Monozygotic (identical) twins have been widely used in genetic studies to determine the relative contributions of heredity and the environment in human diseases. Discordance in disease manifestation between affected monozygotic twins has been attributed to either environmental factors or different patterns of X chromosome inactivation (XCI). However, recent studies have identified genetic and epigenetic differences between monozygotic twins, thereby challenging the accepted experimental model for distinguishing the effects of nature and nurture. Here, we report the genomic and epigenomic sequences in skin fibroblasts of a discordant monozygotic twin pair with Rett syndrome, an X-linked neurodevelopmental disorder characterized by autistic features, epileptic seizures, gait ataxia and stereotypical hand movements. The twins shared the same de novo mutation in exon 4 of the MECP2 gene (G269AfsX288), which was paternal in origin and occurred during spermatogenesis. The XCI patterns in the twins did not differ in lymphocytes, skin fibroblasts, and hair cells (which originate from ectoderm as does neuronal tissue). No reproducible differences were detected between the twins in single nucleotide polymorphisms (SNPs), insertion-deletion polymorphisms (indels), or copy number variations. Differences in DNA methylation between the twins were detected in fibroblasts in the upstream regions of genes involved in brain function and skeletal tissues such as Mohawk Homeobox (MKX), Brain-type Creatine Kinase (CKB), and FYN Tyrosine Kinase Protooncogene (FYN). The level of methylation in these upstream regions was inversely correlated with the level of gene expression. Thus, differences in DNA methylation patterns likely underlie the discordance in Rett phenotypes between the twins.
Although the etiology of autism remains largely unknown, cytogenetic and genetic studies have implicated maternal copy number gains of 15q11–q13 in 1–3% of autism cases. In order to understand how maternal 15q duplication leads to dysregulation of gene expression and altered chromatin interactions, we used microcell-mediated chromosome transfer to generate a novel maternal 15q duplication model in a human neuronal cell line. Our 15q duplication neuronal model revealed that by quantitative RT–PCR, transcript levels of NDN, SNRPN, GABRB3 and CHRNA7 were reduced compared with expected levels despite having no detectable alteration in promoter DNA methylation. Since 15q11–q13 alleles have been previously shown to exhibit homologous pairing in mature human neurons, we assessed homologous pairing of 15q11–q13 by fluorescence in situ hybridization. Homologous pairing of 15q11–q13 was significantly disrupted by 15q duplication. To further understand the extent and mechanism of 15q11–q13 homologous pairing, we mapped the minimal region of homologous pairing to a ∼500 kb region at the 3′ end of GABRB3 which contains multiple binding sites for chromatin regulators MeCP2 and CTCF. Both active transcription and the chromatin factors MeCP2 and CTCF are required for the homologous pairing of 15q11–q13 during neuronal maturational differentiation. These data support a model where 15q11–q13 genes are regulated epigenetically at the level of both inter- and intra-chromosomal associations and that chromosome imbalance disrupts the epigenetic regulation of genes in 15q11–q13.
Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.
Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37–47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.
A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells) by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression.
Type-2 diabetes results from the development of insulin resistance and a concomitant impairment of insulin secretion. Bone morphogenetic protein 4 (Bmp4)-Bmp receptor 1A signaling in β cells has recently been reported to be required for insulin production and secretion. In addition, Bmp4 blocks the differentiation and promotes the expansion of endocrine progenitor cells. Bmp4 therefore regulates the maintenance of homeostasis in the pancreas. In this study, we constructed a reporter plasmid carrying 7-kb enhancer and promoter region of the Bmp4 gene upstream of the firefly luciferase gene. We used this construct to produce transgenic mice by pro-nuclear microinjection, for subsequent in vivo monitoring of Bmp4 expression. The bioluminescent signal was detected mainly in the pancreas in three independent lines of transgenic mice. Furthermore, the bioluminescent signal was enhanced in association with the autophagy response to 24-h fasting. These results suggest that pancreatic expression of Bmp4 is involved in responding to the physiological environment, including through autophagy. These mouse models represent useful tools for toxicological screening, and for investigating the mechanisms responsible for pancreatic Bmp4 functions in vivo, with relevance to improving our understanding of pancreatic diseases.
