Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication

2001 ◽  
Vol 114 (10) ◽  
pp. 1861-1866 ◽  
Author(s):  
J. Postberg ◽  
S.A. Juranek ◽  
S. Feiler ◽  
H. Kortwig ◽  
F. Jonsson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Matrix ◽  
Binding Protein ◽  
Specific Interaction ◽  
Eukaryotic Cells ◽  
Dna Molecules ◽  
Telomere Binding Protein ◽  
The Matrix

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8)individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

ors12, a mammalian autonomously replicating DNA sequence, associates with the nuclear matrix in a cell cycle-dependent manner

1993 ◽  
Vol 105 (3) ◽  
pp. 807-818
Author(s):  
D.C. Mah ◽  
P.A. Dijkwel ◽  
A. Todd ◽  
V. Klein ◽  
G.B. Price ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Matrix ◽  
Pcr Amplification ◽  
S Phase ◽  
Dependent Manner ◽  
The Matrix ◽  
S Period ◽  
Cycle Dependent

Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.

scholarly journals Nuclear Import of the Stem–Loop Binding Protein and Localization during the Cell Cycle

2005 ◽  
Vol 16 (6) ◽  
pp. 2960-2971 ◽  
Author(s):  
Judith A. Erkmann ◽  
Eric J. Wagner ◽  
Jian Dong ◽  
Yanping Zhang ◽  
Ulrike Kutay ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Binding Protein ◽  
Histone Mrna ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Import Receptors ◽  
Mrna Binding

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem–loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impα/Impβ and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impα/Impβ binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impα/Impβ pathway contributes to SLBP nuclear import in HeLa cells.

scholarly journals The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein

2000 ◽  
Vol 14 (14) ◽  
pp. 1777-1788 ◽  
Author(s):  
Haiyan Qi ◽  
Virginia A. Zakian
Keyword(s):  
Dna Polymerase ◽  
Binding Protein ◽  
Point Mutations ◽  
Single Strand ◽  
Catalytic Subunit ◽  
Full Length ◽  
Temperature Sensitive ◽  
Dna Polymerase Α ◽  
Telomere Binding Protein

Saccharomyces telomeres consist of ∼350 bp of C1-3A/TG1-3 DNA. Most of this ∼350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C1-3A strand is degraded to generate a long single strand TG1-3 tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG1-3DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase α, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 orPOL1 that reduced the Cdc13p–Pol1p interaction resulted in telomerase mediated telomere lengthening. Over–expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG1-3 strand lengthening by telomerase and its interaction with Pol1p promotes C1-3A strand resynthesis by DNA polymerase α.

scholarly journals The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

1998 ◽  
Vol 141 (2) ◽  
pp. 335-348 ◽  
Author(s):  
Ian de Belle ◽  
Shutao Cai ◽  
Terumi Kohwi-Shigematsu
Keyword(s):  
Nuclear Matrix ◽  
Cpg Island ◽  
Binding Protein ◽  
Jurkat Cells ◽  
Yeast Cells ◽  
Genomic Sequences ◽  
Specific Gene ◽  
Addition Line ◽  
Chromatin Loops

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation.

scholarly journals Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

2004 ◽  
Vol 3 (5) ◽  
pp. 1185-1197 ◽  
Author(s):  
Bidyottam Mittra ◽  
Dan S. Ray
Keyword(s):  
Cell Cycle ◽  
Binding Protein ◽  
High Specificity ◽  
Binding Activity ◽  
Mrna Levels ◽  
Crithidia Fasciculata ◽  
Target Mrna ◽  
Cycle Dependent

ABSTRACT Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

scholarly journals Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein.

1994 ◽  
Vol 14 (3) ◽  
pp. 1582-1593 ◽  
Author(s):  
A Emili ◽  
J Greenblatt ◽  
C J Ingles
Keyword(s):  
Binding Protein ◽  
Specific Interaction ◽  
Tata Box ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Evolutionarily Conserved ◽  
Drosophila Protein ◽  
Tata Box Binding Protein ◽  
Species Specific

We have used protein-blotting and protein affinity chromatography to demonstrate that each of the two glutamine-rich activation domains of the human transcription factor Sp1 can bind specifically and directly to the C-terminal evolutionarily conserved domain of the human TATA box-binding protein (TBP). These activation domains of Sp1 also bind directly to Drosophila TBP but bind much less strongly to TBP from the yeast Saccharomyces cerevisiae. The abilities of the Sp1 activation domains to interact directly with the TBPs of various species correlate well with their abilities to activate transcription in extracts derived from the same species. We also show that a glutamine-rich transcriptional activating region of the Drosophila protein Antennapedia binds directly to TBP in a species-specific manner that reflects its ability to activate transcription in vivo. These results support the notion that TBP is a direct and important target of glutamine-rich transcriptional activators.

