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Faye M Rosin,
Naohide Watanabe,
Jean-Luc Cacas, Naohiro Kato,
Juana M Arroyo,
Yuda Fang,
Bruce May,
Matthew Vaughn,
Joseph Simorowski,
Umamaheswari Ramu,
Richard W McCombie,
David L Spector,
Robert A Martienssen,
Eric Lam
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ABSTRACT: The interphase nucleus exists as a highly dynamic system, the physical properties of which have functional importance in gene regulation. Not only can gene expression be influenced by the local sequence context, but also by the architecture of the nucleus in three-dimensions (3D), and by the interactions between these levels via chromatin modifications. A challenging task is to resolve the complex interplay between sequence- and genome structure-based control mechanisms. Here, we created a collection of 277 Arabidopsis lines that allow the visual tracking of individual loci in living plants while comparing gene expression potential at these locations, via an identical reporter cassette. Our studies revealed regional gene silencing near a heterochromatin island, via DNA methylation, that is correlated with mobility constraint and nucleolar association. We also found an example of nucleolar association that does not correlate with gene suppression, suggesting that distinct mechanisms exist that can mediate interactions between chromatin and the nucleolus. These studies demonstrate the utility of this novel resource in unifying structural and functional studies towards a more comprehensive model of how global chromatin organization may coordinate gene expression over large scales.
The Plant Journal 07/2008; 55(3):514-25. · 6.16 Impact Factor
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ABSTRACT: Fluorescent protein chromatin tagging as achieved by the lac operator/lac repressor system is useful to trace distinct chromatin domains in living eukaryotic nuclei. To interpret the data correctly, it is important to recognize influences of the tagging system on nuclear architecture of the host cells. Within an Arabidopsis line that carries lac operator/lac repressor/GFP transgenes, the transgene loci frequently associate with each other and with heterochromatic chromocenters. Accumulation of tagged fusion protein further enhances the association frequency. Independent experiments with a transgenic plant carrying another multi-copy transgene also revealed, independent of its transcriptional state, unusually high frequencies of association with each other and with heterochromatin. From these results we conclude that the lac operator/lac repressor chromatin tagging system may alter the spatial chromatin organization in the host nuclei (in particular when more than one insertion locus is present) and also that loci of homologous transgenic repeats associate more often with each other and with endogenous heterochromatin than normal euchromatic regions.
Journal of Cell Science 09/2005; 118(Pt 16):3751-8. · 6.11 Impact Factor
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ABSTRACT: In the pairing-site model, specialized regions on each chromosome function to establish meiotic homolog pairing. Analysis of these sites could provide insights into the mechanism used by Drosophila females to form a synaptonemal complex (SC) in the absence of meiotic recombination. These specialized sites were first established on the X chromosome by noting that there were barriers to crossover suppression caused by translocation heterozygotes. These sites were genetically mapped and proposed to be pairing sites. By comparing the cytological breakpoints of third chromosome translocations to their patterns of crossover suppression, we have mapped two sites on chromosome 3R. We have performed experiments to determine if these sites have a role in meiotic homolog pairing and the initiation of recombination. Translocation heterozygotes exhibit reduced gene conversion within the crossover-suppressed region, consistent with an effect on the initiation of meiotic recombination. To determine if homolog pairing is disrupted in translocation heterozygotes, we used fluorescent in situ hybridization to measure the extent of homolog pairing. In wild-type oocytes, homologs are paired along their entire lengths prior to accumulation of the SC protein C(3)G. Surprisingly, translocation heterozygotes exhibited homolog pairing similar to wild type within the crossover-suppressed regions. This result contrasted with our observations of c(3)G mutant females, which were found to be defective in pairing. We propose that each Drosophila chromosome is divided into several domains by specialized sites. These sites are not required for homolog pairing. Instead, the initiation of meiotic recombination requires continuity of the meiotic chromosome structure within each of these domains.
