Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus.
(1/23)
Infection of bovine leukocytes by the apicomplexan parasite Theileria annulata results in alteration of host cell gene expression and stimulation of host cell proliferation. At present, the parasite-derived factors involved in these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-binding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are located together in a tight cluster, interspersed by two further small open reading frames, all facing head to tail. TashAT2 was shown to be expressed in all T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expressed in the sporozoite stage of the parasite, and also in infected cell lines, where their expression was found to vary between different cell lines. Evidence for transport was provided by antisera raised against TashAT1 and TashAT3 that reacted with the host nucleus of T. annulata-infected cells. Reactivity was particularly strong against the host nuclei of the T. annulata-infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially detected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonstrated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA. (+info)
Synthesis of signals for de novo DNA methylation in Neurospora crassa.
(2/23)
Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such sequences induce methylation, we developed a sensitive in vivo system. Tests of various 25- to 100-bp synthetic DNA sequences revealed that both T and A residues were required on a given strand to induce appreciable methylation. Segments composed of (TAAA)(n) or (TTAA)(n) were the most potent signals; 25-mers induced robust methylation at the special test site, and a 75-mer induced methylation elsewhere. G:C base pairs inhibited methylation, and cytosines 5' of ApT dinucleotides were particularly inhibitory. Weak signals could be strengthened by extending their lengths. A:T tracts as short as two were found to cooperate to induce methylation. Distamycin, which, like the AT-hook DNA binding motif found in proteins such as mammalian HMG-I, binds to the minor groove of A:T-rich sequences, suppressed DNA methylation and gene silencing. We also found a correlation between the strength of methylation signals and their binding to an AT-hook protein (HMG-I) and to activities in a Neurospora extract. We propose that de novo DNA methylation in Neurospora cells is triggered by cooperative recognition of the minor groove of multiple short A:T tracts. Similarities between sequences subjected to repeat-induced point mutation in Neurospora crassa and A:T-rich repeated sequences in heterochromatin in other organisms suggest that related mechanisms control silent chromatin in fungi, plants, and animals. (+info)
The Stigmatella aurantiaca homolog of Myxococcus xanthus high-mobility-group A-type transcription factor CarD: insights into the functional modules of CarD and their distribution in bacteria.
(3/23)
Transcriptional factor CarD is the only reported prokaryotic analog of eukaryotic high-mobility-group A (HMGA) proteins, in that it has contiguous acidic and AT hook DNA-binding segments and multifunctional roles in Myxococcus xanthus carotenogenesis and fruiting body formation. HMGA proteins are small, randomly structured, nonhistone, nuclear architectural factors that remodel DNA and chromatin structure. Here we report on a second AT hook protein, CarD(Sa), that is very similar to CarD and that occurs in the bacterium Stigmatella aurantiaca. CarD(Sa) has a C-terminal HMGA-like domain with three AT hooks and a highly acidic adjacent region with one predicted casein kinase II (CKII) phosphorylation site, compared to the four AT hooks and five CKII sites in CarD. Both proteins have a nearly identical 180-residue N-terminal segment that is absent in HMGA proteins. In vitro, CarD(Sa) exhibits the specific minor-groove binding to appropriately spaced AT-rich DNA that is characteristic of CarD or HMGA proteins, and it is also phosphorylated by CKII. In vivo, CarD(Sa) or a variant without the single CKII phosphorylation site can replace CarD in M. xanthus carotenogenesis and fruiting body formation. These two cellular processes absolutely require that the highly conserved N-terminal domain be present. Thus, three AT hooks are sufficient, the N-terminal domain is essential, and phosphorylation in the acidic region by a CKII-type kinase can be dispensed with for CarD function in M. xanthus carotenogenesis and fruiting body development. Whereas a number of hypothetical proteins homologous to the N-terminal region occur in a diverse array of bacterial species, eukaryotic HMGA-type domains appear to be confined primarily to myxobacteria. (+info)
A Theileria annulata DNA binding protein localized to the host cell nucleus alters the phenotype of a bovine macrophage cell line.
(4/23)
The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (SuAT1) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells. (+info)
The herpesvirus saimiri open reading frame (ORF) 50 (Rta) protein contains an at hook required for binding to the ORF 50 response element in delayed-early promoters.
