Regular ArticleOrganization of Centromeric Domains in Hepatocyte Nuclei: Rearrangement Associated with De Novo Activation of the Vitellogenin Gene Family in Xenopus laevis
Abstract
The existence of a function-dependent, nonrandom organization of chromatin domains within interphase nuclei is supported by evidence which suggests that specific chromatin domains undergo spatial rearrangement under conditions which alter gene expression. Exposure to estrogen of male Xenopus laevis hepatocytes in vitro results in de novo activation of vitellogenin mRNA production and vitellogenin protein synthesis and provides an ideal model to study the association between chromatin organization and changes in gene expression. In a test of the hypothesis that the de novo induction of vitellogenesis in male X. laevis is associated with a spatial rearrangement of specific chromatin domains, centromeric regions were localized by immunofluorescent labeling of associated kinetochore proteins in naive and in estrogen-treated, vitellogenic cells. Analyses by confocal scanning laser microscopy of the three-dimensional spatial distribution of kinetochores in estrogen-treated male hepatocytes showed that a significantly greater proportion of signals was associated with the nuclear periphery than in non-estrogen-treated, naive male cells. In hepatocyte nuclei, quantification of kinetochore signal sizes using image analysis showed that these signals were fewer in number and showed greater variation in size than those of cells in metaphase, with larger signals exhibiting total normalized fluorescence intensities of two, three, four, and five times that associated with kinetochore signals of metaphase cells. These observations are taken to reflect the existence of clustering of kinetochores and, by extension, of centromeres in these cells. In summary, the results show that centromeric domains within interphase nuclei of Xenopus hepatocytes occur as clusters and that these domains undergo spatial rearrangement under conditions which alter the transcriptional state of the cell.
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Evidence for a functional interaction between the ClC-2 chloride channel and the retrograde motor dynein complex
2003, Journal of Biological ChemistryCitation Excerpt :Optical sections were generally 0.7–0.9 μm thick. The extent of co-localization of ClC-2 with EEA1 was quantified using a modification of methods described previously (44, 45). Briefly, background-corrected EEA1-specific immunofluorescence was subtracted from ClC-2-specific immunofluorescence to yield a “difference” image (using Scion Image; Scion Corporation).
The ClC-2 chloride channel has been implicated in essential physiological functions. Analyses of ClC-2 knock-out mice suggest that ClC-2 expression in retinal pigment epithelia and Sertoli cells normally supports the viability of photoreceptor cells and male germ cells, respectively. Further, other studies suggest that ClC-2 expression in neurons may modify inhibitory synaptic transmission via the γ-aminobutyric acid, type A receptor. However, complete understanding of the physiological functions of ClC-2 requires elucidation of the molecular basis for its regulation. Using cell imaging and biochemical and electrophysiological techniques, we show that expression of ClC-2 at the cell surface may be regulated via an interaction with the dynein motor complex. Mass spectrometry and Western blot analysis of eluate from a ClC-2 affinity matrix showed that heavy and intermediate chains of dynein bind ClC-2in vitro. The dynein intermediate chain co-immunoprecipitates with ClC-2 from hippocampal membranes suggesting that they also interact in vivo. Disruption of dynein motor function perturbs ClC-2 localization and increases the functional expression of ClC-2 in the plasma membranes of COS7 cells. Thus, cell surface expression of ClC-2 may be regulated by dynein motor activity. This work is the first to demonstrate an in vivointeraction between an ion channel and the dynein motor complex.
Human satellite 3 (HS3) binding protein from the nuclear matrix: Isolation and binding properties
2000, Biochimica et Biophysica Acta - Molecular Cell ResearchSatellite DNA (satDNA) is the main component of residual DNA in nuclear matrix (NM) preparations. Gel mobility shift assay (GMSA) revealed specific human satellite 3 (HS3) binding activity in NM extracts. An HS3 binding protein was purified using diethylaminoethyl (DEAE)-cellulose and preparative GMSA. The binding was specific, although other satDNA fragments compete to some extent for the binding. DNase I footprinting and methylation interference revealed multiple points of protection distributed throughout the HS3 fragment with periodicity of about 10 bp, mostly inside an AT island. Polyclonal antibodies (AB) were raised against HS3-protein complexes cut from the preparative GMSA gel. On immunoblots, AB recognise a protein, which is not lamin, with apparent molecular mass 70 kDa, the same as revealed by purification (p70). In in situ nuclear matrix preparations combined immunofluorescence (AB) and fluorescent in situ hybridisation (HS3) shows that HS3 and p70 areas correspond to each other. The localisation of this protein detected with AB in interphase nuclei coincides with the heterochromatic regions which surround nucleoli in correspondence with the known HS3 position in the nuclei.
