) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER− (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER− but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the “unoccupied” and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.

The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin β to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin α/β or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.

Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme β-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.  Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.

The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex Function

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23°C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2–1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.