Nuclear organization in differentiating oligodendrocytes

Joseph A. Nielsen1, Lynn D. Hudson2 and Regina C. Armstrong1,3,*

1 Program in Molecular and Cell Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
2 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
3 Department of Anatomy, Physiology and Genetics, Program in Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA

* Author for correspondence (e-mail: rarmstrong{at}usuhs.mil)

Accepted 13 August 2002


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 Materials and Methods
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Many studies have suggested that the 3D organization of chromatin and proteins within the nucleus contributes to the regulation of gene expression. We tested multiple aspects of this nuclear organization model within a primary cell culture system. Oligodendrocyte lineage cells were examined to facilitate analysis of nuclear organization relative to a highly expressed tissue-specific gene, proteolipid protein (PLP), which exhibits transcriptional upregulation during differentiation from the immature progenitor stage to the mature oligodendrocyte stage. Oligodendrocyte lineage cells were isolated from brains of neonatal male rodents, and differentiation from oligodendrocyte progenitors to mature oligodendrocytes was controlled with culture conditions. Genomic in situ hybridization was used to detect the single copy of the X-linked PLP gene within each interphase nucleus. The PLP gene was not randomly distributed within the nucleus, but was consistently associated with the nuclear periphery in both progenitors and differentiated oligodendrocytes. PLP and a second simultaneously upregulated gene, the myelin basic protein (MBP) gene, were spatially separated in both progenitors and differentiated oligodendrocytes. Increased transcriptional activity of the PLP gene in differentiated oligodendrocytes corresponded with local accumulation of SC35 splicing factors. Differentiation did not alter the frequency of association of the PLP gene with domains of myelin transcription factor 1 (Myt1), which binds the PLP promoter. In addition to our specific findings related to the PLP gene, these data obtained from primary oligodendrocyte lineage cells support a nuclear organization model in which (1) nuclear proteins and genes can exhibit specific patterns of distribution within nuclei, and (2) activation of tissue-specific genes is associated with changes in local protein distribution rather than spatial clustering of coordinately regulated genes. This nuclear organization may be critical for complex nucleic-acid—protein interactions controlling normal cell development, and may be an important factor in aberrant regulation of cell differentiation and gene expression in transformed cells.

Key words: Gene expression, Nuclear organization, Oligodendrocyte, Proteolipid protein, Splicing factors


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Within the nucleus, DNA replication and transcription as well as RNA splicing each require coordination among many different proteins interacting with DNA and RNA. Organizing principles within the nucleus have been proposed to facilitate these complex nuclear functions (Lamond and Earnshaw, 1998Go; Misteli, 2000Go). Chromosomes, genes, RNA transcripts, and proteins each localize to discrete yet dynamic domains that may reflect spatial compartmentalization to facilitate nuclear functions. Among the multitude of detectable nuclear domains, it is now important to identify spatial and temporal relationships that have functional significance.

The localization of a gene within the nucleus may be an important regulatory mechanism. For example, targeting of genes to regions of the nucleus containing heterochromatin may be one mechanism of silencing gene expression (Brown et al., 1999Go). Although the peripheral region of the nucleus is known to contain heterochromatin in many cell types, active genes may preferentially distribute to either peripheral or central regions (Marshall et al., 1996Go; Croft et al., 1999Go).

Many nuclear proteins are found concentrated in discrete domains (Lamond and Earnshaw, 1998Go). Numerous studies have identified transcriptionally active genes associated with the periphery of nulcear domains enriched in splicing factors, called splicing factor compartments (SFCs) (Misteli et al., 1997Go; Smith et al., 1999Go; Dundr and Misteli, 2001Go). Additionally, [3H]uridine and Br-UTP incorporation into nascent RNA transcripts labels the periphery of SFCs indicating that this region is a site of active transcription (Misteli and Spector, 1998Go; Wei et al., 1999Go). The periphery of SFCs is also enriched in hyperacetylated chromatin, which is considered a marker for the transcriptionally active state of chromatin (Hendzel et al., 1998Go). SFCs may serve as storage sites from which splicing factors are recruited to adjacent transcriptionally active genes (Misteli et al., 1997Go). Many transcription factors are also concentrated into domains throughout the nucleus, and an unresolved question is whether these sites represent active sites of transcription, storage sites, or other undefined functional accumulations (Elefanty et al., 1996Go; Grande et al., 1997Go; Jolly et al., 1997Go; Schul et al., 1998Go).

