Biomedical subjects

T C Schulz

Publications and source records attributed to T C Schulz.

12 recordsLinked to original sources

PubMed2003

A new mouse model for Down syndrome.

Trisomy 21 (Ts21) is the most common live-born human aneuploidy and results in a constellation of features known as Down syndrome (DS). Ts21 is a frequent cause of congenital heart defects and the leading genetic cause of mental retardation. Although overexpression of a gene(s) or gene cluster on human chromosome 21 (Chr 21) or the genome imbalance by Ts21 has been suggested to play a key role in bringing about the diverse DS phenotypes, little is known about the molecular mechanisms underlying the various phenotypes associated with DS. Four approaches have been used to model DS to investigate the gene dosage effects of an extra copy of Chr 21 on various phenotypes; 1) Transgenic mice overexpressing a single gene from Chr 21, 2) YAC/BAC/PAC transgenic mice containing a single gene or genes on Chr 21, 3) Mice with intact/partial trisomy 16, a region with homology to human Chr 21 and 4) Human Chr 21 transchromosomal (Tc) mice. Here we review our new model system for the study of DS using the Tc technology, including the biological effects of an additional Chr 21 in vivo and in vitro.

Animals↗

PubMed2002

Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.

Formation and differentiation of a pluripotent cell population is central to mammalian development, and the isolation, identification and manipulation of human pluripotent cells is predicted to be of therapeutic use. Within the early mammalian embryo, two distinct populations of pluripotent cells have been described: the inner cell mass (ICM), which differentiates to form a second pluripotent cell populations, the primitive ectoderm. Indirect evidence suggests the existence of temporally distinct intermediate pluripotent cell populations as primitive ectoderm is formed. We coupled an in vitro model of primitive ectoderm formation (the transition of embryonic stem cells to early primitive ectoderm-like (EPL) cells) with ddPCR-based techniques to identify three novel genes, Psc1, CRTR-1 and PRCE, that were expressed differently during pluripotent cell progression. Detailed mapping of these genes with Oct4, Rex1 and Fgf5 on pregastrulation embryos provided the first molecular evidence for the existence of successive, temporally distinct pluripotent cell populations in the embryo between the ICM and primitive ectoderm. No evidence was found for spatial heterogeneity within the Oct4(+) pool. The transition between populations correlated with morphological or developmental alterations in pluripotent cells in vivo. Genes that are temporally expressed during pluripotent cell progression may provide an opportunity for molecular discrimination of pluripotent cells at different stages of maturation in vivo and an understanding of the cellular origins and properties of pluripotent cell lines isolated from diverse sources. Furthermore, the strong correlation of gene expression demonstrated between EPL cell formation in vitro and primitive ectoderm formation in vivo validates EPL cells as a model for primitive ectoderm, thereby providing a model system for the investigation of pluripotent differentiation and an opportunity for directed differentiation of pluripotent cells to therapeutically useful cell populations.

Amino Acid Sequence↗

PubMed2001

Histone variant H2A.Z is required for early mammalian development.

Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding [1]. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones [2]. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1 degrees have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function [3]. Here we show for the first time that a mammalian histone variant, H2A.Z, plays a critical role in early development, and we conclude that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development.

Animals↗

PubMed2001

Construction of 700 human/mouse A9 monochromosomal hybrids and analysis of imprinted genes on human chromosome 6.

As an in vitro assay system for the identification of human imprinted genes, a library of human/mouse A9 monochromosomal hybrids containing a single, intact bsr-tagged human chromosome of known parental origin, derived from normal human fibroblasts, has been previously generated by microcell-mediated chromosome transfer (MMCT). To supplement this assay system, we constructed additional 700 A9 monochromosomal hybrids, using a pSTneo or pPGKneo selection marker. To validate the A9 hybrids, we screened them with chromosome-specific polymorphic markers, and identified the hybrids containing either human chromosome 6, 7, 14, 18, or 21 of known parental origin. Matching paternal and maternal chromosome pairs of A9 hybrids were identified for chromosomes 6, 7, 14, and 18. The paternal-specific expression of ZAC (zinc finger protein, which regulates apoptosis and cell cycle arrest) and HYMAI (hydatidiform mole-associated and imprinted transcript), and the maternal-specific methylation of a CpG island within an imprinted domain on human chromosome 6q24, were maintained in A9 hybrids. For an example, we profiled the expression of expressed sequence tags (ESTs) and the methylation of CpG islands in the 300-kb imprinted domain around 6q24, which may be associated with cancers and transient neonatal diabetes mellitus (TNDM). Thus, the 700 A9 hybrids should be useful for various aspects of imprinting studies.

Animals↗

PubMed2000

Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome.

