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PubMed1988

DNA methylation: a secondary event in globin gene switching?

Changes in gamma-globin gene methylation accompany the fetal to adult globin switch in man. Using somatic cell hybrids made by fusing mouse erythroleukemia and human fetal erythroid cells, we asked whether methylation is a cause or a consequence of gamma-gene inactivation during development. These hybrids initially express human gamma-globin but switch with time in culture to adult globin gene production. In hybrids before the switch, the gamma-genes are unmethylated. After completion of the switch, the hybrids contain methylated gamma-globin genes. However, during the time that the gamma----beta switch is occurring, hybrids are found that no longer express gamma-globin, yet still possess unmethylated gamma-globin genes. This suggests that methylation is not a primary event in gamma-gene inactivation during human development.

Adult↗
PubMed2002

Using a gene-switch transgenic approach to dissect distinct roles of MAP kinases in heart failure.

We have demonstrated that Cre-loxP-mediated gene-switch transgenesis is an effective approach to achieve targeted and temporally regulated gene manipulation in the heart. Using this approach, we have established animal models with targeted activation of different MAPK pathways. From these animal models, we identified distinct features of cardiac pathology associated with individual MAPK branches (summarized in Fig. 8). Specifically, Ras activation appears to promote cardiac hypertrophy, whereas p38 and JNK activation does not. Whereas Ras activation leads to depressed diastolic function associated with suppressed calcium transients and SR calcium uptake, p38 activity seems to modulate cellular contractility without affecting intracellular calcium cycling. Although all three models displayed extensive remodeling in the myocardium, the extent and the composition of interstitial fibrosis are different among them, with Ras- and p38-activated hearts promoting collagen-based fibrosis, and JNK activation leading to induction in fibronectin-based reticular fiber. In addition, JNK activation leads to loss of Cx43 expression and abnormal cell-cell communication. Therefore, ERK, p38, and JNK are three distinct intracellular signaling pathways that contribute to different aspects of cardiac pathology during heart failure. Combining sophisticated genetic manipulation with comprehensive analysis at physiological, molecular, and genomic levels, the transgenic animals established in these studies should serve as valuable model systems to identify and dissect the underlying mechanisms for different aspects of cardiac pathology such as hypertrophy, contractile dysfunction, and abnormal cell-cell communication. The insights learned from these investigations may help to develop novel therapeutic approaches to confront this devastating disease.

Animals↗
PubMed1996

Troponin T gene switching is developmentally regulated by plasma-borne factors in parabiotic chicks.

Myogenesis involves a conserved program of muscle gene isoform switching requiring the synchronized induction and repression of numerous muscle-specific gene family members. Central to understanding the regulation of this process are questions related to the origin and transmission of regulatory signals to the myofiber. We show here that troponin T gene switching can be precociously initiated by extrinsic blood-borne components but also requires other mechanisms that are regulated locally, intrinsically, or posttranscriptionally. We established a chimeric blood circulation by parabiosis between fetal chicks and quails to determine whether signals inducing earlier troponin T mRNA isoform switching in quails could be transduced to chick partners through the serum. While quail fetuses were unaffected by parabiosis, quail serum caused premature troponin T iso-mRNA switching in chick muscle, although initiation remained later than in quails. The onset of repression of a known innervation-dependent acetylcholine receptor mRNA did not coincide with the initiation of troponin T iso-mRNA switching and was not affected by parabiosis. These results support serum-borne factor regulation of isoform switching as an important and distinct mechanism relevant to understanding how extrinsic and intrinsic cues are integrated during muscle differentiation and development.

Animals↗
PubMed1995

Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.

Proper tissue- and developmental stage-specific transcriptional control over the five genes of the human beta-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human beta-globin gene switching. The experiments revealed a number of unexpected properties that are directly attributable to LCR function. First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes. If HS3 is used to replace HS4, gene expression is normal at all developmental stages. Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific beta-type globin gene activation. Although the HS4 duplication leads to alteration in the levels of epsilon- and gamma-globin mRNAs during embryonic erythropoiesis, total beta-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human beta-locus genes are competitively regulated. In summary, the human beta-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.

Animals↗
PubMed2004

Gene switching in Amoeba proteus caused by endosymbiotic bacteria.