The organization of both nuclear proteins and chromatin changes during cell differentiation (Antoniou et al., 1993Go; Santama et al., 1996Go). In this study, we sought to identify changes in nuclear organization occurring during cell differentiation that might contribute to the establishment of terminally differentiated gene expression patterns. Transformed cell lines have been used extensively to study nuclear organization, but established cell lines often have altered differentiation characteristics and may not accurately reflect regulation of tissue-specific gene expression. Therefore, it is important to test relevant nuclear distributions in the context of tissue-specific genes that are activated during differentiation of primary cells. In this study, we used a primary culture system with specific advantages for analysis of nuclear organization relative to cell differentiation. Oligodendrocytes are central nervous system (CNS) cells that produce myelin sheaths, which surround axons to facilitate efficient nerve impulse conduction. During differentiation of oligodendrocytes, there is a simultaneous upregulation of a set of tissue-specific genes that encode the proteins required for synthesis of the myelin sheath. These tissue-specific genes must be appropriately regulated for normal myelination during CNS development and for remyelination associated with CNS regeneration.

This experimental system has several advantages for studying changes in nuclear organization during cell differentiation: (1) primary oligodendrocyte cultures mimic the in vivo progression of differentiation and expression of myelin-specific proteins (Dubois-Dalcq et al., 1986Go); (2) oligodendrocyte upregulation of transcription of the proteolipid protein (PLP) gene during differentiation can be controlled by manipulating the culture conditions; (3) cells isolated from male animals have a single active allele of the X-linked PLP gene; and (4) a second myelin-specific gene, myelin basic protein (MBP), is transcriptionally upregulated at approximately the same time as PLP both in vivo and in vitro.

In this primary culture model system, we used genomic in situ hybridization to monitor the nuclear localization of the PLP and MBP myelin-specific genes relative to differentiation and transcriptional activation within interphase oligodendrocyte nuclei. In addition, genomic in situ hybridization was combined with immunostaining for the splicing factor SC35 (Fu and Maniatis, 1990Go) and the DNA-binding protein myelin transcription factor 1 (Myt1) (Kim and Hudson, 1992Go) to determine the spatial relationship between myelin-specific genes and related nuclear proteins as the cells undergo terminal differentiation.


    Materials and Methods
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 Materials and Methods
 Results
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 References
 
Cell culture
Primary cultures from male neonatal rat brains were prepared as previously described (Armstrong, 1998Go). Briefly, postnatal day 2 rat brains were dissociated, plated in tissue culture flasks, and allowed to grow for 7-10 days. The flasks were placed on a rotary shaker to dislodge immature oligodendrocyte lineage cells, which were then plated onto poly-D-lysine coated chamber slides. Progressive stages of differentiation within the oligodendrocyte lineage can be identified with cell type-specific markers and based upon the characteristic morphology of each stage (Armstrong, 1998Go). To obtain cultures of immature oligodendrocyte lineage phenotypes, cells were grown in medium containing 10 ng/ml of PDGF-AA and 10 ng/ml FGF2 (R and D Systems, Minneapolis, MN) (Armstrong, 1998Go). Preoligodendrocyte progenitors were obtained by allowing these cultures to adhere for only 2 hours before fixation. The majority of plated cells progressed to oligodendrocyte progenitors if allowed to grow, and with PDGF and FGF treatment these cells remained progenitors until fixation at day 3. Differentiated oligodendrocytes were obtained by plating in medium without PDGF and FGF and allowing the cells to mature during 3 days in culture. Astrocytes served as a glial cell control that is not part of the oligodendrocyte lineage. Astrocytes were obtained from the same primary rat brain glial cultures by purification of the population that remained adhered to the initial flasks after the oligodendrocyte lineage cells were dislodged. Primary mouse oligodendrocytes were prepared in a manner similar to the rat oligodendrocyte lineage cells. In experiments to inhibit RNA polymerase II transcription, cells were treated with 5 µg/ml {alpha}-amanitin (Roche Applied Science, Indianapolis, IN) for 2 hours prior to fixation. All animals were handled in accordance with procedures approved by the USUHS Institutional Animal Care and Use Committee. All quantitation was based on data combined from at least 3 independent preparations of cells from separate litters of animals.

