Biomedical subjects

H Leonhardt

Publications and source records attributed to H Leonhardt.

At least 19 recordsLinked to original sources

PubMed2000

Dynamics of DNA replication factories in living cells.

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.

Animals↗

PubMed2000

Structure and function of the mouse DNA methyltransferase gene: Dnmt1 shows a tripartite structure.

Dnmt1 is the predominant DNA methyltransferase (MTase) in mammals. The C-terminal domain of Dnmt1 clearly shares sequence similarity with many prokaryotic 5mC methyltransferases, and had been proposed to be sufficient for catalytic activity. We show here by deletion analysis that the C-terminal domain alone is not sufficient for methylating activity, but that a large part of the N-terminal domain is required in addition. Since this complex structure of Dnmt1 raises issues about its evolutionary origin, we have compared several eukaryotic MTases and have determined the genomic organization of the mouse Dnmt1 gene. The 5' most part of the N-terminal domain is dispensible for enzyme activity, includes the major nuclear import signal and comprises tissue-specific exons. Interestingly, the functional subdivision of Dnmt1 correlates well with the structure of the Dnmt1 gene in terms of intron/exon size distribution as well as sequence conservation. Our results, based on functional, structural and sequence comparison data, suggest that the gene has evolved from the fusion of at least three genes.

Amino Acid Motifs↗

PubMed2000

DNA methylation, nuclear structure, gene expression and cancer.

DNA methylation, chromatin structure, transcription, and cancer have traditionally been studied as separate phenomena. Recent data provide now direct physical and functional links between these processes revealing a complex network of interactions and mutual dependences. Methylated DNA is bound by methyl-CpG binding protein (MeCP) complexes that include histone deacetylases (HDACs). This recruitment of HDACs is suggested to promote local chromatin condensation and thereby repress gene expression. Most recently, also complexes of DNA methyltransferase (Dnmt1) with transcriptional repressors, DMAP1 and pRB, have been described providing a direct link to transcriptional regulation and tumor suppression. Inactivation of the DNA methyltransferase genes (Dnmt1, 3a, and 3b) was found to be lethal in mice and several human diseases (ICF and Rett syndrome) turned out to be linked to DNA methylation. In particular, global hypomethylation has been found in tumor samples together with cancer-type-specific, local hypermethylation. Taken together, these lines of evidence clearly underscore the central role of DNA methylation in the regulation of gene expression and chromatin structure during normal development and diseases like cancer. J. Cell. Biochem. Suppl. 35:78-83, 2000.

Animals↗

PubMed2000

Expression of an alternative Dnmt1 isoform during muscle differentiation.

The methylation pattern of genomic DNA undergoes dramatic changes during mammalian development, with extensive de novo methylation occurring during gametogenesis and after implantation. We identified an alternative Dnmt1 transcript in skeletal muscle by Northern blot analysis and cloned the corresponding cDNA by rapid amplification of cDNA ends and reverse transcription-PCR. Using an in vitro skeletal muscle differentiation system, we show that this alternative Dnmt1 isoform is specifically expressed in differentiated myotubes, whereas the ubiquitously expressed isoform is down-regulated during myogenesis. Sequence analysis showed that this skeletal Dnmt1 isoform is identical to the one present in testis, which had been described as untranslatable. Here we present evidence that this alternative Dnmt1 transcript present in testis and skeletal muscle is translated despite the presence of several out-of-frame upstream ATGs and gives rise to a shorter Dnmt1 isoform, which could play an active role in the change of DNA methylation patterns during gametogenesis and myogenesis.

Alternative Splicing↗

PubMed2000

Targeting regulatory factors to intranuclear replication sites.

Plenty of evidence exists that mammalian nuclei are highly organized. Complex biochemical processes like DNA replication take place at specialized subnuclear sites and proteins directly or indirectly involved are concentrated at these sites. DNA replication is being used as a paradigm to study this functional organization of the nucleus, its underlying principles, and its potential regulatory consequences. In this review we discuss which factors were shown to be localized at nuclear replication sites, how they get there, and what role this might play in the precise, genome-wide regulation and coordination of complex biochemical processes.

Animals↗

PubMed1999

DNA methyltransferase is actively retained in the cytoplasm during early development.

The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.

Alternative Splicing↗

PubMed1999

A mammalian myocardial cell-free system to study cell cycle reentry in terminally differentiated cardiomyocytes.

