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SOX9 and the switch hitting genes.
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Activation of latent Kaposi's sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator.
Kaposi's sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposi's sarcoma, primary effusion lymphomas, and a subset of multicentric Castleman's disease. The mechanism by which this virus establishes latency and reactivation is unknown. KSHV Lyta (lytic transactivator, also named KSHV/Rta), mainly encoded by the ORF 50 gene, is a lytic switch gene for viral reactivation from latency, inasmuch as it is both essential and sufficient to drive the entire viral lytic cycle. Here we show that the Lyta promoter region was heavily methylated in latently infected cells. Treatment of primary effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate caused demethylation of the Lyta promoter and induced KSHV lytic phase in vitro. Methylation cassette assay shows demethylation of the Lyta promoter region was essential for the expression of Lyta. In vivo, biopsy samples obtained from patients with KSHV-related diseases show the most demethylation in the Lyta promoter region, whereas samples from a latently infected KSHV carrier remained in a methylated status. These results suggest a relationship among a demethylation status in the Lyta promoter, the reactivation of KSHV, and the development of KSHV-associated diseases.
ATP- and ADP-dnaA protein, a molecular switch in gene regulation.
DnaA protein functions by binding to asymmetric 9mer DNA sites, the DnaA boxes. ATP-DnaA and ADP-DnaA bind to 9mer DnaA boxes with equal affinity, but only ATP-DnaA protein binds in addition to an as yet unknown 6mer site, the ATP-DnaA box AGATCT, or a close match to it. ATP-DnaA protein binding to ATP-DnaA boxes is restricted to sites located in close proximity to DnaA boxes, suggesting that protein-protein interaction is required for its stabilization. We show that ATP-DnaA represses dnaA transcription much more efficiently than ADP-DnaA. DnaA is thus a regulatory molecule that, depending on the adenosine nucleotide bound, can bind to different sequences and thereby fulfill distinct functions.
Recombinational switch for gene expression.
Flagellar antigens are specified by two genes, H1 and H2. The expression of these genes is regulated such that only one gene activity, or phase, is expressed at a given time. Molecular cloning techniques were used to isolate the segments of Salmonella DNA which contain these genetic loci. Heteroduplex analyses revealed an anomaly in the cloned fragment, that is, and apparent inversion, which was shown to be adjacent to the H2 gene. A correlation was demonstrated between the phase state of the H2 gene and the sequence of the adjacent segment. We propose that an inversion of this region is the phase-determining event in flagellar gene expression in Salmonella.
ZEB negatively regulates the lytic-switch BZLF1 gene promoter of Epstein-Barr virus.
Epstein-Barr virus (EBV) is a human herpesvirus capable of establishing a latent state in B lymphocytes. The product of the immediate-early BZLF1 gene, Zta, is a transcriptional transactivator essential for viral DNA amplification and virion production. Previously, we identified a negative cis-acting element within the BZLF1 promoter termed ZV. ZV contains the sequence 5'-CAGGTA-3' located at nucleotides -17 to -12 relative to the transcription initiation site. It sequence specifically binds a cellular factor, ZVR. Based on sequence binding specificity, we postulated that ZVR may be zinc finger E-box binding factor (ZEB) or a related zinc finger/homeodomain family member. We show here by immunoshift assays that ZVR and human ZEB specifically cross-react with an antibody to deltaEF1, the chicken homolog of ZEB. Competition electrophoretic mobility shift assays confirmed that ZEB binds to the ZV element with the same binding specificity as ZVR. Overexpression of ZEB in either B-lymphocytic DG75 cells or mammary epithelial MCF-7 cells repressed Zta-induced activation of the BZLF1 promoter four- to fivefold via the ZV site. Thus, we conclude that the previously identified cellular repressor ZVR is, in fact, ZEB. We also present evidence that other cellular factors likely affect the transcriptional activity of ZEB. Lastly, we identify a ZEB-binding site within the promoter of the lytic BRLF1 gene of EBV. We postulate that ZEB likely plays an important role in regulating the life cycle of EBV.
Switching metabolic genes to build a better heart.
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[Long distance vectorette PCR for detecting translocation of IgH gene in switch regions of patients with multiple myeloma].
Chromosomal translocations involving immunoglobulin heavy chain (IgH) locus at 14q32 are commonly seen in multiple myeloma (MM), which may involve in pathogenesis and associate with prognosis. To rapidly detect the occurrence of translocation involving IgH locus and the fellow chromosomes and isolate the break points, According to the break points occured in switch regions, patents and references, a long distance vectorette PCR method was developed. The results showed that using this method, a 3.5 kb PCR product from MM cell line U266 was gained, each ends aligned with 11q and IgHalpha 1 switch region, respectively. It is concluded that LDV-PCR is effective detection technique. Detecting China MM patients by using this method can interpret some specific clinic statues and pathogenesis mechanisms, and help to find new therapy targets and to assess the patient prognosis.