PLP mRNA in situ hybridization
In situ hybridization for PLP mRNA was performed as previously described (Redwine and Armstrong, 1998Go). Briefly, cells were fixed with 4% paraformaldehyde, acetylated, and prehybridized with RNA hybridization buffer (DAKO, Carpenteria, CA). A 980 bp cDNA corresponding to the entire coding region of the mouse PLP gene, derived from pLH116 (Hudson et al., 1987Go), served as a template to incorporate digoxigenin-11-UTP (Roche Applied Science, Indianapolis, IN) using in vitro transcription (Ambion, Austin, TX). The probe was denatured and allowed to hybridize overnight. The probe was detected using an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Applied Science, Indianapolis, IN) followed by NBT/BCIP colorimetric detection (DAKO, Carpenteria, CA).

Genomic in situ hybridization
Cells were fixed with 2% paraformaldehyde and processed using a modified protocol for genomic in situ hybridization detection (Johnson et al., 1991Go). The cells were extracted with 0.5% NP40 detergent and dehydrated through graded ethanols. The cells were pretreated with hybridization buffer without probe. The target DNA and probe, labeled with digoxigenin-11-dUTP using nick translation, were denatured and then hybridized overnight. The PLP genomic in situ hybridization probe was generated from a 3.7 kb fragment of the rat PLP promoter (Cambi and Kamholz, 1994Go). Detection of digoxigenin labeled probe was performed using a tyramide signal amplification systemTM (NEN, Boston, MA). Probes were detected with biotinylated anti-digoxin antibody (Jackson ImmunoResearch, West Grove, PA) followed by steptavidin horseradish peroxidase (HRP). HRP was then used to catalyze the deposition of tyramide-FITC at the site of probe binding. The specificity of the hybridization was confirmed by absence of signal using the following conditions: (1) no probe; (2) probe and target not denatured; and (3) hybridization competition with 100-fold excess of non-labeled probe.

For the double genomic hybridization experiments in mouse oligodendrocyte cultures, a mouse PLP probe corresponding to 4.0 kb of the mouse PLP promoter (isolated from an EcoRI and PstI digest of pMuPLP9; L.D.H., unpublished) was labeled with FITC-11-dUTP (Roche Applied Science, Indianapolis, IN). A mouse MBP probe corresponding to 3.0 kb of the mouse MBP promoter (isolated from a XbaI and SalI digest of JCC137; L.D.H., unpublished) was labeled with digoxigenin-11-dUTP (Roche Applied Science, Indianapolis, IN). The probes were hybridized overnight simultaneously, and then detected sequentially using a tyramide signal amplification system. The PLP probe was detected with anti-FITC conjugated with HRP followed by tyramide-dinitrophenyl, anti-dinitrophenyl conjugated with HRP, and then tyramide-FITC. The peroxidase activity was quenched with a 15 minute 0.02 M HCL treatment, and the digoxigenin-MBP probe was detected with anti-digoxin conjugated with biotin, followed by streptavidin-HRP, and tyramide-Cy3. The specificity of each hybridization and detection scheme was confirmed by absence of signal in hybridizations using each single probe followed by combined anti-FITC and anti-digoxin detection protocols. We also confirmed our ability to inactivate the HRP, as required to quench the PLP detection prior to MBP detection. In experiments using the single PLP hybridization protocol, after the incubation with anti-FITC conjugated with HRP, the HRP was inactivated with 0.02 M HCL and the absence of signal was confirmed following the detection protocol.