(5/23)
The herpesvirus saimiri open reading frame (ORF) 50 encodes two proteins, which activate transcription directly, following interactions with delayed-early (DE) promoters containing a specific motif. In this report, we demonstrate that ORF 50 contains a DNA binding domain that has homology to an AT hook DNA binding motif. Deletion analysis of this domain reduces ORF 50-mediated transactivation of the DE ORF 6 and ORF 57 promoters by 100 and 90%, respectively. Furthermore, gel retardation experiments demonstrated that the AT hook motif is required for binding the ORF 50 response element in the promoters of DE genes. Single site-directed mutagenesis of the AT hook revealed that mutation of the glycine residue at position 408 to an alanine reduces ORF 50 transactivation of the ORF 57 promoter by 40%. Moreover, the mutation of multiple basic residues in conjunction with the glycine residue within the core element of the AT hook abolishes ORF 50-mediated transactivation. In addition, p50GFPDeltaAT-hook is capable of functioning as a trans-dominant mutant, leading to a reduction in virus production of approximately 50% compared to that for wild-type ORF 50. (+info)
A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo.
(6/23)
Lens epithelium-derived growth factor p75 (LEDGF/p75) is a DNA-binding, transcriptional co-activator that participates in HIV-1 integration site targeting. Using complementary approaches, we determined the mechanisms of LEDGF/p75 DNA-binding in vitro and chromatin-association in living cells. The binding of highly-purified, recombinant protein was assayed by surface plasmon resonance (SPR) and electrophoretic mobility gel shift. Neither assay revealed evidence for sequence-specific DNA-binding. Residues 146-197 spanning the nuclear localization signal (NLS) and two AT-hook motifs mediated non-specific DNA-binding, and DNA-binding deficient mutants retained the ability to efficiently stimulate HIV-1 integrase activity in vitro. Chromatin-association was assessed by visualizing the localization of EGFP fusion proteins in interphase and mitotic cells. Although a conserved N-terminal PWWP domain was not required for binding to condensed mitotic chromosomes, its deletion subtly affected the nucleoplasmic distribution of the protein during interphase. A dual AT-hook mutant associated normally with chromatin, yet when the mutations were combined with NLS changes or deletion of the PWWP domain, chromatin-binding function was lost. As the PWWP domain did not readily bind free DNA in vitro, our results indicate that chromatin-association is primarily affected through DNA-binding, with the PWWP domain likely contributing a protein interaction to the overall affinity of LEDGF/p75 for human chromatin. (+info)
DNA binding properties of TAF1 isoforms with two AT-hooks.
(7/23)
TATA-binding protein-associated factor 1 (TAF1) is an essential component of the general transcription factor IID (TFIID), which nucleates assembly of the preinitiation complex for transcription by RNA polymerase II. TATA-binding protein and TAF1.TAF2 heterodimers are the only components of TFIID shown to bind specific DNA sequences (the TATA box and initiator, respectively), raising the question of how TFIID localizes to gene promoters that lack binding sites for these proteins. Here we demonstrate that Drosophila TAF1 protein isoforms TAF1-2 and TAF1-4 directly bind DNA independently of TAF2. DNA binding by TAF1 isoforms is mediated by cooperative interactions of two identical AT-hook motifs, one of which is encoded by an alternatively spliced exon. Electrophoretic mobility shift assays revealed that TAF1-2 bound the minor groove of adenine-thymine-rich DNA with a preference for the sequence AAT. Alanine-scanning mutagenesis of the alternatively spliced AT-hook indicated that Lys and Arg residues made essential DNA contacts, whereas Gly and Pro residues within the Arg-Gly-Arg-Pro core sequence were less important for DNA binding, suggesting that AT-hooks are more divergent than previously predicted. TAF1-2 bound with variable affinity to the transcription start site of several Drosophila genes, and binding to the hsp70 promoter was reduced by mutation of a single base pair at the transcription start site. Collectively, these data indicate that AT-hooks serve to anchor TAF1 isoforms to the minor groove of adenine-thymine-rich Drosophila gene promoters and suggest a model in which regulated expression of TAF1 isoforms by alternative splicing contributes to gene-specific transcription. (+info)
Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone beta subunit (FSHbeta) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone alpha subunit (alphaGSU), and luteinizing hormone beta subunit (LHbeta). A series of deletion mutants of the porcine alphaGSU (up to -1059 bp) and LHbeta (up to -1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine alphaGSU and LHbeta promoters by Prop-1, which was found to activate the alphaGSU promoter of -1059/+12 bp up to 11.7-fold but not the LHbeta promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, -1038/-1026, -942/-928, -495/-479, -338/-326, -153/-146, and -131/-124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of alphaGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for alphaGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of alphaGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHbeta gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of alphaGSU and FSHbeta gene expressions. (+info)