Cardiomyocytes withdraw from the cell cycle in the early neonatal period, rendering the adult heart incapable to regenerate after injury. In the present study, we report the establishment of a cell-free system to investigate the control of cell cycle reentry in mammalian ventricular cardiomyocyte nuclei and to specifically address the question of whether nuclei from terminally differentiated cardiomyocytes can be stimulated to reenter S phase when incubated with extracts from S-phase cells. Immobilized cardiomyocyte nuclei were incubated with nuclei and cytoplasmic extract of synchronized H9c2 muscle cells or cardiac nonmyocytes. Ongoing DNA synthesis was monitored by biotin-16-dUTP incorporation as well as proliferating cell nuclear antigen expression and localization. Nuclei and cytoplasmic extract from S-phase H9c2 cells but not from H9c2 myotubes induced DNA synthesis in 92% of neonatal cardiomyocyte nuclei. Coincubation in the presence of cycloheximide indicated that de novo translation is required for the reinduction of S phase. Similar results were obtained with adult cardiomyocyte nuclei. When coincubated with both cytoplasmic extract and nuclei or nuclear extracts of S-phase cells, >70% of adult cardiomyocyte nuclei underwent DNA synthesis. In conclusion, these results demonstrate that postmitotic ventricular myocyte nuclei are responsive to stimuli derived from S-phase cells and can thus bypass the cell cycle block. This cell-free system now makes it feasible to analyze the molecular requirements for the release of the cell cycle block and will help to engineer strategies for regenerative growth in cardiac muscle.

Animals↗

PubMed1999

Structure and function in the nucleus: subnuclear trafficking of DNA replication factors.

The traditional view of the eukaryotic cell nucleus as a more or less amorphous milieu in which proteins and nucleic acids are freely floating has been challenged by an ever increasing number of reports uncovering highly organized structures where biological processes are concentrated together with their corresponding factors. The identification and utilization of protein domains that are necessary and sufficient for targeting to different subnuclear compartments have begun to elucidate the molecular principles underlying this structural organization and its dynamic behavior. The combination of biochemical, cell biology, and biophysical approaches to study nuclear structure and function should help to elucidate how these higher-order structures organize and coordinate countless enzymatic activities in time and space within the mammalian nucleus. J. Cell Biochem. Suppls. 32/33:15-23, 1999.

Animals↗

PubMed1999

A novel isoform of the smooth muscle cell differentiation marker smoothelin.

Studies on smooth muscle cell differentiation and those on vascular development in mouse and humans have long been hampered by the lack of suitable markers. Here we describe a novel, large isoform of smoothelin, a structural protein of differentiated, contractile smooth muscle cells. The protein, which is highly conserved in mouse and humans, shows homology with other cytoskeleton-associated smooth muscle cell proteins and contains an actinin-type actin-binding domain. Northern blot analysis from various mouse organs identified short and long smoothelin mRNA forms, which exhibit distinct tissue expression patterns. The short form is highly expressed in visceral muscle tissues such as intestine and stomach and is not detectable in brain, while the long mRNA form is expressed in all vascularized organs. These results may provide new tools and approaches to study both smooth muscle cell differentiation and proliferative vascular disease.

Actinin↗

PubMed1999

Direct protein transfer to terminally differentiated muscle cells.

Recently, a new approach for direct protein transfer to mammalian cells based on the herpes simplex virus type 1 protein VP22 has been described. This protein has the remarkable property of intercellular trafficking, which is independent of direct cell contacts and is also retained when fused to heterologous proteins. However, the spreading has only been described for proliferating cells and has also been controversially discussed. In this study we describe the generation of a GFP-VP22 fusion protein which is able to spread in COS-7 cells after transient transfection. Moreover, we show in coculture experiments with transfected COS-7 cells and C2C12 myotubes that this fusion protein is also able to spread into terminally differentiated skeletal muscle cells. These results suggest that VP22 might be a novel therapeutic tool for direct protein transfer not only in proliferating but also in terminally differentiated cells.

Animals↗

PubMed1999

Functional links between nuclear structure, gene expression, DNA replication, and methylation.

Over the last decades it became clear that mammalian nuclei are highly organized. Nuclear processes like DNA replication and RNA metabolism take place in distinct subnuclear foci, which are enriched for enzymes involved in the corresponding biochemical reactions. This colocalization of functions with their respective factors is often referred to as functional organization of the nucleus. This organization is achieved by assembly of different enzymes and regulatory factors into high-molecular-weight complexes that are tethered to insoluble nuclear structures. Recently, several links between nuclear structure, gene expression, DNA replication, and methylation have been described that illustrate the interrelation of higher-order structures and nuclear functions. New insights into the functional organization of the nucleus and how it could explain the high precision and overall coordination of nuclear processes are discussed.

Animals↗

PubMed1998

A short DNA methyltransferase isoform restores methylation in vivo.

