Possible involvement of LKB1-AMPK signaling in non-homologous end joining.

LKB1/STK11 is a tumor suppressor gene responsible for Peutz-Jeghers syndrome, an inherited cancer disorder associated with genome instability. The LKB1 protein functions in the regulation of cell proliferation, polarization and differentiation. Here, we suggest a role of LKB1 in non-homologous end joining (NHEJ), a major DNA double-strand break (DSB) repair pathway. LKB1 localized to DNA ends upon the generation of micro-irradiation and I-SceI endonuclease-induced DSBs. LKB1 inactivation either by RNA interference or by kinase-dead mutation compromised NHEJ-mediated DNA repair by suppressing the accumulation of BRM, a catalytic subunit of the SWI/SNF complex, at DSB sites, which promotes the recruitment of an essential NHEJ factor, KU70. AMPK2, a major substrate of LKB1 and a histone H2B kinase, was recruited to DSBs in an LKB1-dependent manner. AMPK2 depletion and a mutation of H2B that disrupted the AMPK2 phoshorylation site impaired KU70 and BRM recruitment to DSB sites. LKB1 depletion induced the formation of chromosome breaks and radials. These results suggest that LKB1-AMPK signaling controls NHEJ and contributes to genome stability.

Transcription-dependent rearrangements of actin and nuclear myosin I in the nucleolus.

Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.

Antiangiogenic activity of BAI1 in vivo: implications for gene therapy of human glioblastomas.

Glioblastomas are the most common primary brain tumors in adults. These tumors exhibit a high degree of vascularization, and malignant progression from astrocytoma to glioblastoma is often accompanied by increased angiogenesis and the upregulation of vascular endothelial growth factor and its receptors. In this study, we investigated the in vivo antiangiogenic and antitumor effects of brain-specific angiogenesis inhibitor 1 (BAI1) using human glioblastoma cell lines. Glioblastoma cells were transduced with an adenoviral vector encoding BAI1 (AdBAI1), and Northern and Western blot analyses, respectively, demonstrated BAI1 mRNA and protein expression in the transduced tumor cells. Using an in vivo neovascularization assay, we found that angiogenesis surrounding AdBAI1-transduced glioblastoma cells transplanted into transparent skinfold chambers of SCID mice was significantly impaired compared to control treated cells. Additionally, in vivo inoculation with AdBAI1 of established subcutaneous or intracerebral transplanted tumors significantly impaired tumor growth and promoted increased mouse survival. Morphologically, the tumors exhibited signs of impaired angiogenesis, such as extensive necrosis and reduced intratumoral vascular density. Taken together, these data strongly indicate that BAI1 may be an excellent gene therapy candidate for the treatment of brain tumors, especially human glioblastomas.

Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation.

Since the discovery of SRY/SRY as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/SRY and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/SRY, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination.

Identification of two cDNAs for human actin-related proteins (Arps) that have remarkable similarity to conventional actin.

We identified two cDNAs coding for the novel human actin-related proteins (Arps) hArpM1 and hArpM2. Both of them show remarkable similarity to conventional actin, and the ATP-binding motif and nuclear-export signals of actin are highly conserved. Their mRNAs are expressed in all tested human tissues, but in smaller amounts than that of actin. These features suggest that hArpM1 and M2 are involved in cytoskeletal organization like other cytoplasmic Arp subfamilies.

Absence of Z-chromosome inactivation for five genes in male chickens.