Immunostaining of nuclear proteins
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X 100. Mouse anti-SC35 monoclonal antibody (ATCC, Manassas, VA) and rabbit anti-Myt1-His polyclonal antibody (Armstrong et al., 1995Go) were added to slides, and incubated overnight. Following a blocking step with 5% normal donkey serum, the primary antibodies were detected with donkey anti-mouse FITC and donkey anti-rabbit Cy3 (Jackson ImmunoResearch, West Grove, PA). For the in situ hybridization in combination with immunostaining experiments, a 5-minute post-fixation with 4% paraformaldehyde was included before the addition of the primary antibody, and the immunostaining was then carried out as described above.

Image analysis
Two-dimensional images were collected with an Olympus IX70 epifluorescence microscope equipped with a 40x objective using a Spot2 digital camera. 3D images were collected in 0.25 µm sections through the Z-dimension with a 63x objective (1.4 NA) on a Ziess Axiophot epifluorescence microscope using a Sensicam digital camera. The haze was removed from 3D images using the deconvolve algorithm and point spread functions generated for the red and green channels within Slidebook (Intelligent Imaging Innovations, Denver, CO). The images were analyzed using Metamorph software (Universal Imaging Corporation, West Chester, PA) and domains were scored as co-localized when there was pixel overlap in the red and green channels. A micrometer was used to calculate XY resolution with one pixel=0.18 µm at 40x. Figures were assembled in Adobe Photoshop (Adobe, San Jose, CA). As a control for image registration, fiduciary markers that fluoresce in both the red and green wavelengths were imaged. In experiments where images were collected of a single sub-resolution bead (0.17 µm diameter) in both channels, the merged image had complete overlap of the red and green pixels.


    Results
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 Materials and Methods
 Results
 Discussion
 References
 
The tissue-specific PLP gene is transcriptionally upregulated during differentiation of oligodendrocytes in primary cultures.
Transcription of the PLP gene increases developmentally as oligodendrocytes differentiate and form myelin (Macklin et al., 1991Go; Shiota et al., 1991Go). In our culture system, we demonstrate the difference in PLP gene expression in progenitors versus differentiated oligodendrocytes. Oligodendrocyte progenitors were isolated from postnatal day 2 male rat pups, and differentiation was inhibited by addition of platelet derived growth factor-AA (PDGF) and fibroblast growth factor 2 (FGF). The progenitors exhibited a characteristic bipolar morphology (Fig. 1A). Only background signal was detected by in situ hybridization for PLP mRNA at this progenitor stage (Fig. 1B). Parallel cultures grown without the addition of PDGF and FGF terminally differentiated into mature oligodendrocytes. After three days in medium without PDGF and FGF, there were high levels of PLP mRNA transcripts detected in cells with the characteristic highly branched morphology of differentiated oligodendrocytes (Fig. 1C,D). PLP mRNA in situ hybridization was also performed on astrocyte cultures as a glial cell control that does not express detectable levels of PLP mRNA (Fig. 1E,F). These data confirm our ability to control oligodendrocyte differentiation and the associated upregulation of myelin-specific gene expression.



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Fig. 1. PLP mRNA in situ hybridization in primary rat oligodendrocyte cultures. Phase contrast image analysis shows bipolar processes that are characteristic of progenitors (A), multiple branched processes characteristic of oligodendrocytes (C), and flat fibroblastic morphology of astrocytes (E). Brightfield images show that PLP mRNA was not detectable in cultured progenitors (B, same field as A) or astrocytes (F, same field as E). High levels of PLP mRNA (blue/black signal) accumulated in differentiated oligodendrocytes (D, same field as C). Bar, 50 µm.

