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PubMed1998

Mechanisms of developmental regulation in globin loci.

Recent advances in the study of globin gene switching in the context of the complete locus have contributed greatly to our understanding of the mechanisms of developmental regulation. It has become clear that the interactions between the distant locus control region and the individual globin genes, as well as the trans-acting factors and physical parameters that affect these interactions, are crucial determinants in the developmental modulation of globin gene expression. This review concentrates on recent advances in the highly studied human beta-globin locus and will compare and contrast data from the human alpha-globin locus as well as the alpha and beta loci from other species where appropriate.

Animals↗

PubMed2001

Comparative whole-genome analyses reveal over 100 putative phase-variable genes in the pathogenic Neisseria spp.

Previously, a complete genome analysis of Neisseria meningitidis strain MC58 revealed the largest repertoire of putative phase-variable genes described in any species to date. Initial comparisons with two incomplete Neisseria spp. genome sequences available at that time revealed differences in the repeats associated with these genes in the form of polymorphisms, the absence of the potentially unstable elements in some alleles, and in the repertoire of the genes that were present. Analyses of the complete genomes of N. meningitidis strain Z2491 and Neisseria gonorrhoeae strain FA1090 have been performed and are combined with a comprehensive comparative analysis between the three available complete genome sequences. This has increased the sensitivity of these searches and provided additional contextual information that facilitates the interpretation of the functional consequences of repeat instability. This analysis identified: (i) 68 phase-variable gene candidates in N. meningitidis strain Z2491, rather than the 27 previously reported; (ii) 83 candidates in N. gonorrhoeae strain FA1090; and (iii) 82 candidates in N. meningitidis strain MC58, including an additional 19 identified through cross-comparisons with the other two strains. In addition to the 18 members of the opa gene family, a repertoire of 119 putative phase-variable genes is described, indicating a huge potential for diversification mediated by this mechanism of gene switching in these species that is central to their interactions with the host and environmental transitions. Eighty-two of these are either known (14) or strong (68) candidates for phase variation, which together with the opa genes make a total of 100 identified genes. The repertoires of the genes identified in this analysis diverge from the different species groupings, indicating horizontal exchange that significantly affects the species and strain complements of these genes.

Bacterial Proteins↗

PubMed1999

Strategy of liver-directed gene therapy: present status and future prospects.

The liver is particularly amenable to gene therapy as it is the site of many metabolic diseases and malignancies. Thus, liver-directed gene therapy is being actively pursued and developed as a method of treatment for various liver diseases. Strategies of liver-directed gene therapy include drug delivery to the liver, compensation of the defective gene(s), anti-tumor activity, anti-viral therapy, and immunomodulation. The strategy chosen for liver-directed gene therapy depends on the genetic basis of the disease. Many aspects are key factors to the success of the chosen strategy: intervention of genes, efficient gene delivery system, stable transgene expression, transgene regulation, target cell transfection, and timing of transgene expression. Several tactics can be used to overcome problems in the above, and these include the use of a gene switch to exogenously regulate transgene expression, targeting at the transcriptional level, circumvention of the immune response (as in the use of adenovirus vector to achieve long-term correction of genetic diseases), and genetically engineered antibodies in gene transfer. At the present rate of research activity and development, gene therapies may soon be more efficient than current standard treatments for some liver diseases.

Animals↗

PubMed1991

Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.

Animals↗

PubMed1991

Analysis of the v-myb structural components important for transactivation of gene expression.

In order to define the domains of the v-myb protein that are important for transactivation of gene expression, we have studied transactivation by the v-myb gene and a set of v-myb deletion mutants using transient transfection assays in NIH 3T3 cells. Analysis of the set of v-myb deletion products demonstrated that a previously unidentified region in the carboxyl-terminal portion of the protein is required for transactivation. This region lies between amino acids 295-356 with respect to the 5' end of the v-myb gene. Switching the v-myb DNA binding domain with the DNA binding domain of the rat glucocorticoid receptor (rGR) switched the cis-element requirement for v-myb action: only reports containing glucocorticoid response elements were activated by myb-rGR fusion proteins. The carboxyl terminal region essential for transactivation by the intact v-myb gene was also necessary for transactivation by the rGR-fusion gene. Carboxyl-terminal deletion mutations that encompassed the novel transactivation region were able to block wild-type v-myb transactivation when tested in transient co-expression assays. In an unexpected sidelight to our studies, we could demonstrate that the lacZ gene present in the prokaryotic vector sequences contained a DNA element that fortuitously can act as a v-myb-dependent enhancer element, and that v-myb protein can bind to this element in vitro. The lacZ enhancer contains the myb consensus DNA binding site YAAC(G/T)G.

