Cohen M, Lee KK, Wilson KL, Gruenbaum Y. Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina. Trends Biochem Sci. 2001;26 (1) :41-7.Abstract
The number and complexity of genes encoding nuclear lamina proteins has increased during metazoan evolution. Emerging evidence reveals that transcriptional repressors such as the retinoblastoma protein, and apoptotic regulators such as CED-4, have functional and dynamic interactions with the lamina. The discovery that mutations in nuclear lamina proteins cause heritable tissue-specific diseases, including Emery-Dreifuss muscular dystrophy, is prompting a fresh look at the nuclear lamina to devise models that can account for its diverse functions and dynamics, and to understand its enigmatic structure.
Kakhlon O, Gruenbaum Y, Cabantchik ZI. Repression of the heavy ferritin chain increases the labile iron pool of human K562 cells. Biochem J. 2001;356 (Pt 2) :311-6.Abstract
The role of ferritin in the modulation of the labile iron pool was examined by repressing the heavy subunit of ferritin in K562 cells transfected with an antisense construct. Repression of the heavy ferritin subunit evoked an increase in the chemical levels and pro-oxidant activity of the labile iron pool and, in turn, caused a reduced expression of transferrin receptors and increased expression of the light ferritin subunit.
Tour E, Pillemer G, Gruenbaum Y, Fainsod A. The two Xenopus Gbx2 genes exhibit similar, but not identical expression patterns and can affect head formation. FEBS Lett. 2001;507 (2) :205-9.Abstract
Gbx2 homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. Two Gbx2 genes were identified in Xenopus laevis, differing in 13 amino acids, including a change in the homeodomain. Xgbx2a is activated earlier during gastrulation and reaches higher levels of expression while Xgbx2b is expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns.
Lee KK, Gruenbaum Y, Spann P, Liu J, Wilson KL. C. elegans nuclear envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of nuclear envelope breakdown during mitosis. Mol Biol Cell. 2000;11 (9) :3089-99.Abstract
Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes.
Liu J, Rolef Ben-Shahar T, Riemer D, Treinin M, Spann P, Weber K, Fire A, Gruenbaum Y. Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes. Mol Biol Cell. 2000;11 (11) :3937-47.Abstract
Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.
Gruenbaum Y, Wilson KL, Harel A, Goldberg M, Cohen M. Review: nuclear lamins--structural proteins with fundamental functions. J Struct Biol. 2000;129 (2-3) :313-23.Abstract
The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed of both peripheral and integral membrane proteins, including lamins and lamina-associated proteins. Lamins can interact with one another, with lamina-associated proteins, with nuclear scaffold proteins, and with chromatin. Likewise, most of the lamina-associated proteins are likely to interact directly with chromatin. The nuclear lamina is required for proper cell cycle regulation, chromatin organization, DNA replication, cell differentiation, and apoptosis. Mutations in proteins of the nuclear lamina can disrupt these activities and cause genetic diseases. The structure and assembly of the nuclear lamina proteins and their roles in chromatin organization and cell cycle regulation were recently reviewed. In this review, we discuss the roles of the nuclear lamina in DNA replication and apoptosis and analyze how mutations in nuclear lamina proteins might cause genetic diseases.
Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y, Horvitz HR. Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science. 2000;287 (5457) :1485-9.Abstract
The Caenorhabditis elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localized to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells had been induced to die, CED-4 assumed a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 was blocked by a gain-of-function mutation in ced-9 but was not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death.
Goldberg M, Harel A, Brandeis M, Rechsteiner T, Richmond TJ, Weiss AM, Gruenbaum Y. The tail domain of lamin Dm0 binds histones H2A and H2B. Proc Natl Acad Sci U S A. 1999;96 (6) :2852-7.Abstract
In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain was found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo.
Bercovich D, Plotsky Y, Gruenbaum Y. Improved protocol for using avian red blood cells as substrates for the polymerase chain reaction. Biotechniques. 1999;26 (6) :1080-2.
Goldberg M, Harel A, Gruenbaum Y. The nuclear lamina: molecular organization and interaction with chromatin. Crit Rev Eukaryot Gene Expr. 1999;9 (3-4) :285-93.Abstract
The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed mainly of nuclear lamins and lamina-associated proteins. The nuclear lamina is involved in nuclear organization, cell cycle regulation, and differentiation. As such, impairment in its architecture and/or function leads to genetic diseases and apoptosis. This article describes the molecular organization of the nuclear lamins, their assembly into filaments, their distribution within the nucleus, and the complex network of interactions between them and other proteins of the inner nuclear membrane. Recent findings unraveled evidence for specific interactions between proteins of the nuclear lamina and the chromatin. These include interactions between nuclear lamins and core histones, Lamina Associated Polypeptide 2 (LAP2), and the Barrier to Autointegration Factor (BAF) and interactions between lamin B receptor (LBR) and the chromodomain protein HP1. Taken together, these studies attribute a role for both the nuclear lamins and the lamina-associated proteins, LAP2 and LBR, in nuclear organization and nuclear assembly.