 

Active PLP transcription induces adjacent splicing factor compartments
The spatial relationship between the PLP gene and SFCs was examined as cells differentiated and upregulated transcription from the PLP gene locus. The splicing required for processing the PLP gene is typical of mammalian genes; the PLP gene encompasses approximately 17 kb of DNA with 7 exons (Macklin, 1992Go; Lewin, 1994Go). Oligodendrocyte lineage cell cultures and astrocyte cultures were prepared, as described in the methods. Genomic in situ hybridization with a probe directed against the promoter and upstream regulatory regions of the PLP gene was used to detect a single site corresponding to the X-linked PLP gene in a given nucleus. This genomic in situ hybridization was combined with immunofluorescence to simultaneously detect the splicing factor SC35 which labels SFCs (Fig. 2). Astrocytes, which do not express detectable PLP mRNA (Fig. 1F), exhibited co-localization of the PLP gene with a discrete SC35 domain in 15±3% of the cells examined (Fig. 2A, Fig. 3). Progenitor cells, which under these culture conditions are not expressing marked levels of PLP mRNA (Fig. 1B), exhibited co-localization of the PLP gene with SC35 in 22±9% of the cells (Fig. 2B, Fig. 3). In contrast, in differentiated oligodendrocytes in which the PLP gene should be highly expressed (Fig. 1D), the PLP gene was co-localized with SC35 in 63±5% of the cells (Fig. 2C, Fig. 3).



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Fig. 2. SC35 immunostaining combined with genomic in situ hybridization. Merged images for PLP genomic in situ hybridization (green) combined with SC35 immunostaining (red) in astrocytes (A), progenitors (B), oligodendrocytes (C) and oligodendrocytes treated with {alpha}-amanitin (D). IRBP genomic in situ hybridization (green) merged with SC35 immunostaining (red) in progenitors (E). Bar, 10 µm.

 


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Fig. 3. Quantitation of genomic in situ hybridization combined with SC35 immunostaining. The PLP gene and SC35 domains were scored as co-localized when there was pixel overlap in the red and green channels (see Fig. 2). SC35 domains are more frequently co-localized with the PLP gene in oligodendrocytes (oligos, n=110) compared with cells that do not transcribe detectable levels of PLP, i.e. astrocytes (astros, n=55), progenitors (OPs, n=116), and oligodendrocytes treated with {alpha}-amanitin (oligos+AM, n=110) (P<0.05; Chi-square). As a control, the IRBP gene, which is not expressed in oligodendrocytes, had a frequency of PLP/SC35 co-localization similar to cells that do not express PLP (n=50). Error bars=standard error of the proportion.

 

The spatial relationship between the PLP gene and SFCs was confirmed in the Z-axis (Fig. 4). Digital optical sections were used to generate 3D reconstructions for 10% of each oligodendrocyte lineage population sampled in the 2D analysis. A similar spatial relationship and frequency of association was found between the PLP gene and SC35 in this 3D analysis as with the 2D analysis.



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Fig. 4. 3D reconstruction of PLP genomic in situ hybridization merged with SC35 immunostaining in oligodendrocytes. The PLP gene (green) co-localizes with SC35 immunostaining (red) to appear as yellow at the periphery of nuclear domains enriched in SC35 in oligodendrocytes. Oligodendrocytes are shown as a 3D reconstruction imaged in the XY plane (A) and rotated 90 degrees to the XZ view (B). In progenitors shown as a 3D reconstruction, the PLP gene does not co-localize with SC35 imaged in the XY plane (C) or when rotated 90 degrees to the XZ view (D). The blue line outlines the nuclear periphery. Bar, 5 µm.

 

As an additional control, the spatial relationship of SFCs was examined relative to a gene that is not expressed in mature oligodendrocytes. The interphotoreceptor retinoid binding protein (IRBP) gene was selected since IRBP is expressed by photoreceptor cells and the pineal gland (van Veen et al., 1986Go), but is not expressed in oligodendrocytes (D. Borst, personal communication). The IRBP gene co-localized with an SC35 domain in 26±4% of the oligodendrocytes examined (Fig. 2E, Fig. 3).