The Salicylamide Derivative, Niclosamide, Inhibits CREB Function in Acute Myeloid Leukemia Cells In Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1647-1647
Author(s):  
Hee-Don Chae ◽  
Nick Cox ◽  
Xiaohua Zhang ◽  
Jae Wook Lee ◽  
David Morgens ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Therapeutic Window ◽  
Hl60 Cells ◽  
Acute Myeloid

Abstract CREB (cAMP Response Element Binding protein) is a transcription factor that is overexpressed in primary Acute Myeloid Leukemia (AML) cells and is associated with a decreased event-free survival and increased risk of relapse. We previously demonstrated that CREB overexpression increases leukemia cell growth and survival. Transgenic mice overexpressing CREB in myeloid cells develop a myeloproliferative neoplasm and myelodysplasia. CREB knockdown inhibits AML cell proliferation but not normal hematopoietic stem cell activity in vivo. To demonstrate the feasibility of targeting CREB for treatment for AML, we recently described a small molecule inhibitor of CREB, N-(4-cyanophenyl)-3-hydroxy-2-naphthamide (XX-650-23), which is a compound originally based on naphthol AS-E phosphate first identified as an inhibitor of CREB interaction with its coactivator, CBP (CREB Binding Protein). To identify a lead candidate with improved potency and physicochemical properties, we performed structure-activity relationships (SAR) studies for a series of salicylamides derived from naphthol AS-E phosphate. Development of this series led to the identification of the anthelmintic niclosamide as a potent agent that suppresses cell viability of five AML cell lines (IC50= 280 nM (HL60), 340 nM (KG1), 420 nM (MOLM13), 560 nM (MV411), 360 nM (U937), without a significant decrease in colony forming activity of normal bone marrow cells up to 10 μM (18- to 36-fold therapeutic window). Niclosamide binds CBP with a KD of 22.3 nM by Surface Plasmon Resonance (Biacore) analysis. To determine whether niclosamide specifically inhibits CREB-mediated gene expression in cells, luciferase reporter gene activity under the control of a promoter containing two CRE elements was measured after treatment of niclosamide for 6 hours. Niclosamide inhibited CREB-driven luciferase activity in HL60 cells with an IC50 of 1.09 μM. We also examined the efficacy of niclosamide in an AML patient-derived xenograft (PDX) mouse model. Niclosamide significantly inhibited the progression of AML in mice injected with primary AML cells. The percentage of circulating AML cells in the peripheral blood (%), vehicle vs. niclosamide treatment 5 weeks after engraftment were 28.75 ± 3.507 vs. 0.5363 ± 0.2744 (n=8, p< 0.001, mean ± SEM). In Kaplan Meier analysis, the median survival of PDX mice was 41 days vs. 51.5 days (p = 0.0015, log-rank test). To characterize the cellular effects of niclosamide, we analyzed the DNA profile, apoptosis, DNA-damage, cell cycle regulators, and other signaling molecules using flow cytometry. Niclosamide treatment increased DNA-damaged and apoptosis populations during the G1/S cell cycle phase, which also showed reduced phosphorylated CREB levels. To examine the functional requirement of CREB, we determined the effects of CREB knockdown in HL60 cells treated with niclosamide. CREB knockdown protected HL60 cells from niclosamide treatment-mediated cytotoxic effects (IC50=670 nM for CREB knockdown vs. 200 nM for vector control cells). Furthermore, combination treatment of niclosamide with XX-650-23 in HL60 cells showed an additive antiproliferative effect, suggesting that niclosamide and XX-650-23 regulate the same targets or pathways to inhibit viability of AML cells. To further identify genes that confer resistance or sensitivity to niclosamide, we performed a functional shRNA screen using subsets of whole genomic shRNA libraries (apoptosis, motility, other cancer; 35154 elements). We identified 53 genes, including tumor necrosis factor receptor superfamily members, which when knocked downed conferred resistance to niclosamide at a 10% false discovery rate. Taken together, our results demonstrate that niclosamide is a potential drug to treat AML by inducing DNA-damage, apoptosis and cell cycle arrest through the inhibition of CREB-dependent pathways in AML cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel DNA-binding Protein Coordinates Asymmetric Chromosome Replication and Chromosome Partitioning

2016 ◽  
Author(s):  
James A. Taylor ◽  
Gaël Panis ◽  
Patrick H. Viollier ◽  
Gregory T. Marczynski
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Replication Origin ◽  
Caulobacter Crescentus ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Chromosome Replication ◽  
Chromosome Partitioning ◽  
Chromosome Separation