Human chromosome 11p15.5 harbors an intriguing imprinted gene cluster of 1 Mb. This imprinted domain is implicated in a wide variety of malignancies and Beckwith-Wiedemann syndrome (BWS). Recently, several lines of evidence have suggested that the BWS-associated imprinting cluster consists of separate chromosomal domains. We have previously identified LIT1, a paternally expressed antisense RNA within the KvLQT1 locus through a positional screening approach using human monochromosomal hybrids. KvLQT1 encompasses the translocation breakpoint cluster in BWS and patients exhibit frequent loss of maternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster. Here we generated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent with the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal alleles of multiple imprinted loci at the centromeric domain including KvLQT1 and p57(KIP2). The deletion had no effect on imprinting of H19 located at the telomeric end of the cluster. Our findings demonstrate that the LIT1 CpG island can act as a negative regulator in cis for coordinate imprinting at the centromeric domain, thereby suggesting a role for the LIT1 locus in a BWS pathway leading to functional inactivation of p57(KIP2). Thus, the targeting and precise modification of human chromosomal alleles using the DT40 cell shuttle system can be used to define regulatory elements that confer long-range control of gene activity within chromosomal domains.

Alleles↗

PubMed2000

Cellular senescence of a human bladder carcinoma cell line (JTC-32) induced by a normal chromosome 11.

Human chromosome 11 is expected to carry tumor suppressor genes for a variety of human cancers, including bladder carcinoma. To examine the functional role of a putative tumor suppressor gene(s) on this chromosome in the development of bladder carcinoma, we performed microcell-mediated transfer of chromosome 11 into the bladder carcinoma cell line, JTC-32. Fifteen of 20 colonies formed by the transfer experiment showed a remarkable change in cell morphology. They flattened and ceased growing, or senesced, prior to 10 population doublings. The presence of transferred chromosome 11-derived fragments in the growth-arrested cells was confirmed by PCR-based polymorphism analyses. The remaining 5 microcell hybrid clones exhibited a parental cell-like morphology, and presumably escaped from senescence, which was accompanied by deletions and/or rearrangements of the transferred chromosome 11. On the other hand, a transferred normal chromosome 7 neither changed the cell morphology nor arrested the cell growth. These results support the hypothesis that chromosome 11 contains a gene or genes which restore the senescence program lost during the immortalization process of JTC-32 cells.

Aged↗

PubMed2000

Multipoint imprinting analysis in sporadic colorectal cancers with and without microsatellite instability.

Disrupted imprinting is implicated in certain tumorigenesis. Since aberrant methylation has been described for a majority of microsatellite instability (MSI)-positive sporadic colorectal cancers, we have investigated alteration to the imprinting in 55 sporadic colorectal cancers with or without MSI. Loss of imprinting (LOI) of IGF2 and PEG1/MEST was observed in 42% and 35% of informative cancers, respectively. H19 expression was not detected in 24% of informative cancers. SNRPN and NDN retained monoallelic expression in all the cancers examined. These findings indicate no simultaneous disruption of the imprinted genes. LOI of IGF2 and PEG1/MEST was also observed in colorectal mucosa from almost all the patients with LOI in tumor tissue. Moreover, MSI-positive colorectal cancers exhibit LOI of IGF2 with a high frequency compared to MSI-negative cancers (P=0.013). These observations, consistent with a previous report, establish an association between LOI of IGF2 and MSI in colorectal cancers and provide insight into susceptibility of tumor development.

Alleles↗

PubMed1999

Mouse A9 cells containing single human chromosomes for analysis of genomic imprinting.

To develop an systematic in vitro approach for the study of genomic imprinting, we generated a new library of human/mouse A9 monochromosomal hybrids. We used whole cell fusion and microcell-mediated chromosome transfer to generate A9 hybrids containing a single, intact, bsr-tagged human chromosome derived from primary fibroblasts. A9 hybrids were identified that contained either human chromosome 1, 2, 4, 5, 7, 8, 10, 11, 15, 18, 20, or X. The parental origin of these chromosomes was determined by polymorphic analysis using microsatellite markers, and matched hybrids containing maternal and paternal chromosomes were identified for chromosomes 5, 10, 11 and 15. The imprinted gene KVLQT1 on human chromosome 11p15.5 was expressed exclusively from the maternal chromosome in A9 hybrids, and the parental-origin-specific expression patterns of several other imprinted genes were also maintained. This library of human monochromosomal hybrids is a valuable resource for the mapping and cloning of human genes and is a novel in vitro system for the screening of imprinted genes and for their functional analysis.

Animals↗

PubMed1999

LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids.