The increased frequency of association of the PLP gene with SFCs in mature cells was dependent on transcriptional activity. Differentiated oligodendrocytes were treated for 2 hours with 5 µg/ml {alpha}-amanitin to inhibit RNA polymerase II activity. In the presence of {alpha}-amanitin, the frequency that the PLP gene was associated with an SC35 domain was significantly decreased (Fig. 2D, Fig. 3). This finding suggests that transcriptional activity of the PLP gene is required to induce adjacent SC35 accumulation.

These data demonstrate that in differentiated oligodendrocytes the PLP gene is frequently associated with nuclear compartments containing the splicing factor SC35. This association is not simply the result of cell differentiation, but is dependent upon PLP transcriptional activity.

The PLP gene does not exhibit radial translocation during oligodendrocyte differentiation
The location of genes could be related to the partitioning of heterochromatin within the nucleus and might serve as a mechanism of transcriptional control. In our experiments, the PLP gene appeared to more typically localize within a peripheral region of the nucleus (see examples in Figs 2, 4) whereas the IRBP gene alleles, which are both inactive in these cells, did not have a notable nuclear localization (see example in Fig. 2). This apparent differential distribution was substantiated using phase contrast microscopy to image individual nuclei combined with fluorescence imaging of the genomic in situ hybridization signal for each gene (Fig. 5A). A gene was classified as within the peripheral region of the nucleus if the measured distance between the center of the in situ hybridization signal and the edge of the nucleus was less than 1.5 µm. Since the IRBP gene has two alleles, the cells were also scored by the relative location of both alleles as a set in each cell, which demonstrated that at least one allele was located in the central region in the majority of the cells examined (Fig. 5B). This difference in localization between the PLP gene and the IRBP gene supported the interpretation that the PLP gene may be non-randomly localized within the nuclear periphery. The PLP gene location was more carefully examined using DAPI nuclear stain to identify the nuclear volume (Fig. 6). Based upon preliminary measurements of the nuclear volume with imaging of DAPI fluorescence, a 1.5 µm border inside the nuclear periphery comprised approximately 50% of the nuclear area (data not shown). In differentiated oligodendrocytes, as well as progenitors and astrocytes, the PLP gene was localized within this peripheral border in approximately 75% of the cells examined for each cell type (Fig. 5C). When compared to a random distribution, using the calculated average area of the nucleus, in each cell type the PLP gene was found non-randomly associated with the peripheral region of the nucleus. The similar preferential localization in oligodendrocytes and astrocytes indicates that this peripheral localization does not correlate with transcriptional status of the PLP gene. Accordingly, the PLP gene does not undergo a large-scale change in radial position as progenitors differentiate into mature oligodendrocytes and upregulate transcription from the PLP locus.



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Fig. 5. Quantitation of PLP and IRBP gene nuclear localization. The PLP and IRBP genes were classified as within the peripheral region of the nucleus if the measured distance between the center of the in situ hybridization signal and the edge of the nucleus was less than 1.5 µm. When the center of the in situ hybridization signal was located within the remaining nuclear volume, the localization was counted as central. In panel A, phase-contrast microscopy was used to identify the nuclear boundary. The PLP gene was more frequently associated with the peripheral region of the nucleus in oligodendrocyte progenitors (OPs, n=22) and oligodendrocytes (oligos, n=37). Analysis of individual IRBP alleles within each cell did not indicate a preferential association of IRBP alleles with the peripheral region of the nucleus (OPs, n=38; oligos, n=30). In panel B, both IRBP alleles were analyzed within each cell and were categorized as central-central (CC), central-peripheral (CP), or peripheral-peripheral (PP). In panel C, DAPI was used to identify the nuclear periphery. The PLP gene was non-randomly associated with a peripheral localization in astrocytes (astros, n=52), oligodendrocyte progenitors (OPs, n=103), or oligodendrocytes (oligos, n=101) (*P<0.05, chi-square). The PLP gene localization was not significantly different when comparing across cell types. Error bars=standard error of the proportion.

 


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Fig. 6. PLP gene nuclear localization. PLP genomic in situ hybridization (green or red) to detect the PLP gene combined with the nuclear stain DAPI (blue) to show the nuclear volume in astrocytes (A), progenitors (B) and oligodendrocytes (C). Bar, 10 µm.