In order to examine if Z-chromosome inactivation, which is analogous to X-chromosome inactivation in mammals, takes place in male birds having ZZ sex chromosomes, five Z-linked genes of chickens which are expressed in both sexes in certain tissues were selected: i.e. genes for growth hormone receptor, nicotinic acetylcholine receptor beta3, aldolase B, beta1,4-galactosyltransferase I, and iron-responsive element-binding protein (also known as cytosolic aconitase). Antisense or sense riboprobe was prepared from an intronic sequence of each gene and subjected to fluorescence in situ hybridization to nascent transcripts of each gene in a nucleus. Each antisense riboprobe hyridized to two spots of nascent RNA which corresponded to its gene loci on the two Z chromosomes in a majority of nuclei in a tissue of the male. The efficiency of detection of two spots per nucleus was comparable to that for the glyceraldehyde-3-phosphate dehydrogenase gene, an autosomal housekeeping gene. These results suggest strongly that Z-chromosome inactivation, i.e. virtual silence of transcription at one of the alleles, does not take place for these five Z-linked genes in male chickens.

The N-terminal internal region of BLM is required for the formation of dots/rod-like structures which are associated with SUMO-1.

Bloom Syndrome (BS) is a human autosomal genetic disorder characterized by a predisposition to a variety of malignant tumors. The gene responsible for BS encodes a protein (BLM) consisting of 1417 amino acids with a nuclear localization signal in the C-terminal region, which is a member of the RecQ helicase family. We previously showed, using a yeast two-hybrid system, that BLM interacted with Ubc9, which is the conjugating enzyme of SUMO-1 (small ubiquitin-related modifier-1). In the present study, we exogenously expressed a green fluorescent protein-tagged Bloom syndrome protein, GFP-BLM, in human 293EBNA cells and found that it formed dots/rod-like structures associated with SUMO-1 in the nucleus. Deletion experiments indicated that the region from amino acids 238 to 586 of BLM is required for the formation of dots/rod-like structures associated with SUMO-1, and the DNA helicase domain, but not the helicase activity itself, slightly affected the formation and/or stability of these structures. Expression of a GFP-BLM which contained the 238-586 region, but lacked the C-terminal nuclear localization signal, resulted in localization to the cytoplasm without the formation of dots/rod-like structures and association with SUMO-1, indicating that these events occur only in the nucleus.

Co-localization of chicken DNA topoisomerase IIalpha, but not beta, with sites of DNA replication and possible involvement of a C-terminal region of alpha through its binding to PCNA.

Clones for DNA topoisomerase IIalpha and beta (topo-IIalpha and beta) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIalpha and beta were about 80% identical for the N-terminal ATPase domain and the central core domain but only 37% for the C-terminal domain. Polyclonal antibodies were raised against C-terminal polypeptides specific to topo-IIalpha and beta. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIalpha was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2'-deoxyuridine, whereas topo-IIbeta was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIalpha and beta suggested that a sequence region (residues 1280-1294) in the C-terminal domain of topo-IIalpha was effective in co-localization with sites of DNA replication. This region consists of a QTxhxF motif (x, any residue; h, hydrophobic residue) followed by a KR-rich sequence, which resembles those found in several proteins known to associate with proliferating cell nuclear antigen (PCNA) or targeted to the replication factory. An in vitro pull-down assay with glutathione-S-transferase-PCNA and (His)6-tagged truncated forms of topo-IIalpha demonstrated that polypeptides containing the above region (residues 1158-1553 or 1158-1294) bound to PCNA in vitro.

Novel actin-related proteins in vertebrates: similarities of structure and expression pattern to Arp6 localized on Drosophila heterochromatin.

Actin-related proteins (Arps), which share a basal structure with actin isoforms but possess different functions, have been identified in a wide variety of organisms. The Arps are classified into subfamilies based on the relatedness of their sequences and functions. Recently, several Arp subfamilies have been shown to be localized in the nucleus and included in protein complexes involved in the organization of chromatin structure, for example, in chromatin remodeling and histone acetyltransferase complexes. A member of the Arp6 subfamily in Drosophila, dArp6, is localized on centric heterochromatin together with heterochromatin protein 1 (HP1). We have identified the first examples of the Arp6 subfamily in vertebrates, novel human and chicken Arps, hArp6 and gArp6, respectively. They are closely related to each other (98% similar) and show apparent similarity to dArp6 (70%). In addition, the hArp6 gene possesses evolutionarily conserved exon/intron structures compared with genes for members of the Arp6 subfamily in invertebrates. Like Drosophila dArp6, gArp6 is expressed abundantly in the early developmental stages, when heterochromatin condensation and nuclear maturation occur. The finding of a conserved Arp6 subfamily in vertebrates will contribute to the understanding of molecular mechanisms of heterochromatin organization.