The inactivation of the XP-C gene does not affect somatic hypermutation or class switch recombination of immunoglobulin genes.
The mechanism of somatic hypermutation of immunoglobulin genes is not known, but appears to be linked to transcription and perhaps DNA repair. In order to determine if global DNA repair or the repair of the nontranscribed DNA strand is required for somatic mutation, we have analysed mice whose XP-C gene was inactivated by homologous recombination. Our study shows that hypermutation occurs in XP-C knockout mice with a normal frequency, suggesting that the XP-C gene product is not required for somatic hypermutation. Furthermore, we found that Ig gene switch recombination also is normal in these mice.
Gene expression profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene expression.
The global pattern of growth-phase-dependent gene expression of Helicobacter pylori during in vitro culture was analyzed by using a high-density DNA microarray. To detect consistent coordinated gene expression in this bacterium, temporal changes in transcription were assessed in two independent time courses. Cluster analysis of the expression profiles highlighted a major switch in gene expression during the late log-to-stationary phase transition that we have termed the Log-Stat switch. Statistical analysis of the genes that were significantly induced or repressed during the Log-Stat switch revealed that many of these genes were related to virulence. Among these, expression of the genes for the neutrophil activating protein (napA) and the major flagellin subunit (flaA) were significantly induced. Additionally, the expression of a number of genes involved in iron homeostasis changed dramatically at this switch; the gene for the iron-storage protein, pfr, was induced, while the genes for two putative iron uptake proteins, fecA and frpB, were significantly repressed. These data suggest that the late log phase may correspond to the most virulent phase of growth in H. pylori and may be intimately related to its pathogenesis. The use of microarrays to analyze the kinetics of the transcriptional response of a bacterial pathogen to a changing environment has enabled the discovery of previously unappreciated relationships between genes by elucidation of coordinated gene expression profiles.
Effective long range mapping in Schizosaccharomyces pombe with the help of swi5.
The switching gene swi5 has a function in mating-type switching. In addition, the swi5 mutation causes an increased radiation sensitivity and reduces meiotic recombination about ten-fold. Based on the latter property, an experimental protocol was developed for using swi5 in long-range mapping in S. pombe. It is suitable for a speedy mapping of any new gene which has not yet been cloned. The procedure was used to clarify the map positions of some genes.
MATa donor preference in yeast mating-type switching: activation of a large chromosomal region for recombination.
During mating-type gene switching in Saccharomyces cerevisiae, DNA at the MAT locus is replaced by sequences copied from one of two unexpressed donor loci, HML or HMR, located near the two ends of the same chromosome and > or = 90 kb from MAT. MATa cells recombine nearly 90% of the time with HML, whereas MAT alpha cells select HMR. MATa donor preference was examined by deleting HML and inserting a donor at other chromosome III locations. MATa activated a large (> or = 40 kb) region near the left end of chromosome III, such that a donor placed at several sites within this domain was strongly preferred over HMR. When inserted outside of this domain, the donor was used equally with HMR. MATa donor preference for HML was abolished by the expression of the negative regulator, MAT alpha 2; however, HML regained its preferred status when the donor was unsilenced. Mating-type-dependent activation of the left end of the chromosome is also observed for other types of recombination that do not involve MAT switching. Spontaneous recombination between two leu2 alleles is 20-30 times higher in MATa than in MAT alpha when one of the leu2 alleles is inserted in place of HML. Transcription in this donor activation region is not affected by mating type. We conclude that MATa donor preference involves a mating-type-regulated change in the accessibility of a large chromosomal domain for recombination.
Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching.
IgM+ cells cultured from the I.29 B cell lymphoma can be induced with lipopolysaccharide (LPS) or, to a greater extent, with LPS plus anti-idiotype antibody to switch to IgG2a, IgE or IgA expression. The isotype switch is accompanied by rearrangement of immunoglobulin (Ig) heavy (H) chain genes. Here we demonstrate that the commitment of the I.29 IgM+ cells to switch to IgA appears to be manifested by hypomethylation of the alpha constant region genes in IgM+ cells, and by the presence of small amounts of RNAs transcribed from non-rearranged alpha gene(s) in IgM+ cells. The commitment to switch to IgE or IgG2a is also in accord with the presence of small amounts of RNA transcripts from the non-rearranged epsilon and gamma 2a genes, although the hypomethylation of the epsilon and gamma 2a genes is not as dramatic as that of the alpha genes. These results suggest that I.29 cells switch specifically to IgA, IgE or IgG2a due to the activation of the corresponding H chain constant region genes in IgM+ cells prior to the actual switch recombination event.
GENE impedance: a natural process for control of gene expression and the origin of RNA interference.