Nuclear topology of murine, cerebellar Purkinje neurons: Changes as a function of development
2000, Experimental Cell ResearchThe interphase nucleus is a structurally ordered, three-dimensional structure, in which specific chromatin domains occupy distinct spatial positions that can, in turn, be modified with changes in cell function. A fundamental goal in developmental neurobiology is the identification of mechanisms that dictate the orderly expression of genes in a cell-specific manner. Given that different neuronal populations feature a characteristic spatial topology of centromeric sequences, the positioning of specific DNA sequences may constitute such a mechanism. We tested the hypothesis that the cell-specific nuclear topology in fully differentiated neurons is acquired before or during that stage at which neuron-specific sequences are first expressed. For this, we assessed the number and spatial distribution of centromeric domains in the murine, cerebellar Purkinje neuron as a function of postnatal development. Centromeric domains were localized by immunofluorescence of centromere-associated kinetochore proteins and visualized by confocal microscopy. Kinetochores are known to cluster in Purkinje neurons. Thus, the number of signals discerned is always less than the chromosome complement of the species. The number of signals observed in adults (10.8 ± 0.46) (mean ± SEM) is established by postnatal day 15 (P15), after a transient decrease from 11.44 ± 0.44 at P0 to 8.78 ± 0.24 at P3. The distribution of signals characteristic of the adult, with the majority located at the nucleolus, is established by P5 and is associated with a decrease in the fraction of signals at the nuclear periphery. These changes are temporally associated with the onset of processes such as dendritic differentiation and synaptic maturation and might serve the process of differentiation by placing specific sequences into transcriptionally competent, nuclear sites.
Dynamics of structure-function relationships in interphase nuclei
1999, Life SciencesThe interphase nucleus is a topologically ordered, three-dimensional structure. While it remains unclear whether this structural organization also represents compartmentalization of function, the presence of the latter would likely be reflected in the spatial coupling of molecular factors involved in related events. This review summarizes morphological evidence, derived from in situ experiments, which indicates the existence of compartmentalization of both chromatin and non-chromatin components in the interphase nucleus. Moreover, the review addresses the spatial relationships of these components relative to each other and correlates these spatial relationships with such nuclear functions as transcription, splicing and nucleo-cytoplasmic transport of pre-mRNA. Given that it is increasingly recognized that such spatial relationships are dynamic, the review also addresses the emerging concept that the spatial intranuclear organization changes with changes in cell function, a concept which supports the hypothesis that the spatial organization of the interphase nucleus may represent one of the fundamental control mechanisms in gene expression.
The cellular organization of gene expression
1998, Current Opinion in Cell BiologyRecent cell biological observations have provided new insights into how transcription, pre-mRNA splicing and 3′ processing are organized and coordinated with each other in the mammalian cell nucleus. Morphological observations are supported by biochemical evidence that suggests physical interactions between components of the transcription and RNA processing machineries. A working model of the cellular organization of gene expression is now emerging.
Repositioning of human interphase chromosomes by nucleolar dynamics in the reverse transformation of HT1080 fibrosarcoma cells
1998, Experimental Cell ResearchAn experimental system which should be valuable for studying the role of spatial positioning of the nuclear genome in human cell function has been developed. Reverse transformation of the malignant HT1080 fibrosarcoma cell line upon treatment with 8-chloro-cAMP results in growth inhibition, cytoskeletal reorganization, changes in nuclear shape and chromatin accessibility, and formation of prominent nucleoli. Fluorescentin situhybridization was used to study DNA positioning during nuclear remodeling. Morphometric analysis of the hybridization sites for both repetitive sequences and “painting probes” for whole chromosomes indicated dispersal of acrocentric chromosomes in untreated cells and a highly organized central location of these ribosome gene-containing chromosomes in association with one or a few large nucleoli in nondividing treated cells. The results suggest that there was a directed movement of interphase chromosomes during a response which normalized a malignant cell line. These large-scale repositionings may serve two functions in restoring a normal transcriptional setup to the nucleus. First, ribosome genes are placed in the nucleolus, their transcriptional suborganelle. Second, nucleolar anchorings together with additional perinucleolar centromeric associations orient the domain shapes of entire chromosomes, installing gene-rich chromosomal regions into pockets of (accessible) DNAse I-sensitive chromatin populated by spliceosomes.