Mammalian imprinted genes are frequently arranged in clusters on particular chromosomes. The imprinting cluster on human chromosome 11p15 is associated with Beckwith-Wiedemann syndrome (BWS) and a variety of human cancers. To clarify the genomic organization of the imprinted cluster, an extensive screen for differentially expressed transcripts in the 11p15 region was performed using monochromosomal hybrids with a paternal or maternal human chromosome 11. Here we describe an imprinted antisense transcript identified within the KvLQT1 locus, which is associated with multiple balanced chromosomal rearrangements in BWS and an additional breakpoint in embryonal rhabdoid tumors. The transcript, called LIT1 (long QT intronic transcript 1), was expressed preferentially from the paternal allele and produced in most human tissues. Methylation analysis revealed that an intronic CpG island was specifically methylated on the silent maternal allele and that four of 13 BWS patients showed complete loss of maternal methylation at the CpG island, suggesting that antisense regulation is involved in the development of human disease. In addition, we found that eight of eight Wilms' tumors exhibited normal imprinting of LIT1 and five of five tumors displayed normal differential methylation at the intronic CpG island. This contrasts with five of six tumors showing loss of imprinting of IGF2. We conclude that the imprinted gene domain at the KvLQT1 locus is discordantly regulated in cancer from the imprinted domain at the IGF2 locus. Thus, this positional approach using human monochromosomal hybrids could contribute to the efficient identification of imprinted loci in humans.

Animals↗

PubMed1998

Epigenetic reprogramming of the human H19 gene in mouse embryonic cells does not erase the primary parental imprint.

BACKGROUND: Genomic imprinting in mammals is thought to result from epigenetic modifications to chromosomes during gametogenesis, which leads to differential allelic expression during development. There is a requirement for an appropriate experimental system to enable the analysis of the mechanisms of genomic imprinting during embryogenesis. RESULTS: To develop a novel in vitro system for studying the molecular basis of genomic imprinting, we constructed mouse cell lines containing either a paternal or maternal human chromosome 11, by microcell-mediated chromosome transfer. Allele-specific expression and DNA methylation studies revealed that the imprinting status of the human H19 gene was maintained in mouse A9 mono-chromosomal hybrids. Each parental human chromosome was introduced independently into mouse near-diploid immortal fibroblasts (m5S) and two embryonal carcinoma (EC) cell lines (OTF9-63 and P19). The paternal allele of human H19 remained in a repressed state in m5S cells, but was de-repressed in both EC cells. The paternal H19 allele was demethylated extensively in OTF9-63 cells, whereas the only alteration in P19 hybrids was de novo methylation on both alleles in the 3' region. Following in vitro differentiation, the expressed paternal H19 allele was selectively repressed in differentiated derivatives of EC hybrids. CONCLUSION: These results indicated that human imprint marks could function effectively in mouse cells, and that the imprinting process was epigenetically reprogrammed in embryonal carcinoma cells, without erasure of the primary imprint that marked the parental origin. Therefore, these mono-chromosomal hybrids could provide a valuable in vitro system to study the mechanisms involved in the regulation of imprinted gene expression.

Animals↗

PubMed1997

The mouse homeobox gene, Gbx2: genomic organization and expression in pluripotent cells in vitro and in vivo.

The Gbx2 homeodomain is widely conserved in metazoans. We investigated the mouse Gbx2 locus by isolation and characterization of genomic clones and by physical localization to the genome. The Gbx2 gene contained a single intron that separated the proposed functional protein domains. This organization was conserved with human GBX2. Physical localization of Gbx2 to Chromosome 1C5-E1 indicated that the genomic relationship between the linked Gbx2 and En1 genes differs between mouse and human, making it unlikely to be of functional significance. We also extended the known expression pattern of Gbx2 beyond the gastrulation stage embryo and the developing CNS to pluripotent cells in vitro and in vivo. Gbx2 expression was demonstrated in undifferentiated embryonic stem cells but was downregulated in differentiated cell populations. In the embryo, Gbx2 expression was detected before primitive streak formation, in the inner cell mass of the preimplantation embryo. Gbx2 is therefore a candidate control gene for cell pluripotency and differentiation in the embryo.

Amino Acid Sequence↗

PubMed1995

An unusual arrangement of 13 zinc fingers in the vertebrate gene Z13.

The zinc finger is a protein domain that imparts specific nucleic acid-binding activity on a wide range of functionally important proteins. In this paper we report the molecular cloning and characterization of a novel murine zinc-finger gene, mZ13. Analysis of mZ13 cDNAs revealed that the gene expresses a 794-amino-acid protein encoded by a 2.7 kb transcript. The protein has an unusual arrangement of 13 zinc fingers into a 'hand' of 12 tandem fingers and a single isolated finger near the C-terminus. This structural organization is conserved with the probable chicken homologue, cZ13. mZ13 also contained an additional domain at the N-terminus which has previously been implicated in the regulation of zinc-finger transcription factor DNA-binding, via protein-protein interactions. mZ13 expression was detected in a wide range of murine embryonic and adult tissues. The structural organization of mZ13 and its expression profile suggest that it may function as a housekeeping DNA-binding protein that regulates the expression of specific genes.

Amino Acid Sequence↗