The expression of genes for S-adenosylmethionine synthetase (SAMS), which catalyzes the synthesis of S-adenosylmethionine (AdoMet), a major methyl donor in cells, was studied in symbiont-free (D) and symbiont-bearing (xD) amoeba strains to determine the effect of bacterial endosymbionts. The symbionts suppressed the expression of the gene in host xD amoebae, but amoebae still exhibited about half the enzyme activity found in symbiont-free D amoebae. The study was aimed at elucidating mechanisms of the suppression of the amoeba's gene and determining the alternative source for the gene product. Unexpectedly, we found a second sams (sams2) gene in amoebae, which encoded 390 amino acids. Results of experiments measuring SAMS activities and amounts of AdoMet in D and xD amoebae showed that the half SAMS activity found in xD amoebae came from the amoeba's SAMS2 and not from their endosymbionts. The expression of amoeba sams genes was switched from sams1 to sams2 as a result of infection with X-bacteria, raising the possibility that the switch in the expression of sams genes by bacteria plays a role in the development of symbiosis and the host-pathogen interactions. This is the first report showing such a switch in the expression of host sams genes by infecting bacteria.

Amino Acid Sequence↗
PubMed1990

Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice.

Transgenic mice containing a human fetal (gamma-) or adult (beta-) globin gene linked to the beta-globin gene locus activation region (LAR) express the gene throughout development. By contrast, transgenic mice containing LAR linked to both a fetal and an adult globin gene display the normal developmental switch from fetal to adult gene expression. This suggests that the human fetal-to-adult globin gene switch is controlled through a mutually exclusive interaction between LAR and either the gamma- or beta-globin gene, resulting in the expression of only one gene at any given moment.

Animals↗
PubMed2002

Agrobacterium flagellar switch gene fliG is liquid inducible and important for virulence.

Agrobacterium tumefaciens C58 was mutagenized with a mini-Tn5 transposon containing a promoterless gene encoding the green fluorescent protein (GFP). A mutant, CGS74, exhibited a higher GFP expression level in liquid media than on solid media. The ability of the mutant to cause tumors on plants was attenuated. Sequence analysis showed that the transposon was inserted at the fliG gene, which encodes a flagellar motor switch protein required for flagellar movement. Studies of the fliG-gfp fusion gene indicated that the promoter activity of the fliG gene was higher in liquid than in solid media. Electron microscopy studies demonstrated that the mutant was nonflagellate. This suggests that the A. tumefaciens motility is important for virulence and that bacterial flagellar synthesis occurs at a higher level in a liquid environment than in a solid environment, perhaps resulting in a higher motility.

Agrobacterium tumefaciens↗
PubMed2002

Chromatin structure and control of beta-like globin gene switching.

The human beta-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional beta-like globin genes, epsilon, (G)gamma, (A)gamma, delta, and beta, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNaseI-hypersensitive sites located 6-22 Kb upstream of the epsilon -globin gene. During ontogeny two switches occur in beta-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic epsilon - to fetal gamma-globin occurs at six weeks of gestation. The second switch from gamma- to adult delta- and beta-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching.

Animals↗
PubMed1989

MB-02 cells undergo fetal to adult globin gene switching in response to erythropoietin.

The recently described MB-02 human cell line requires Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) for continuous growth and terminally differentiates into enucleate, hemoglobinized cells in response to erythropoietin. Here, analysis of globin production now demonstrates that uninduced MB-02 cells produce alpha globin and the fetal globin chains G gamma and A gamma in a ratio of 1:3. Addition of erythropoietin results in de novo synthesis of beta globin chains and a marked increase in total Hb/cell. Thus, the MB-02 cell line partially recapitulates the fetal to adult globin switch that occurs during erythroid and human fetal development and provides a new clonal human erythroid progenitor system for investigating the biochemical and molecular events involved in globin gene switching.

Cell Line↗
PubMed1992

The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe.

The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h90) strains of Schizosaccharomyces pombe. The MT region of h90 comprises three cassette genes: the expression site mat1:1 and two silent loci, mat2:2 and mat3:3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1:1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2:2 and mat3:3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.

Blotting, Southern↗
PubMed1995

Role of erythroid Kruppel-like factor in human gamma- to beta-globin gene switching.

Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that contains zinc finger domains similar to the Kruppel protein of Drosophila melanogaster. Previous studies demonstrated that EKLF binds to the CACCC box in the human beta-globin gene promoter and activates transcription. CACCC box mutations that cause severe beta-thalassemias in humans inhibit EKLF binding. Results described in this paper suggest that EKLF functions predominately in adult erythroid tissue. The EKLF gene is expressed at a 3-fold higher level in adult erythroid tissue than in fetal erythroid tissue, and the EKLF protein binds to the human beta-globin promoter 8-fold more efficiently than to the human gamma-globin promoter. Co-transfection experiments in the human fetal-like erythroleukemia cell line K562 demonstrate that over-expression of EKLF activates a beta-globin reporter construct 1000-fold; a linked gamma-globin reporter is activated only 3-fold. Mutation of the beta-globin CACCC box severely inhibits activation. These results demonstrate that EKLF is a developmental stage-enriched protein that preferentially activates human beta-globin gene expression. The data strongly suggest that EKLF is an important factor involved in human gamma- to beta-globin gene switching.

