Single Copy Of Human Chromosome Research Articles

Latent Regulatory Potential of Human-Specific Repetitive Elements

SummaryAt least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it.

EMS mutant spectra generated by multi-parameter flow cytometry

The CHO A L cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59 − regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59 − clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

Genetic control of telomerase and replicative senescence in human and rodent cells.

The ribonucleoprotein telomerase is detectable in most human cancer cells and immortalized cells but is absent or inactive in the vast majority of normal counterparts. Repression of telomerase activity in human somatic cells, which leads to telomere shortening and replicative senescence, may have evolved as a protective mechanism against immortalization, unfettered clonal evolution and cancer. Rodent cells in culture are far more susceptible to immortalization and malignant progression than human cells. This can be explained by our observation that normal diploid rodent (hamster) fibroblasts possess active telomerase throughout their proliferative life span, and therefore they do not require a telomerase activation step during immortalization. Monochromosome transfer techniques have enabled us to identify powerful telomerase repressive activity specifically associated with the introduction of a single copy of human chromosome 3 into human carcinoma cells. Fine-structure deletion analysis of non-repressed hybrids has permitted us to map the position of the candidate telomerase repressor gene to 3p21.1-3p21.3. A strategy for isolating the gene has been developed involving a combination of fine-structure deletion mapping and functional gene transfer approaches. The availability of cloned telomerase repressor genes will advance our understanding of telomerase regulation in the human soma and its disruption during human cancer development.

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A L). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59 − and CD59 + populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.

The binding of the ubiquitous transcription factor Sp1 at the locus control region represses the expression of β-like globin genes

To investigate the function of transcription factor Sp1 in beta-like globin gene activation, we analyzed the recruitment of Sp1, fetal Krüppel-like factor 2 (FKLF2), and related factors at the human beta-globin locus in a human fetal liver and mouse erythroleukemia hybrid cell (A181gamma cell) that contains a single copy of human chromosome 11. Sp1 binds at the GT boxes of the cis-elements throughout the beta-locus, but it is phosphorylated and lost over DNase I hypersensitive site (HS)2, HS3, HS4, and the human beta-globin gene promoter after A181gamma cell differentiation. The binding of FKLF2 at HS2 and HS3 was unchanged. Histone deacetylase 1, which could be recruited by Sp1, is also lost over HS2 and HS3 after differentiation, resulting in the acetylation of histones 3 and 4 across the human beta-globin locus. We previously detected in vivo GT footprints over the beta-globin locus after A181gamma differentiation. Here, we report that after differentiation, the p300/CREB-binding protein-associated factor is recruited by FKLF2 to the locus control region to acetylate histones 3 and 4 at the human beta-globin gene locus. Our results suggest that Sp1 is an inhibitor of beta-like globin gene transcription during erythroid terminal differentiation. Its phosphorylation and release allow the erythroid-specific FKLF2 or erythroid Krüppel-like factor to interact with other erythroid-specific transcription factors to initiate the transcription of beta-like globin genes.

A role for long-lived radicals (LLR) in radiation-induced mutation and persistent chromosomal instability: counteraction by ascorbate and RibCys but not DMSO

Miazaki, Watanabe, Kumagai and their colleagues reported that induction of HPRT − mutants by X-rays in cultured human cells was prevented by ascorbate added 30 min after irradiation. They attributed extinction of induced mutation to neutralization by ascorbate of radiation-induced long-lived mutagenic radicals (LLR), found using spectroscopy to have half-lives of minutes or hours. We find that post-irradiation treatment with ascorbate reduces, but does not eliminate, induction of CD59 − mutants in human-hamster hybrid A L cells exposed to high-LET carbon-ions (LET of 100 KeV/μm). A L cells contain a standard set of Chinese hamster ovary (CHO) chromosomes and a single copy of human chromosome 11 containing the CD59 gene which encodes the CD59 cell surface antigen, a convenient marker for mutation. RibCys [2(R, S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid] a ‘prodrug’ of l-cysteine which also scavenges LLR, had a similar but lesser effect on induced mutation. DMSO, which scavenges classical radicals like H and OH but not LLR, also reduced mutation, but only when it was present during irradiation. The lethality of carbon-ions was not altered by ascorbate, RibCys no matter when added. Post-radiation addition of ascorbate and RibCys also affected the quality of CD59 − mutations induced by carbon-ions. The major change in mutant spectra was a reduction in the prevalence of small, intragenic mutations (mutations not detected by PCR) and in the prevalence of unstable, complicated mutants, which display high levels of persistent chromosomal instability. Thus, ascorbate and RibCys may suppress some kinds of mutations induced by ionizing radiation including those displaying aspects of radiation-induced genomic instability. Countering the effects of both classical radicals and LLR may be important in preventing genetic diseases.