Two murine DNA methyltransferase isoforms (MTases) have been observed, a longer form in somatic and embryonic stem (ES) cells and a shorter form in oocytes and preimplantation embryos. While the longer MTase is associated with maintenance methyltransferase activity in replicating cells, little is known about the shorter form. We present genetic and biochemical evidence that both isoforms are expressed from the same Dnmt1 gene by using different translation initiation sites in exons 1 and 4. We further demonstrate that the shorter isoform can functionally rescue Dnmt1 null ES cells that have a hypomethylated genome. These rescued ES cells differentiate in vivo into a variety of cell types, unlike the Dnmt1 null ES cells that die upon induction of differentiation. These results show that the shorter isoform can substitute for the longer maintenance MTase in ES and differentiated cells. Our data further indicate that the shorter MTase isoform found in oocytes is fully functional in vivo and may play an active role in the regulation of DNA methylation and the establishment of imprinting patterns.

Amino Acid Sequence↗

PubMed1998

Protein targeting to subnuclear higher order structures: a new level of regulation and coordination of nuclear processes.

Though there are no separating membranes within the nucleus, different factors are often concentrated at sites where their respective function is required, a phenomenon referred to as functional organization of the nucleus. How is then this organization achieved and how are the different metabolic processes integrated in the nucleus? One emerging principle was revealed by the identification of protein domains that, though not involved in catalysis, regulate enzyme activity at a higher order level by targeting enzymes to the right place at the right time. These targeting sequences constitute an assembly code for nuclear 'protein factories,' which ensure the extremely high efficiency and accuracy needed in a complex and competitive environment as the living mammalian cell.

Animals↗

PubMed1998

Intranuclear targeting of DNA replication factors.

Mammalian nuclei are highly organized into functional compartments. Major nuclear processes like DNA replication and RNA processing take place in distinct foci. These microscopically visible foci are formed by the assembly of, for example, DNA replication factors and associated proteins into megadalton complexes often referred to as protein machines or factories. Thus far, two proteins, DNA ligase I and DNA methyltransferase (DNA MTase), have been analyzed in greater detail. In both cases, the assembly process appears to be controlled by distinct targeting sequences that were attached to the catalytic protein core in the course of evolution and mediate the association with replication factories in mammalian cells. The dynamics of these nuclear structures throughout the cell cycle are analyzed using green fluorescent protein (GFP). Further studies are needed to elucidate the architecture, regulation, and role of these subnuclear structures.

Animals↗

PubMed1997

Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo.

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

Amino Acid Sequence↗

PubMed1996

Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene.

Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS/PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.

Amino Acid Sequence↗

PubMed1995

Targeting and association of proteins with functional domains in the nucleus: the insoluble solution.

The mammalian nucleus is highly organized into distinct functional domains separating different biochemical processes such as transcription, RNA processing, DNA synthesis, and ribosome assembly. A number of proteins known to participate in these processes were found to be specifically localized at their corresponding functional domains. A distinct targeting sequence, necessary and sufficient for the localization to DNA replication foci, was identified in the N-terminal, regulatory domain of DNA methyltransferase and DNA ligase I and might play a role in the coordination of DNA replication and DNA methylation. The fact that the targeting sequence is absent in lower eukaryotic and prokaryotic DNA ligase I homologs suggests that "targeting" is a rather recent development in evolution. Finally, targeting sequences have also been identified in some splicing factors and in viral proteins, which are responsible for their localization to the speckled compartment and to the nucleolus, respectively. These higher levels of organization are likely to contribute to the regulation and coordination of the complex and interdependent biochemical processes in the mammalian nucleus.

Animals↗

PubMed1993

[Eosinophilic fasciitis (Shulman syndrome) as a rare differential diagnosis of sclerodermiform skin changes].

For 3 months, a 70-year-old woman had been affected by painless, symmetrical swellings in the limbs, spreading proximally, causing hardening of the skin and impaired joint movements, but no fever. Her general condition and findings on physical examination were according to age. ESR was 22/42 mm and there was a thrombocytosis (533,000/microliters) and eosinophilia (16%, absolute 2,140/microliters). Radiological and ultrasound examinations were normal. Skin biopsy revealed marked oedema of the connective tissue septa and perivascular lymphohistiocytic infiltrations, which--together with the other findings--suggested eosinophilic fasciitis. She was treated with prednisone, 100 mg daily, reduced gradually to 8 mg daily, as well as cimetidine, 800 mg daily, as immunomodulator, and intensive physiotherapy. The swellings and eosinophilia regressed within 8 weeks, but the hardening and impaired mobility persisted. Maintenance glucocorticoids are being continued.

Aged↗