The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.
Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoidtet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.
human artificial chromosome; conditional centromere; gene delivery
Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human chromosome 3, via microcell-mediated chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated chromosome 3 regions produced by a novel chromosomal engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.
Microcell-mediated chromosome transfer (MMCT) is a technique by which a chromosome(s) is moved from donor to recipient cells by microcell fusion. Polyethylene glycol (PEG) has conventionally been used as a fusogen, and has been very successful in various genetic studies. However, PEG is not applicable for all types of recipient cells, because of its cell type-dependent toxicity. The cytotoxicity of PEG limits the yield of microcell hybrids to low level (10-6 to 10-5 per recipient cells). To harness the full potential of MMCT, a less toxic and more efficient fusion protocol that can be easily manipulated needs to be developed.
Microcell donor CHO cells carrying a human artificial chromosome (HAC) were transfected with genes encoding hemagglutinin (H) and fusion (F) proteins of an attenuated Measles Virus (MV) Edmonston strain. Mixed culture of the CHO transfectants and MV infection-competent human fibrosarcoma cells (HT1080) formed multinucleated syncytia, suggesting the functional expression of the MV-H/F in the CHO cells. Microcells were prepared and applied to HT1080 cells, human immortalized mesenchymal stem cells (hiMSC), and primary fibroblasts. Drug-resistant cells appeared after selection in culture with Blasticidin targeted against the tagged selection marker gene on the HAC. The fusion efficiency was determined by counting the total number of stable clones obtained in each experiment. Retention of the HAC in the microcell hybrids was confirmed by FISH analyses. The three recipient cell lines displayed distinct fusion efficiencies that depended on the cell-surface expression level of CD46, which acts as a receptor for MV. In HT1080 and hiMSC, the maximum efficiency observed was 50 and 100 times greater than that using conventional PEG fusion, respectively. However, the low efficiency of PEG-induced fusion with HFL1 was not improved by the MV fusogen.
Ectopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.
Genomic imprinting is widely conserved amongst placental mammals. Imprinted expression of IGF2R, however, differs between mice and humans. In mice, Igf2r imprinted expression is seen in all fetal and adult tissues. In humans, adult tissues lack IGF2R imprinted expression, but it is found in fetal tissues and Wilms' tumors where it is polymorphic and only seen in a small proportion of tested samples. Mouse Igf2r imprinted expression is controlled by the Air (Airn) ncRNA whose promoter lies in an intronic maternally-methylated CpG island. The human IGF2R gene carries a homologous intronic maternally-methylated CpG island of unknown function. Here, we use transfection and transgenic studies to show that the human IGF2R intronic CpG island is a ncRNA promoter. We also identify the same ncRNA at the endogenous human locus in 16–40% of Wilms' tumors. Thus, the human IGF2R gene shows evolutionary conservation of key features that control imprinted expression in the mouse.
Genomic imprinting; IGF2R; AIR (AIRN) ncRNA; Wilms' tumor
Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individual's own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.
Microsatellite instability (MSI) is a hallmark of mismatch repair deficiency. High levels of MSI at mono- and dinucleotide repeats in colorectal cancer (CRC) are attributed to inactivation of the mismatch repair genes, hMLH1 and hMSH2. CRC with low levels of MSI (MSI-L) exists; however its molecular basis is unclear. There is another type of MSI - “elevated microsatellite alterations at selected tetranucleotide repeats” - (EMAST) where loci containing [AAAG]n or [ATAG]n repeats are unstable. EMAST is frequent in non-colorectal cancers; however the incidence of EMAST and its cause in CRC is not known. Here, we report that MSH3-knock-down or MSH3-deficient cells exhibit the EMAST phenotype and low levels of mutations at dinucleotide repeats. About 60% of 117 sporadic CRC cases exhibit EMAST. All of the cases defined as MSI-H (16 cases) exhibited high levels of EMAST. Among 101 non-MSI-H cases, all 19 cases of MSI-L and 35 of 82 cases of MSS exhibited EMAST. Although non-MSI-H CRC tissues contained MSH3-negative tumor cells ranging from 2-50% of the total tumor cell population, the tissues exhibiting EMAST contained more MSH3-negative cells (average 31.5%) than did the tissues not exhibiting EMAST (8.4%). Taken together, our results support the idea that MSH3-deficiency causes EMAST or EMAST with low levels of MSI at the loci with dinucleotide repeats in CRC.