Animals↗
PubMed2003

Transcriptional activation of the metallothionein I gene by electric pulses in vivo: basis for the development of a new gene switch system.

BACKGROUND: In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscular disorders and for the systemic delivery of therapeutic proteins. Nevertheless, for a safe and effective protein production, the spatial and temporal control of gene expression is critical. The existing regulating systems rely on the use of an exogenously regulatory protein and/or an inducer drug whose pharmacological properties are of major concerns for therapeutic applications in humans. Therefore, new strategies based on endogenous regulatable elements have been explored. METHODS: Gene expression profiles of skeletal muscle submitted or not to electrical pulses and harvested at different times were compared using the Affymetrix GeneChip technology. The endogenous metallothionein promoter was studied by Northern blot and semiquantitative and quantitative RT-PCR. The inducibility of the metallothionein I promoter placed in a plasmid exogenous context was studied using the murine SEAP reporter gene. RESULTS: The expression of metallothionein I mRNA is significantly increased 6 h after electric pulses delivery. This induction is transient. Identical MT-I expression level is observed after several sequential series of pulses delivery. We demonstrated as well that the MT-II promoter was sensitive to electric pulses delivery. Moreover, the metallothionein I promoter, placed in a plasmid context in front of a reporter gene, was also activated by the application of transient electric field. CONCLUSIONS: We identified a promoter highly inducible by the controlled electric stimuli applied for electrotransfer experiments. The use of the metallothionein promoter is promising for the time-control by physical stimuli of the expression of a therapeutic gene.

Animals↗
PubMed1992

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

Aging↗
PubMed1999

Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis.

Previous attempts to establish transgenic mouse models to study the functions of transforming growth factor beta1 (TGFbeta1) in the skin revealed controversial roles for TGFbeta1 in epidermal growth (inhibition vs. stimulation) and resulted in neonatal lethality in one instance. To establish a viable transgenic model for studying functions of TGFbeta1 in the skin, we have now developed transgenic mice, which allow focal induction of the TGFbeta1 transgene in the epidermis at different expression levels and at different developmental stages. This system, termed "gene-switch," consists of two transgenic lines. The mouse loricrin vector targets the GLVPc transactivator (a fusion molecule of the truncated progesterone receptor and the GAL4 DNA binding domain), and a thymidine kinase promoter drives the TGFbeta1 target gene with GAL4 binding sites upstream of the promoter. These two transgenic lines were mated to generate bigenic mice, and TGFbeta1 transgene expression was controlled by topical application of an antiprogestin. On epidermal-specific induction of the TGFbeta1 transgene, the BrdUrd labeling index in the transgenic epidermis decreased 6-fold compared with controls. Induction of the TGFbeta1 transgene expression also caused epidermal resistance to phorbol 12-myristate 13-acetate-induced hyperplasia, with a reduction in both epidermal thickness and BrdUrd labeling compared with those in controls. In addition, TGFbeta1 transgene expression induced an increase in angiogenesis in the dermis. Given that the TGFbeta1 transgene can affect both the epidermis and dermis, this transgenic model will provide a useful tool for studying roles of TGFbeta1 in wound-healing and skin carcinogenesis in the future.

Animals↗
PubMed1993

Human fetal-to-adult globin gene switching in transgenic mice: persistent expression of the G gamma-globin gene in the Japanese HPFH.

We linked a 3.3 kb fragment containing the human gamma-globin gene together with a 5.5 kb fragment containing the human beta-globin gene. This construct was introduced into the germ line of mice. Analysis of the resulting transgenic mice showed that expression of the human gamma- and beta-globin genes were regulated as that of the mouse embryonic and adult globin genes and human gamma-to-beta globin switching was reconstructed in the mice. By replacing the gamma-globin gene in the construct with modified counterparts, this transgenic mouse system enables us to analyze cis-acting elements essential for erythroid-specific and developmental stage-specific expression of the test gene under the condition in which human beta-globin gene expression is regulated. In this system expression of the G gamma-globin gene bearing the point mutation found in a Japanese patient of hereditary persistence of fetal hemoglobin (HPFH) (1) persisted at a equivalent level to beta-globin expression in fetal and adult mice.