Multiple actin-related proteins of Saccharomyces cerevisiae are present in the nucleus.

An increasing number of actin-related proteins (Arps), which share the basal structure with skeletal actin but possess distinct functions, have been found in a wide variety of organisms. Individual Arps of Saccharomyces cerevisiae were classified into Arps 1-10 based on the relatedness of their sequences and functions, where Arp1 is the most similar to actin, and Arp10 is the least similar. While Arps 1-3 and their orthologs in other organisms are localized exclusively in the cytoplasm, Arp4 (also known as Act3) is localized in the nucleus and is involved in transcriptional regulation. Here we examined the more divergent Arps for possible nuclear functions. We show that Arps 5-9 are localized in the nucleus, but Arp10 is not. The nuclear export signals identified in actin are well conserved in the cytoplasmic Arps, Arps 1-3, but less conserved in the nuclear Arps. Gel filtration chromatography experiments show that the nuclear Arps are larger than monomer in size and thus are present in multi-protein complexes. Since nuclear protein complexes containing Arps are found to be responsible for histone acetylation and chromatin remodeling, it is suggested that most of the divergent Arps are involved in the !transcriptional regulation through chromatin modulation.

The nuclear actin-related protein of Saccharomyces cerevisiae, Act3p/Arp4, interacts with core histones.

Act3p/Arp4, an essential actin-related protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.

Two isoforms of a human actin-related protein show nuclear localization and mutually selective expression between brain and other tissues.

Actin-related proteins (Arps), which are divergent, but apparently homologues to actin, are categorized into 10 classes. While Arps belonging to classes 1-3 were found to be localized in the cytoplasm across eukaryotic phyla, other classes of Arps were found mostly in invertebrates and suggested to contribute to structural modulation of chromatin. Here we report the identification and the characterization of two human isoforms of an Arp not belonging to classes 1-3, which we designated hArpN alpha and hArpN beta. Both proteins were expressed in HeLa cells and they were found localized within the nucleus. Most interestingly, in different human tissues, hArpN alpha and beta were found to be expressed mutually exclusively, and the expression of hArpN alpha was absolutely restricted to the brain. These findings suggest that, in vertebrates, members of distantly related Arps might have tissue-specific functions in the nucleus, possibly through structural modulation of chromatin.

Triplet repeat-containing ribosomal protein L14 gene in immortalized human endothelial cell line (t-HUE4).

A cDNA encoding human 60S ribosomal subunit protein L14 (hRL14) was isolated from a human immortal endothelial cell line, t-HUE4. This cell line was established via a series of cell lines cultured in a serum-free and a protein-free medium, and a directional cDNA library has been constructed and screened in search for the genes modulating protein synthesis machinery in cell proliferation. A putative full-length clone with an open reading frame of 220 amino acids; predicted molecular weight of 23.6 kDa. A significant identity for hRL14 was observed with rat RL14 (85% identity), with exception of COOH-terminal region, but not with any eukaryote amino acid sequences so far deposited to database. The typical features of ribosomal proteins were observed in hRL14, as seen in nuclear targeting sequences necessary for the transport from cytoplasm to nucleolus, a bZIP like (basic region-leucine zipper) element for the binding to rRNA, and the internal repeat sequences; the pentapeptide QKA(A/S)X. The COOH-terminal region of the transcripts contained fifteen triplet repeats (GCT; alanine) at nucleotide 465 to 509, which is significantly expanded compared to the rat RL14. However, the repeat number was all the same among the normal human endothelial cell line and the cell lines established in the course of t-HUE4 establishment. A single band with about 800 bases was identified by Northern blot analysis without tissue specificity. This GCT repeat was found to be one of the longest uninterrupted repeats in a coding sequence, which were associated with the highest degree of polymorphism.