Microsatellite Instability; MSH3; Colorectal Cancer; DNA Mismatch Repair; Elevated Microsatellite Alterations at Selected Tetranucleotide Repeats (EMAST)
The CTCF paralog BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein thought to play a role in chromatin organization and gene expression. Under normal physiologic conditions, BORIS is predominantly expressed during embryonic male germ cell development; however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or cancer-testis gene. It has been suggested that BORIS may be a pro-proliferative factor, whereas CTCF favors antiproliferation. BORIS and CTCF share similar zinc finger DNA-binding domains and seem to bind to identical target sequences. Thus, one critical question is the mechanism governing the DNA-binding specificity of these two proteins when both are present in tumor cells. Chromatin immunoprecipitation (ChIP) in HCT116 cells and their hypermethylated variant showed that BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using both whole-cell extracts and in vitro translated CTCF and BORIS protein, and methylation-specific ChIP PCR showed that BORIS is a methylation-independent DNA-binding protein. Finally, experiments in murine hybrid cells containing either the maternal or paternal human chromosome 11 showed that BORIS preferentially binds to the methylated paternal H19 differentially methylated region, suggesting a mechanism in which the affinity of CTCF for the unmethylated maternal allele directs the DNA binding of BORIS toward the paternal allele.
Glioma includes astrocytoma, oligodendroglioma, ependymoma and glioblastoma. We previously reported the epigenetic silencing of paternally expressed gene 3 (PEG3) in glioma cell lines. In this study, we investigated methylation of an exonic CpG island in the promoter region and the expression of PEG3 gene in 20 glioma and 5 non-tumor tissue samples. We found wide variations in the methylation level. Hypomethylaiton and hypermethylation was found in 3 and 4 glioma tissue samples, respectively. Monoallelic expression, which is an evidence of an imprinted gene, was maintained in eight out of nine informative cases which have T/C polymorphisms in PEG3. The lower gene expression, which suggested epigenetic silencing of PEG3, was confirmed statistically in glioblastoma using quantitative reverse-transcription polymerase chain reaction. Interestingly, we found higher expression of PEG3 in two out of three oligodendrogliomas. A negative correlation between the methylation level and gene expression was shown by regression analysis. These results suggest that the abnormal regulation of PEG3 is associated with several glioma subtypes and that it plays an important role in tumorigenesis.
glioma; PEG3; DNA methylation; epigenetic silencing
We attempted to clone candidate genes on 10p14–15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid.
RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells.
These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT.
Loss of genomic imprinting is involved in a number of developmental abnormalities and cancers. ZAC is an imprinted gene expressed from the paternal allele of chromosome 6q24 within a region known to harbor a tumor suppressor gene for several types of neoplasia. p57KIP2 (CDKN1C) is a maternally expressed gene located on chromosome 11p15.5 which encodes a cyclin-dependent kinase inhibitor that may also act as a tumor suppressor gene. Mutations in ZAC and p57KIP2 have been implicated in transient neonatal diabetes mellitus (TNDB) and Beckwith–Wiedemann syndrome, respectively. Patients with these diseases share many characteristics. Here we show that mouse Zac1 and p57Kip2 have a strikingly similar expression pattern. ZAC, a sequence-specific DNA-binding protein, binds within the CpG island of LIT1 (KCNQ1OT1), a paternally expressed, anti-sense RNA thought to negatively regulate p57KIP2 in cis. ZAC induces LIT1 transcription in a methylation-dependent manner. Our data suggest that ZAC may regulate p57KIP2 through LIT1, forming part of a novel signaling pathway regulating cell growth. Mutations in ZAC may, therefore, contribute to Beckwith–Wiedemann syndrome. Furthermore, we find changes in DNA methylation at the LIT1 putative imprinting control region in two patients with TNDB.
Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER−/− cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.