Disruption of the Mechanism of Long Range Activation within the Human α-Globin Complex

The human alpha-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5'-zeta2-alpha2-alpha1-3'), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. This DNase I-hypersensitive site is the only known regulatory element displaying strong erythroid-specific enhancer activity within the human alpha-globin complex. How this enhancer activity is shared among different erythroid genes present in the same cluster without affecting the ubiquitous genes present within and around the complex is poorly understood. To address this issue, we used hybrid murine erythroleukemia cells containing a single copy of human chromosome 16 and targeted the insertion of different sequences downstream of HS-40 by recombinase-mediated cassette exchange. We thus demonstrate that (i). HS-40-mediated erythroid-specific activation of the alpha-globin genes is impaired solely by the insertion of a promoter sequence and not a coding sequence, unless it is methylated, and that (ii). the degree of transcriptional repression observed seems to be related directly to the transcriptional rate of the inserted promoter. Taken together, these results emphasize the importance of promoter sequences as the main targets for the activation mechanism of the human alpha-globin genes by HS-40.

Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer.

The methylation status of binding sites of the insulator protein, CTCF, in the H19 promoter has been suggested as being critical to the regulation of imprinting of the H19/IGF2 locus located in chromosome 11p15. In this study, we have analyzed the methylation of all of seven potential CTCF-binding sites in the human H19 promoter since the methylation status of these sites has not been reported. We found that all the binding sites except the sixth were hypermethylated whereas only the sixth binding site showed allele-specific methylation in normal human embryonic ureteral tissue. We also analyzed the methylation status of these sites in human-mouse somatic-cell-hybrid clones containing a single copy of human chromosome 11 and which were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) to yield clones which expressed human IGF2 and H19 mutually exclusively of each other. In most of the clones, a correlation between methylation of the sixth CTCF-binding site and expression of IGF2 was observed. Therefore, we analyzed the methylation status of this site in human bladder cancer and found hypomethylation of the paternal allele in two of six informative cases. These results demonstrate that only the sixth CTCF-binding site acts as a key regulatory domain for switching between H19 or IGF2 expression, whereas the other sites are not subject to allele-specific methylation. Loss of methylation imprinting of H19 is linked to hypomethylation of the paternal allele in human bladder cancer, unlike the situation in Wilms' tumor and colon cancer where the maternal allele becomes hypermethylated.

Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene.

Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001). We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.

Targeted inactivation of the major positive regulatory element (HS-40) of the human alpha-globin gene locus

We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha- globin gene expression.

Targeted Inactivation of the Major Positive Regulatory Element (HS-40) of the Human α-Globin Gene Locus

We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha-globin gene expression.

An "in-out" strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the beta-globin locus control region.

The human beta-globin locus control region (LCR) is a complex DNA regulatory element that controls the expression of the cis-linked beta-like globin genes located in the 55 kilobases 3' of the LCR. We have initiated the functional analysis of the LCR by homologous recombination in murine erythroleukemia cell somatic hybrids that carry a single copy of human chromosome 11 on which the beta-globin locus is situated. High-level expression of the human beta-globin gene normally occurs when these hybrid cells are induced to differentiate. We have reported that the insertion of an expressed selectable marker gene (driven by the Friend virus enhancer/promoter) into the LCR disrupts the LCR-mediated regulation of globin transcription. In these cells, beta-globin is no longer expressed when the cells differentiate; instead, expression of the selectable marker gene increases significantly after differentiation. Since present techniques for homologous recombination require the insertion of a selectable marker, further progress in using homologous recombination to analyze the LCR depends on deletion of the selectable marker and demonstration that the locus functions normally after the insertion, expression, and deletion of the selectable marker. Here we show that after precise deletion of the selectable marker by using the FLP recombinase/FRT (FLP recombinase target) system, the locus functions as it did before the homologous recombination event. These studies demonstrate the feasibility of using homologous recombination to analyze the LCR in particular, and other complex cis-regulatory DNA elements in general, in their normal chromosomal context.

The structure and function of interferon-γ receptors

Interferon-γ exerts its pleiotropic immunomodulatory effects by interacting with a single type of IFNγ receptor expressed on nearly all cells. Human and murine IFNγ receptors have been purified, their cDNAs cloned and expressed, and their primary structure elucidated. In addition, a considerable amount of data is available that defines the life cycle of the receptor including the kinetics of its biosynthesis, its constitutive- and ligand-induced phosphorylation, and its recycling behavior. Recent transfection experiments have revealed that an additional species-specific component is necessary to form functionally active IFNγ receptors in heterologous cells. On the basis of complementation assays, the gene for the human accessory molecule has been localized to human chromosome 21. However, the nature of the gene product(s) remains unknown. Using murine fibroblasts that contain a single copy of human chromosome 21 and eukaryotic cell expression vectors that contain a series of human IFNγ receptor intracellular domain deletion mutants, we have been able to demonstrate that the receptor's intracellular domain plays an obligatory role in mediating IFNγ-dependent biologic responses. Subsequent studies showed that two distinct regions of the intracellular domain are functionally important: a membrane proximal region of 48 amino acids needed for both internalization and induction of biologic responses and the carboxy terminal 39 amino acids needed only for function and not for internalization. Moreover, receptor-mediated ligand internalization was found to be insufficient for biologic response induction. These results thus demonstrate that the IFNγ receptor's intracellular domain plays a critical functional role in mediating IFNγ-dependent cellular responses and provides new insights into the mechanism of IFNγ receptor-mediated signal transduction.