Purification and nucleic-acid-binding properties of a Saccharomyces cerevisiae protein involved in the control of ploidy.

Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum. The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kühne, C. & Karwan, A. (1995) Yeast 11, 929-944]. Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins. In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scp160p. Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellulose and anti-Scp160p affinity chromatography. In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA. Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes. Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm.

A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of chicken and marked decrease of its immunoreactivity during the progression of S phase.

A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunoreactivity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.

The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus.

Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known. ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa. Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal. An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract. Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus. Nuclear staining was observed in all cells regardless of the stage of the cell cycle. Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction. We suggest that Act3p is an essential constituent of yeast chromatin.

An essential gene of Saccharomyces cerevisiae coding for an actin-related protein.

Actin filaments provide the internal scaffold of eukaryotic cells; they are involved in maintenance of cell shape, cytokinesis, organelle movement, and cell motility. The major component of these filaments, actin, is one of the most well-conserved eukaryotic proteins. Recently genes more distantly related to the conventional actins were cloned from several organisms. In the budding yeast, Saccharomyces cerevisiae, one conventional actin gene, ACT1 (coding for the filament actin), and a so-called actin-like gene, ACT2 (of unknown function), have so far been identified. We report here the discovery of a third member of the actin gene family from this organism, which we named ACT3. The latter gene is essential for viability and codes for a putative polypeptide, Act3, of 489 amino acids (M(r) = 54,831). The deduced amino acid sequence of Act3 is less related to conventional actins than is the deduced amino acid sequence of Act2, mainly because of three unique hydrophilic [corrected] segments. These segments are found inserted into a part of the sequence corresponding to a surface loop of the known three-dimensional structure of the actin molecule. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3. Our findings demonstrate that, unexpectedly, there exist three members of the diverse actin protein family in budding yeast that obviously provide different essential functions for survival.

W-heterochromatin of chicken; its unusual DNA components, late replication, and chromatin structure.

About 65% of DNA in the chicken W chromosome has been shown to consist of XhoI and EcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kb XhoI and the 1.2-kb EcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescence in situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed along XhoI and EcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the beta-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of the HaeIII-digested MSB-1 nuclei yielded a chromatin fraction in which XhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for the XhoI family sequence were present in this fraction.

Presence of female-specific bent-repetitive DNA sequences in the genomes of turkey and pheasant and their interactions with W-protein of chicken.

Two female-specific repetitive DNA units, the 0.4 kb PstI and 0.5 kb TaqI sequences, were detected in the genomic DNA of turkey and pheasant, respectively, by Southern blot hybridization under non-stringent conditions with the W chromosome-specific 0.7 kb XhoI repetitive unit of chicken as a probe. Cloning and sequencing of these two repetitive units revealed that they shared features with the XhoI family repetitive unit of chicken although the overall similarities of the nucleotide sequences were less than 60%. In common with the chicken XhoI family they consisted of tandem repeats of about 21 bp, the majority of which contained (A)3-5 and (T)3-5 clusters separated by six or seven relatively G + C-rich sequences, and they behaved as bent DNA molecules on polyacrylamide gel electrophoresis at room temperature. W-protein, purified from chicken liver nuclei and shown to bind with high affinity to the XhoI family repetitive unit, also bound with the cloned repetitive units from turkey and pheasant DNase I footprint analysis suggested that the mode of interaction of W-protein with these units was similar to that with the 0.7 kb XhoI sequence. On the other hand, W-protein did not bind to the female-specific 0.4 kb BamHI repetitive unit from the Bobwhite quail. The 0.4 kb BamHI sequence contained some A and T clusters but these clusters did not appear in phase with the pitch of DNA helix and the repetitive unit did not show DNA bending.