Animals↗

PubMed1998

Epstein-Barr viral gene expression in B-lymphocytes.

The strategy of the Epstein-Barr virus to persist lifelong in the host depends on establishing a reservoir, which cannot be detected by the immune system but allows reactivation of the virus for shedding and transmission to a new host. Epithelial cells and B-cells play a major role in this viral strategy of EBV, since differentiating epithelial tissues were shown to be permissive for lytic replication in vivo, whereas the B-lymphocytes become predominantly latently infected. However, which cells are the reservoir and which the sites of lytic replication are not quite clear. With the technique of reverse transcription, PCR and immunohistochemistry, we demonstrated that the B-cells of the peripheral blood are a major site of virus production during the primary infection during infectious mononucleosis. These permissive B-cells were also detected after convalescence, however, the absence of any lytic transcripts suggested an efficient immunological control very early in the viral lytic cycle. Serological data on reactivation of EBV correlated with the detection of lytic cycle transcripts in the blood and thus demonstrated that the site of virus production during infectious mononucleosis must be different from that of the persistent state. In those cases, where the infection takes a chronic active course, control of lytic replication is insufficient, either on the level of immune surveillance or of viral gene regulation. We have demonstrated a virus strain with a lytic phenotype in an individual suffering chronic active infection. The impaired capability of this virus to immortalise B-cells correlated with an enhanced expression of the lytic switch gene BZLF-1 and down-regulation of latent regulatory genes in the early phase of infection.

B-Lymphocytes↗

PubMed1999

Cellular, intracellular, and developmental expression patterns of murine SWAP-70.

SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.

Animals↗

PubMed1984

A homologous protein-coding sequence in Drosophila homeotic genes and its conservation in other metazoans.

The homeotic genes of the bithorax complex (BX-C) and the Antennapedia complex (ANT-C) of Drosophila appear to specify the developmental fate of segments or parts of segments of the fly. We have previously reported weak DNA sequence homology between 3' portions of the Antennapedia and fushi tarazu genes of the ANT-C and the Ultrabithorax gene of the BX-C. Here we show that this DNA homology (the homeo box) is due to a conserved protein-coding sequence present in these three pattern-formation genes. Thus the functional homology between these developmental controlling genes is reflected in a structural homology in their gene products. The homeo box sequence is also present in a few copies in the genomes of some other invertebrates, and is even conserved in vertebrate genomes, including the human genome. Apparently at least a part of these developmental switch genes from Drosophila is highly conserved during evolution, and might perform an analogous function in many metazoans .

Amino Acid Sequence↗

PubMed1996

An element upstream from the human delta-globin-encoding gene specifically enhances beta-globin reporter gene expression in murine erythroleukemia cells.

We have previously shown that a DNA-binding factor specific to adult hematopoietic cells (polypryrimidine-binding factor, PYBF) binds to a pyrimidine-rich region 1 kb upstream from the human delta-globin-encoding gene (HBD). The developmental stage-specificity of PYBF and the location of its binding site between the fetal and adult beta-globin (HBB)-like genes suggest that PBYF and its binding site may function in fetal-to-adult globin gene switching. Here, we describe the effect of 383-bp (delta383) and 99-bp (delta99) sequences containing the PYBF-binding site on transcription from various globin and non-globin promoters, using a transient assay with the cat reporter gene in murine erythroleukemia (MEL) cells, a cell line with abundant PYBF activity. We show that both delta383 and delta99 specifically enhance expression of cat for plasmids containing a human adult globin (HBB) promoter, whereas expression of similar constructs using human fetal (A gamma-) globin (HBG1) or simian virus 40 (SV40) promoters is not enhanced. The results suggest that PYBF and the pyrimidine-rich region upstream from HBD can specifically enhance HBB transcription in adult erythroid cells.

Adult↗

PubMed2004

Hematopoietic gene expression profile in zebrafish kidney marrow.