Gene expression is controlled by coordinated transcriptional and post-transcriptional mechanisms. Normally, expression of a gene switches on and off in response to specific physiological signals that are triggered by cellular demand for the gene products at a given time. Based on our previous studies and the scientific literature, we hypothesize that when a gene promoter switches to transcriptional repression mode, transcription of the gene ceases, and a small amount of double-stranded RNA (dsRNA) is synthesized by the RNA polymerase switching to the opposite DNA strand at the termination region of the gene. These dsRNA structures, which result from normal transcriptional repression, can then be processed into short interfering RNAs (siRNAs) within the nucleus. These molecules subsequently direct specific cleavage of the cognate mRNAs and interfere with their translation through sequence complementarily. We further hypothesize that cellular defense mechanisms invoked by invading genetic elements could be rooted in this fundamental regulatory pathway that we call "GENE impedance", or simply, GENEi. Here, we present a working model that illustrates how transcription-termination and transcription-arrest can contribute to the regulation of gene expression via GENEi. In our model RNAi is only one component of GENEi, which is a more generalized mechanism of gene regulation.
Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However, later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50 xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.
The candidate sex-reversing DAX1 gene is autosomal in marsupials: implications for the evolution of sex determination in mammals.
The human X-linked DAX1 gene was cloned from the region of the short arm of the human X found in duplicate in sex-reversed Xdup Y females (E. Zanaria et al., 1994, Nature 372: 635-641). DAX1 is suggested to be required for ovarian differentiation and to play an important role in mammalian sex determination or differentiation pathways. Its proposed dose-dependent effect on sexual development suggests that DAX1 could represent an evolutionary link with an ancestral sex-determining mechanism that depended on the dosage of an X-linked gene. Furthermore, DAX1 could also represent the putative X-linked switch gene, which independently controls sexual dimorphisms in marsupial mammals in an X-dose-dependent manner (D.W. Cooper et al., 1993, Semin. Dev. 4: 117-128). If DAX1 has a present role in marsupial sexual differentiation or had an ancestral role in mammalian sex determination, it would be expected to lie on the marsupial X chromosome, despite the autosomal localization of other human Xp genes. We therefore cloned and mapped the DAX1 gene in the tammar wallaby (Macropus eugenii). DAX1 was located on wallaby chromosome 5p near other human Xp genes, indicating that it was originally autosomal and that it is not involved in X-linked dose-dependent sex determination in an ancestral mammal nor in marsupial sexual differentiation.
Helicobacter pylori infection in mice: Role of outer membrane proteins in colonization and inflammation.
BACKGROUND & AIMS: Mouse models of Helicobacter pylori infection have yielded variable results with respect to colonization and inflammation. We examined whether outer membrane proteins (OMPs) or the cag pathogenicity island (PAI) was responsible for some of this variability. METHODS: C57BL/6 mice received clinical H. pylori isolates with different genotypes for the cag PAI and OMP gene switch status, as well as isogenic gene knockout mutants for cagE, oipA, babA2, hopZ, cagE/oipA, or oipA/hopZ. Bacterial density, histology, and mucosal cytokine/chemokine levels were measured after 4 and 12 weeks. RESULTS: oipA, hopZ, hopO, and hopP switch status influenced both H. pylori density and colonization ability in mice. When 2 or more of the genes were "off," colonization rates were markedly reduced compared with those for strains with all genes "on" (from 58% to 0% after 12 weeks). oipA knockout mutants caused reduced inflammation and decreased mucosal interleukin 6 messenger RNA and KC messenger RNA and protein levels. H. pylori density and colonization ability were reduced if hopO or hopP switch status was changed from "on" to "off." There was a close relationship (r > 0.7) between the H. pylori density of the gastric mucosa of humans and mice when using the same H. pylori strains. CONCLUSIONS: Many of the differences reported with mouse models may reflect subtle unrecognized differences (e.g., switch status of an OMP gene), emphasizing the necessity of characterizing isolates before and after experiments. The mouse model may be suitable for investigating factors related to colonization ability, H. pylori density, and gastric mucosal inflammation.
Purification and characterization of the immunoglobulin switch sequence-specific endonuclease (Endo-SR) from bovine spleen.
Mature B lymphocytes are able to specifically alter their Ig isotype expression in response to extracellular stimuli via a highly regulated, deletional recombination process called isotype switch recombination. Switch recombination breakpoints predominantly map to large (1-10 kb), G-rich and highly repetitive switch regions that are located directly upstream of immunoglobulin heavy-chain constant region genes. Switch region repeat structures vary considerably both within and between species, but all switch regions contain disproportionate numbers of two pentamer motifs, TGGGN and TGAGC, that are found at or directly adjacent to most analysed switch junctions. We have recently identified an endonuclease activity, Endo SR, that preferentially cleaves TGGGN and TGAGC switch motifs. We have purified the bovine endonuclease activity to homogeneity and have identified a protein with a molecular weight of approximately 32,000 that directly correlates with enzyme activity. As discussed in this report, we have found that murine and bovine Endo-SR are preferentially enriched in lymphoid tissue nuclear extracts and that both enzymes demonstrate highly similar physical and biochemical characteristics. However, each enzyme demonstrates related but distinctive specificities for consensus and degenerate TGGGN and TGAGC switch pentamer motifs.