Aging↗
PubMed1981

Recombination events near the immunoglobulin Cmu gene join variable and constant region genes, switch heavy-chain expression, or inactivate the locus.

Immunoglobulin heavy-chain expression is initiated by recombination between a variable region (VH) gene and one of several joining region (JH) genes located near the mu constant region (Cmu) gene, and the active VH gene can subsequently switch to another CH gene. That the general mechanism for CH switching involves recombination between sites within the JH-Cmu intervening sequence and the 5' flanking region of another CH gene is supported here by Southern blot hybridization analysis of eight IgG- and IgA-secreting plasmacytomas. An alternative model requiring successive VH linkage to similar JH clusters near each CH gene is shown to be very unlikely since the mouse genome appears to contain only one complement of the JH locus and no JH gene was detectable within large cloned sequences flanking germline C gamma 3 and C gamma 1 genes. Thus, VH-JH joining and CH switching are mediated by separate regions of "the joining-switch" or J-S element. In each plasmacytoma examined, the J-S element had undergone recombination within both the JH locus and the switch region and was shown to be linked to the functional CH gene in an IgG3, and IgG1, and three IgA secretors. Both JH joining and CH switching occurred by deletion of DNA. Switch recombination occurred at more than one site within the J-S element in different lines, even for recombination with the same CH gene. Significantly, although heavy-chain expression is restricted to one allele ("allelic exclusion"), all rearranged in each plasmacytoma. Some rearrangements were aberrant, involving, for example, deletion of all JH genes from the allele. Hence, an error-prone recombination machinery may account for allelic exclusion in many plasmacytomas.

Animals↗
PubMed1990

An in vitro globin gene switching model based on differentiated embryonic stem cells.

We used mouse embryonic stem (ES) cells to study globin gene expression and switching in vitro. We show that ES-derived embryoid bodies express the full complement of mouse embryonic globin genes in the correct temporal order and that on further differentiation, a switch occurs to the fetal/adult genes. In addition, the erythroid-specific transcription factor NF-E1 was shown to be expressed coordinately with that of globin in embryoid bodies. We conclude from these experiments that the ES cell system provides a good model to study hematopoietic development. When the human epsilon- or beta-globin genes driven by the dominant control region (DCR) are introduced into this system, the human epsilon-globin gene, in contrast to the beta-globin gene, is not deregulated by the presence of the DCR and is expressed strictly as an embryonic gene. We conclude from this that the epsilon-globin gene is not regulated by competition with other genes in the human beta-globin locus.

Animals↗
PubMed1998

Expression and processing of G0/G1 switch gene 24 (G0S24/TIS11/TTP/NUP475) RNA in cultured human blood mononuclear cells.

The human G0/G1 switch (G0S) gene, G0S24, and its rodent immediate-early homolog (TIS11, TTP, NUP475) are part of a mammalian gene family whose members encode CCCH zinc finger domains and domains similar to part of the large subunit of RNA polymerase II and to the Mei2 regulator of G1 arrest in fission yeast. We compared the RNA expression of G0S24 with that of other G0S genes in cultured blood mononuclear cells and examined the levels of various RNA processing intermediates. Freshly isolated cells contained high levels of several G0S RNAs, which declined by 24 h, suggesting transient spontaneous stimulation during cell purification (Heximer et al., 1996). However, in cells preincubated for 24 h, G0S24 RNA levels remained much higher than those of other G0S genes (107+/-42 x 10(6) molecules/microg of RNA); stimulation with lectin (Con-A) further increased G0S24 RNA, much of which remained nuclear. Like those of FOS/G0S7, EGR1/G0S30 and of the gene encoding the regulator of G protein signalling 1 (RGS1), G0S24 RNA levels increased more in response to a protein kinase C activator than to a calcium ionophore, whereas the opposite held for FOSB/G0S3 and RGS2/G0S8. With appropriate PCR primer pairs, we showed a G0S24 RNA processing intermediate, which crossed the exon-1/intron boundary, and nonpolyadenylated nuclear RNA extending into the 3' flank, where there is a second CpG island. The concentration of the latter intermediate (1.2+/-0.2 x 10(6) molecules/microg of RNA), which increased transiently on cell stimulation, did not account for all G0S24 nuclear RNA. The levels of G0S24 RNA and both intermediates were increased by the protein synthesis inhibitor cycloheximide, consistent with regulation by a labile repressor.

Base Sequence↗