The zebrafish kidney marrow is considered to be the organ of definitive hematopoiesis, analogous to the mammalian bone marrow. We have sequenced 26,143 ESTs and isolated 304 cDNAs with putative full-length ORF from a zebrafish kidney marrow cDNA library. The ESTs formed 7,742 assemblies, representing both previously identified zebrafish ESTs (56%) and recently discovered zebrafish ESTs (44%). About 30% of these EST assemblies have orthologues in humans, including 1,282 disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database. Comparison of the effective and regulatory molecules related to erythroid functions across species suggests a good conservation from zebrafish to human. Interestingly, both embryonic and adult zebrafish globin genes showed higher homology to the human embryonic globin genes than to the human fetal/adult ones, consistent with evo-devo correlation hypothesis. In addition, conservation of a whole set of transcription factors involved in globin gene switch suggests the regulatory network for such remodeling mechanism existed before the divergence of the teleost and the ancestor of mammals. We also carried out whole-mount mRNA in situ hybridization assays for 493 cDNAs and identified 80 genes (16%) with tissue-specific expression during the first five days of zebrafish development. Twenty-six of these genes were specifically expressed in hematopoietic or vascular tissues, including three previously unidentified zebrafish genes: coro1a, nephrosin, and dab2. Our results indicate that conserved genetic programs regulate vertebrate hematopoiesis and vasculogenesis, and support the role of the zebrafish as an important animal model for studying both normal development and the molecular pathogenesis of human blood diseases.

Animals↗

PubMed2002

Phytochrome mediates the external light signal to repress FT orthologs in photoperiodic flowering of rice.

Phytochromes confer the photoperiodic control of flowering in rice (Oryza sativa), a short-day plant. To better understand the molecular mechanisms of day-length recognition, we examined the interaction between phytochrome signals and circadian clocks in photoperiodic-flowering mutants of rice. Monitoring behaviors of circadian clocks revealed that phase setting of circadian clocks is not affected either under short-day (SD) or under long-day (LD) conditions in a phytochrome-deficient mutant that shows an early-flowering phenotype with no photoperiodic response. Non-24-hr-light/dark-cycle experiments revealed that a rice counterpart gene of Arabidopsis CONSTANS (CO), named PHOTOPERIOD SENSITIVITY 1 (Heading date 1) [SE1 (Hd1)], functions as an output of circadian clocks. In addition, the phytochrome deficiency does not affect the diurnal mRNA expression of SE1 upon floral transition. Downstream floral switch genes were further identified with rice orthologs of Arabidopsis FLOWERING LOCUS T (FT). Our RT-PCR data indicate that phytochrome signals repress mRNA expression of FT orthologs, whereas SE1 can function to promote and suppress mRNA expression of the FT orthologs under SD and LD, respectively. This SE1 transcriptional activity may be posttranscriptionally regulated and may depend on the coincidence with Pfr phytochromes. We propose a model to explain how a short-day plant recognizes the day length in photoperiodic flowering.

Amino Acid Sequence↗

PubMed1992

Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid.

The gene coding for apolipoprotein AI (apoAI), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine. Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAI gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXR alpha over the previously characterized RA receptors RAR alpha and RAR beta. In this study we investigated the combined effects of ARP-1 and RXR alpha on apoAI gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXR alpha-mediated transactivation of the apoAI gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXR alpha bind to site A with similar affinities. These assays also revealed that ARP-1 and RXR alpha bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXR alpha alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAI gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXR alpha has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXR alpha together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXR alpha together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAI gene responsiveness to RXR alpha and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXR alpha. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.

Apolipoprotein A-I↗

PubMed1993

Transcription factor GATA-1 and erythroid development.

In summary, we derived an experimental system that allows us to dissect the function of GATA-1 in red cell development at a genetic level. We have established the essential nature of GATA-1 during both primitive and definitive erythropoiesis. By ablating the expression of the endogenous GATA-1 gene, we are in a position to introduce a variety of constructs that harbor subtle modifications in flanking or protein-coding sequences. We can now study regulatory regions and functional domains of the protein in the context of a true erythroid environment, experiments that have not been possible heretofore. Although the assay involves the dramatic loss of red cell production, it should be possible to define important regulatory domains that can then be assayed using less stringent systems, such as cell-free extracts for in vitro transcription. The ideal situation would be analyses conducted in GATA-1- erythroid cells. However, these cells have been impossible to generate given the requirement of GATA-1 for Epo receptor expression and red cell viability (C. Simon and S. Orkin, unpublished observations). It may be possible to produce such cells by first expressing the Epo receptor under the influence of a constitutive promoter and then targeting the GATA-1 gene. If GATA-1- red cells were available, the analyses would involve the actual transcription of or chromatin structure surrounding the globin genes. Structure-function studies of the GATA-1 protein could be greatly simplified and a larger number of mutants studied. However, the ES cell system can be used as an alternative until targeted erythroleukemia cells become available. Other applications involve the introduction of other GATA-binding protein family members to determine whether they rescue the mutation. If they cannot, chimeric proteins can be tested to identify which amino acids distinguish the different family members. We feel that these experiments are vital to understanding the function of GATA-1 during erythroid ontogeny. How does GATA-1 regulate red cell genes like globin or the Epo receptor? Once we identify the functional domains of the GATA-binding proteins, we hope to learn what proteins GATA-1 binds to in the basic transcription machinery or in chromatin. Is GATA-1 necessary for globin gene switching? GATA-1 may be modified differently during development so that the locus control region can interact with different globin promoters. We may find that one region of the protein is required for embryonic expression and another for adult globin gene expression.