The Extracellular Domain of the Human Interferon Gamma Receptor Interacts with a Species-Specific Signal Transducer

At least two species-specific gene products are required for signal transduction by interferon gamma (IFN-gamma). The first is the IFN-gamma receptor, which binds ligand with high affinity in a species-specific manner. The second is an undetermined species-specific signal transducer(s). To determine whether the human IFN-gamma receptor (hIFN-gamma R) interacts directly with this signal transducer(s) and, if so, with what functional domain(s), we constructed expression vectors for the hIFN-gamma R and three hybrid human-murine IFN-gamma receptors. The hybrid receptors contained the extracellular, human IFN-gamma (hIFN-gamma) binding domain of the hIFN-gamma R, either the human or murine transmembrane domain, and either the human or murine intracellular domain. The vectors encoding these receptors were stably transfected into two mouse cell lines, one of which (SCC-16-5) contains a single copy of human chromosome 21. The resulting cell lines were treated with hIFN-gamma, and murine major histocompatibility complex class I antigen expression was analyzed by immunofluorescence flow cytometry. All transfected cell lines lacking human chromosome 21 remained insensitive to hIFN-gamma. However, all four of the IFN-gamma receptors were able to signal when expressed in the cell line containing human chromosome 21. We conclude that the extracellular domain of the IFN-gamma receptor is involved not only in the species specificity of IFN-gamma binding but also in signalling through interaction with an as yet unidentified species-specific factor(s) encoded by a gene(s) on human chromosome 21.

The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer.

At least two species-specific gene products are required for signal transduction by interferon gamma (IFN-gamma). The first is the IFN-gamma receptor, which binds ligand with high affinity in a species-specific manner. The second is an undetermined species-specific signal transducer(s). To determine whether the human IFN-gamma receptor (hIFN-gamma R) interacts directly with this signal transducer(s) and, if so, with what functional domain(s), we constructed expression vectors for the hIFN-gamma R and three hybrid human-murine IFN-gamma receptors. The hybrid receptors contained the extracellular, human IFN-gamma (hIFN-gamma) binding domain of the hIFN-gamma R, either the human or murine transmembrane domain, and either the human or murine intracellular domain. The vectors encoding these receptors were stably transfected into two mouse cell lines, one of which (SCC-16-5) contains a single copy of human chromosome 21. The resulting cell lines were treated with hIFN-gamma, and murine major histocompatibility complex class I antigen expression was analyzed by immunofluorescence flow cytometry. All transfected cell lines lacking human chromosome 21 remained insensitive to hIFN-gamma. However, all four of the IFN-gamma receptors were able to signal when expressed in the cell line containing human chromosome 21. We conclude that the extracellular domain of the IFN-gamma receptor is involved not only in the species specificity of IFN-gamma binding but also in signalling through interaction with an as yet unidentified species-specific factor(s) encoded by a gene(s) on human chromosome 21.

Induction of Cellular Senescence in Immortalized Cells by Human Chromosome 1

The control of cellular senescence by specific human chromosomes was examined in interspecies cell hybrids between diploid human fibroblasts and an immortal, Syrian hamster cell line. Most such hybrids exhibited a limited life span comparable to that of the human fibroblasts, indicating that cellular senescence is dominant in these hybrids. Karyotypic analyses of the hybrid clones that did not senesce revealed that all these clones had lost both copies of human chromosome 1, whereas all other human chromosomes were observed in at least some of the immortal hybrids. The application of selective pressure for retention of human chromosome 1 to the cell hybrids resulted in an increased percentage of hybrids that senesced. Further, the introduction of a single copy of human chromosome 1 to the hamster cells by microcell fusion caused typical signs of cellular senescence. Transfer of chromosome 11 had no effect on the growth of the cells. These findings indicate that human chromosome 1 may participate in the control of cellular senescence and further support a genetic basis for cellular senescence.

Tumorigenicity of Mouse-Human Diploid Hybrids in Nude Mice

Somatic cell hybrids between normal mouse cells and simian virus 40 (SV40)-transformed human cells, which contained a diploid complement of mouse chromosomes and the human chromosome 7 carrying the genome of SV40, were tumorigenic in nude mice. One single copy of human chromosome 7 per hybrid cell appeared to be sufficient for the tumorigenicity of the hybrids.