Genetics 03/2005; 169(2):767-81. · 4.01 Impact Factor
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ABSTRACT: With the rapid development of sequencing technologies in the past decade, many eukaryotic genomes have been resolved at the primary sequence level. However, organization of the genome within nuclei and the principles that govern such properties remain largely unclear. Optimization of fluorescence probe-based hybridization technologies combined with new advances in the instrumentation for microscopy has steadily yielded more structural information on chromosome organization in eukaryote model systems. These studies provide static snapshots of the detailed organization of chromatin. More recently, the successful application of a chromatin tagging strategy utilizing auto fluorescent fusion proteins opened a new era of chromatin studies in which the dynamic organization of the genome can be tracked in near real time. This review focuses on these new approaches to studying chromatin organization and dynamics in plants, and on future prospects in unraveling the basic principle of chromosome organization.
Annual review of plant biology 02/2004; 55:537-54. · 25.96 Impact Factor
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ABSTRACT: In the current model of chromatin organization in the interphase cell nucleus, chromosomes are organized into territories. Although constrained diffusion of chromatin in interphase cells has been confirmed in all cell types examined, little is known about chromatin dynamics in plant interphase cells. In this work, we measured for the first time interphase chromatin dynamics in plants using the green-fluorescent-protein-mediated chromatin-tagging system. Moreover, we compared the dynamics of diploid guard cells and endoreduplicated pavement cells. The movement of tagged loci in live seedlings shows constrained behavior in both types of nuclei. However, we found that the apparent confinement area for tagged loci in pavement cells is over 6 times larger than it is in guard cells. These findings suggest that chromatin is anchored to some component of the nucleus and that this might be responsible for the different dynamics of chromatin diffusion between diploid cells and endoreduplicated cells.
Journal of Cell Science 07/2003; 116(Pt 11):2195-201. · 6.11 Impact Factor
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Naohiro Kato
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ABSTRACT: The nucleus is the cellular organelle in which the bulk of the genomic information is stored. From studies using fluorescence microscopy with optical sections of fixed cells, a picture of an organized nuclear structure has emerged. Recently, the application of the green fluorescent protein (GFP) as a fluorescent dye allows the visualization of nuclear dynamics in live cells. Using four-dimensional fluorescence microscopy, the nuclear structures within an interphase nucleus are perceived to have dynamic domains. Structural analyses of a living plant nucleus contribute to our understanding of the genome information process in a particular cell in multicelluar systems.
Genetic engineering 02/2003; 25:65-90.
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ABSTRACT: AGAMOUS, a key player in floral morphogenesis, specifies reproductive organ identities and regulates the timely termination of stem cell fates in the floral meristem. Here, we report that strains carrying mutations in three genes, HUA1, HUA2, and HUA ENHANCER4 (HEN4), exhibit floral defects similar to those in agamous mutants: reproductive-to-perianth organ transformation and loss of floral determinacy. HEN4 codes for a K homology (KH) domain-containing, putative RNA binding protein that interacts with HUA1, a CCCH zinc finger RNA binding protein in the nucleus. We show that HUA1 binds AGAMOUS pre-mRNA in vitro and that HEN4, HUA1, and HUA2 act in floral morphogenesis by specifically promoting the processing of AGAMOUS pre-mRNA. Our studies underscore the importance of RNA processing in modulating plant development.
Developmental Cell 02/2003; 4(1):53-66. · 14.03 Impact Factor
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ABSTRACT: Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.
Genetics 10/2002; 162(1):245-58. · 4.01 Impact Factor
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ABSTRACT: The control of subcellular localization of proteins and their interaction with other partners in vivo are important parameters that provide clues to their function and regulation. The ability to simultaneously track multiple protein species with high resolution should provide a valuable assay system to study and characterize various types of posttranslational control pathways. In this work, we established the system and a method involving "spectral profiling" for the resolution of four different fluorescent protein tags in the same viewing field using digital imaging technology. With these techniques, we have (a) developed new derivatives of mGFP5, which is commonly used in the plant field, that are about three times brighter; (b) demonstrated that four spectrally distinct fluorescent proteins (cyan, green, yellow, and red) that are fused to a transcription factor could be stably expressed in nuclei and distinguished in tobacco (Nicotiana tabacum) mesophyll cells; and (c) shown that interaction between partners of a dimeric transcription factor can be detected by measuring fluorescence resonance energy transfer. These technologies should help one to study protein-protein interactions efficiently, especially for nuclear proteins under in vivo conditions.
Plant physiology 08/2002; 129(3):931-42. · 6.53 Impact Factor