AbstractBacterial chromosome replication is regulated from a single replication origin (ori) that receives cell cycle signals. Following replication, bacteria often use theparABSpartition system with a centromere-likeparSlocus to place the chromosomes into the daughter cells. Our knowledge of cell cycle regulation is incomplete and we searched for novel regulators of chromosome replication. Here we show that in the cell cycle modelCaulobacter crescentusa novel DNA-binding protein promotes both the initiation of chromosome replication and the earliest step of chromosome partitioning. We used biochemical fractionation to identify a protein (OpaA) that preferentially binds to mutatedoriDNA that also increasesori-plasmid replicationin vivo. OpaA represents a previously unknown class of DNA-binding proteins.opaAgene expression is essential and sufficient OpaA levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified the genomic binding sites for OpaA, with the strongest associations at theparABSlocus nearori. Using molecular-genetic and fluorescence microscopy experiments, we showed that OpaA also promotes the first step of chromosome partitioning, the initial separation of the duplicatedparSloci followingorireplication. This separation occurs before theparABSmechanism and it coincides with the regulatory step that splits the symmetry of the chromosomes so that they are placed at distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that OpaA coordinates replication with the poorly understood mechanism of early chromosome separation.opaAlethal suppressor and antibiotic experiments argue that future studies be focused on the mechanistic roles for transcription and translation at this critical step of the cell cycle.Author SummaryLike all organisms, bacteria must replicate their chromosomes and move them into the newly dividing cells. Eukaryotes use non-overlapping phases, first for chromosome replication (S-phase) followed by mitosis (M-phase) when the completely duplicated chromosomes are separated. However, bacteria combine both phases so chromosome replication and chromosome separation (termed chromosome “partitioning”) overlap. In many bacteria, includingCaulobacter crescentus, chromosome replication initiates from a single replication origin (ori) and the first duplicated regions of the chromosome immediately begin “partitioning” towards the cell poles long before the whole chromosome has finished replication. This partitioning movement uses the centromere-like DNA called“parS”that is located near theori. Here we identify a completely novel type of DNA-binding protein called OpaA and we show that it acts at bothoriandparS. The timing and coordination of overlapping chromosome replication and partitioning phases is a special regulatory problem for bacteria. We further demonstrate that OpaA is selectively required for the initiation of chromosome replication atoriand likewise that OpaA is selectively required for the initial partitioning ofparS. Therefore, we propose that OpaA is a novel regulator that coordinates chromosome replication with the poorly understood mechanism of early chromosome separation.

scholarly journals Distinct gene-selective roles for a network of core promoter factors in Drosophila neural stem cell identity

2018 ◽  
Author(s):  
Alexandre Neves ◽  
Robert N. Eisenman
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Brain Stem ◽  
Cell Polarity ◽  
Neurological Disorders ◽  
Associated Factors ◽  
Binding Protein ◽  
Core Promoter ◽  
Tata Binding Protein

AbstractThe transcriptional mechanisms that allow neural stem cells (NSC) to balance self-renewal with differentiation are not well understood. Employing an in vivo RNAi screen we identify here NSC-TAFs, a subset of nine TATA-binding protein associated factors (TAFs), as NSC identity genes in Drosophila. We found that depletion of NSC-TAFs results in decreased NSC clone size, reduced proliferation, defective cell polarity and increased hypersensitivity to cell cycle perturbation, without affecting NSC survival. Integrated gene expression and genomic binding analyses revealed that NSC-TAFs function with both TBP and TRF2, and that NSC-TAF-TBP and NSC-TAF-TRF2 shared target genes encode different subsets of transcription factors and RNA-binding proteins with established or emerging roles in NSC identity and brain development. Taken together, our results demonstrate that core promoter factors are selectively required for NSC identity in vivo by promoting cell cycle progression and NSC cell polarity as well as by restraining premature differentiation. Because pathogenic variants in a subset of TAFs have all been linked to human neurological disorders, this work may stimulate and inform future animal models of TAF-linked neurological disorders.Author summaryThe brains of many animal species are built with brain stem cells. Having too many brain stem cells can lead to brain tumors whereas too few can lead to birth defects such as microcephaly. A number of next generation sequencing studies have implicated proteins referred to as TATA-box-binding protein associated factors (TAFs) in human neurological disorders including microcephaly, but prior to this study, their function in brain development was unknown. Here we use brain stem cells, known as neural stem cells (NSCs), from the fruit fly Drosophila melanogaster as a model system to decipher how TAFs control brain stem cell identity. By combining genetics and low-input genomics, we show that TAFs directly control NSC cell division and cell polarity but do not appear to be required for NSC survival. We further show that TAFs accomplish these functions by associating either with their canonical partner TBP (TATA-binding protein) or the related protein TRF2. In summary, our study reveals unexpected and gene-selective functions of a unique subset of TAFs and their binding partners, which could inform future studies that seek to model human neurological disorders associated with TAFs.

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