Amino Acid Sequence↗

PubMed1994

Genomic footprinting and sequencing of human beta-globin locus. Tissue specificity and cell line artifact.

In order to gain further insights of the regulatory mechanisms of human beta-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult beta and fetal gamma globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the beta, but not gamma or epsilon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non-erythroid human cell lines. Protein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human beta globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human beta globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human beta-like globin switch during erythroid development.

Adult↗

PubMed1986

trans Activation of the simian virus 40 enhancer.

We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth.

Animals↗

PubMed2006

Isolation and expression profiling of genes upregulated in bone marrow-derived mononuclear cells of rheumatoid arthritis patients.

We have comprehensively identified the genes whose expressions are augmented in bone marrow-derived mononuclear cells (BMMC) from patients with Rheumatoid Arthritis (RA) as compared with BMMCs from Osteoarthritis (OA) patients, and named them AURA after augmented in RA. Both stepwise subtractive hybridization and microarray analyses were used to identify AURA genes, which were confirmed by northern blot analysis and/or reverse transcription polymerase chain reaction (RT-PCR). We also assessed their expression levels in individual patients by quantitative real-time RT-PCR. Of 103 AURA genes we have identified, the mRNA levels of the following 10 genes, which are somehow related to immune responses, were increased in many of the RA patients: AREG (=AURA9), FK506-binding protein 5 (FKBP5 = AURA45), C-type lectin superfamily member 9 (CLECSF9 = AURA24), tyrosylprotein sulfotransferase 1 (TPST1 = AURA52), lymphocyte G0/G1 switch gene (G0S2 = AURA8), chemokine receptor 4 (CXCR4 = AURA86), nuclear factor-kappa B (NF-kappaB = AURA25) and two genes of unknown function (FLJ11106 = AURA1, BC022398 = AURA2 and XM_058513 = AURA17). Since AREG was most significantly increased in many of the RA patients, we subjected it to further analysis and found that AREG-epidermal growth factor receptor signaling is highly activated in synovial cells isolated from RA patients, but not in OA synoviocytes. We propose that the expression profiling of these AURA genes may improve our understanding of the pathogenesis of RA.

Amphiregulin↗

PubMed1996

Both loss-of-function and gain-of-function mutations in snf define a role for snRNP proteins in regulating Sex-lethal pre-mRNA splicing in Drosophila development.

The Drosophila snf gene encodes a protein with functional homology to the mammalian U1A and U2B" snRNP proteins. Studies, based on the analysis of three viable alleles, have suggested a role for snf in establishing the female-specific splicing pattern of the sex determination switch gene, sex-lethal. Here, we show that the non-sex-specific lethal null allele is required for female sex determination, arguing against the formal possibility that the viable alleles disrupt a function unrelated to snf's wild-type function. Moreover, we find snf is required for normal cell growth and/or survival, as expected for a protein involved in a cell-vital process such as RNA splicing. We also show that of the three viable alleles only one, snfJA2, is a partial loss-of-function mutation. The other two viable alleles, snf1621 and snfe8H, encode antimorphic proteins. We find the antimorphic proteins are mislocalized and correlate their mislocalization with their molecular lesions and mutant phenotypes. Finally, we provide genetic evidence that the antimorphic alleles interfere with the autoregulatory splicing function of the Sex-lethal protein. Based on these studies we suggest a model in which the snRNP protein, Snf, functions with Sex-lethal to block recognition of the regulated male-specific exon.

Alleles↗

PubMed2006

Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals.

Zooecdysteroids (arthropod steroid hormones) regulate the development of arthropods and probably many other invertebrates. Phytoecdysteroids are analogues occurring in a wide range of plant species, where they contribute to the deterrence of phytophagous invertebrates. The purpose of this short review is to summarise findings on the occurrence, metabolism and pharmacological effects of ecdysteroids in mammalian systems and to draw attention to their potential applications, particularly in gene-switch technology, where ecdysteroid analogues (steroidal and non-steroidal) can be used as effective and potent elicitors.

Animals↗