Bercovich D, Regev Z, Ratz T, Luder A, Plotsky Y, Gruenbaum Y. Quantitative ratio of primer pairs and annealing temperature affecting PCR products in duplex amplification. Biotechniques. 1999;27 (4) :762-4, 766-8, 770.Abstract
The quantity of PCR products that are simultaneously amplified from two different loci in a duplex amplification (DA) are significantly lower for one of the loci, as compared to identical PCR amplification in separate single-band amplifications (SBA). This difference in amplification probably occurs already after the second cycle of amplification. To further analyze this phenomenon, we tested different reaction conditions, including annealing times, a wide range of temperatures, various quantities of the template, several nucleotide concentrations, different amounts of TaqI DNA Polymerase, number of amplification cycles and various amounts of primers and primers ratio. Changing the ratio between the sets of primers in DA had the most significant effect on the relative levels of amplification of the loci with an optimal ratio of 4:1 in favor of the set of primers used to amplify the underrepresented fragment. The optimal annealing temperatures for the tested sets of primers were identical in SBA and different in DA. Possible reasons for this phenomenon are discussed.
Goldberg M, Lu H, Stuurman N, Ashery-Padan R, Weiss AM, Yu J, Bhattacharyya D, Fisher PA, Gruenbaum Y, Wolfner MF. Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA. Mol Cell Biol. 1998;18 (7) :4315-23.Abstract
The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.
Khatib H, Sagiv N, Gruenbaum Y. Research notes: Fresh and frozen pools of chicken red blood cells as substrates for direct polymerase chain reaction. Poult Sci. 1998;77 (6) :902-4.Abstract
A method is presented for reliable use of pooled chicken blood samples for estimation of microsatellite frequencies by direct polymerase chain reaction (PCR) amplification of DNA. This method overcomes the variability of hematocrit values in individual chickens and eliminates the step of DNA preparation. The estimated frequencies of polymorphic alleles in fresh and frozen pooled blood samples were similar to those obtained by calculating these frequencies from the individual genotyping. When frozen pooled blood samples are used, pools should be prepared prior to their freezing.
Ashery-Padan R, Weiss AM, Feinstein N, Gruenbaum Y. Distinct regions specify the targeting of otefin to the nucleoplasmic side of the nuclear envelope. J Biol Chem. 1997;272 (4) :2493-9.Abstract
Otefin is a 45-kDa nuclear envelope protein with no apparent homology to other known proteins. It includes a large hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of 17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the nuclear envelope. Chemical extraction of nuclei from Drosophila embryos revealed that otefin is a peripheral protein whose association with the nuclear envelope is stronger than that of lamin. Deletion mutants of otefin were expressed in order to identify regions that direct otefin to the nuclear envelope. These experiments revealed that the hydrophobic sequence at the carboxyl terminus is essential for correct targeting to the nuclear envelope, whereas additional regions in the hydrophilic domain of otefin are required for its efficient targeting and stabilization in the nuclear envelope.
Goldberg M, Gruenbaum Y. [Knockout and knockin mice]. Harefuah. 1997;132 (4) :269-74.
Ashery-Padan R, Ulitzur N, Arbel A, Goldberg M, Weiss AM, Maus N, Fisher PA, Gruenbaum Y. Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development. Mol Cell Biol. 1997;17 (7) :4114-23.Abstract
Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.
Ulitzur N, Harel A, Goldberg M, Feinstein N, Gruenbaum Y. Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system. Mol Biol Cell. 1997;8 (8) :1439-48.Abstract
A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.
Zhao K, Harel A, Stuurman N, Guedalia D, Gruenbaum Y. Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state. FEBS Lett. 1996;380 (1-2) :161-4.Abstract
The nuclear matrix maintains specific interactions with genomic DNA at sites known as matrix attachment regions (M/SARs). M/SARs bind in vitro to lamin polymers. We show that the polymerized alpha-helical rod domain of lamin Dm0 provides by itself the specific binding to the ftz M/SAR. In contrast, unpolymerized rod domain does not bind specifically to this M/SAR. Non-specific binding to DNA is also observed with Dm0 containing a point mutation that impairs its ability to polymerize or with the isolated tail domain. These data suggest that the specific binding of lamins to M/SARs requires the rod domain and depends on the lamin polymerization state.
Schubert FR, Fainsod A, Gruenbaum Y, Gruss P. Expression of the novel murine homeobox gene Sax-1 in the developing nervous system. Mech Dev. 1995;51 (1) :99-114.Abstract
We have isolated the novel murine Sax-1 gene, a member of the NK-1 class of homeobox genes, and report its expression pattern in the developing central nervous system (CNS) in comparison to two other homeobox genes, Evx-1 and Pax-6. Sax-1 was found to be transiently expressed in the developing posterior CNS. First seen in the ectoderm lateral to the primitive streak, the signal later encompassed the neural plate. Posteriorly, the expression domain overlapped with the Evx-1 expression in the streak, while anteriorly it was delimited by the Pax-6 signal in the neural tube. This early phase starting at day 9.5 pc, Sax-1 was expressed in distinct areas of spinal cord, hindbrain and forebrain. Particularly strong signals were detected in rhombomere 1 and in the pretectum. In these areas, subsets of neurons may be marked and specified. In addition to the normal pattern of Sax-1 during development, the expression in different mouse mutants was analysed. In Brachyury curtailed homozygotes, the expression of Sax-1 was found to be reduced during neurulation and even lost at day 9.0 pc. Ventral shift and finally loss of the signal in the ventral spinal cord was observed in Danforth's short tail homozygotes.
Khatib H, Berkovitz D, Ratz T, Plotzki Y, Fainsod A, Gruenbaum Y. Mapping the CdxA gene to a new linkage group in chicken. Anim Genet. 1995;26 (3) :211.