 

The transcription factor Myt1 localizes to different nuclear domains from splicing factors
Splicing can occur as a co-transcriptional event (Misteli and Spector, 1999Go). Therefore, nuclear proteins involved in splicing and transcription may exhibit specific relative nuclear distributions that facilitate availability at sites of ongoing transcription and splicing. Two-color immunofluorescence was used to detect the nuclear distribution of a representative splicing factor, SC35, and a representative DNA-binding protein Myt1. Myt1 was used for this example because Myt1 binds to the PLP promoter and Myt1 is distributed in discrete nuclear domains in oligodendrocyte lineage cells (Armstrong et al., 1995Go). SC35 and Myt1 immunoreactivities exhibited very different nuclear patterns (Fig. 7A,B). As previously described in other systems (Fu and Maniatis, 1990Go), SC35 was found in a pattern characteristic of SFCs. Myt1 immunoreactivity appeared as more numerous punctate domains scattered throughout the nucleus. Myt1 immunoreactivity was predominately excluded from the interior regions of SC35 domains, but frequently was observed in discrete accumulations associated with the periphery of SC35 domains.



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Fig. 7. Nuclear localization of SC35 splicing factors and Myt1 DNA-binding proteins in oligodendrocyte progenitors. Progenitor cultures were double immunostained using anti-SC35 detected with anti-mouse FITC (green) and anti-Myt1 detected with anti-rabbit Cy3 (red). 3D reconstruction of SC35/Myt1 double immunostain imaged in XY plane (A) and with the 3D image rotated 90 degrees to show XZ view (B). Bar, 5 µm.

 

The nuclear distribution of Myt1 DNA-binding protein is independent of PLP promoter activity
Accumulations of DNA-binding proteins near their genomic targets may contribute to the regulation of relative interactions. Given our independent observations of PLP gene localization and Myt1 domains each being associated with the periphery of SFCs, it was important to examine whether the PLP gene and Myt1 domains were co-localized. PLP genomic in situ hybridization was combined with Myt1 immunostaining to determine the spatial relationship between Myt1 nuclear domains and the PLP gene. We analyzed three progressive stages of differentiation within the oligodendrocyte lineage: pre-oligodendrocyte progenitors, oligodendrocyte progenitors, and differentiated oligodendrocytes (Figs 8, 9). At each stage in the lineage, Myt1 immunoreactivity was associated with the PLP gene in approximately 50% of the cells observed. The IRBP gene, which is not expressed in oligodendrocytes (D. Borst, personal communication), also exhibited a similar approximately 50% frequency of association with Myt1. These data indicate that while discrete domains of Myt1 DNA-binding protein are present in nuclei, these domains do not preferentially accumulate to detectable levels near presumptive gene targets, such as the PLP gene, when these targets are transcriptionally active.



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Fig. 8. PLP genomic in situ hybridization combined with Myt1 immunostaining. Merged images for PLP genomic in situ hybridization (green) combined with Myt1 immunostaining (red) in pre-oligodendrocyte progenitors (A), progenitors (B) and oligodendrocytes (C). IRBP genomic in situ hybridization (green) merged with Myt1 immunostaining (red) in progenitors (D). Bar, 10 µm.

 


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Fig. 9. Quantitation of PLP genomic in situ hybridization combined with Myt1 immunostaining. The PLP gene and Myt1 domains were scored as co-localized when there was pixel overlap in the red and green channels (see Fig. 8). Myt1 nuclear domains were found associated with the PLP gene in approximately 50% of the cells analyzed (pre-oligodendrocyte progenitors (preOP, n=50), progenitors (OPs, n=101), and oligodendrocytes (oligos, n=100). The IRBP gene, which is not expressed in oligodendrocyte progenitors, was also found associated with Myt1 nuclear domains in approximately 50% of the progenitor cells analyzed (n=50). No statistical difference was found between any of the groups using the chi-square statistical test. Error bars=standard error of the proportion.

 

Two coordinately regulated myelin genes remain spatially separated during oligodendrocyte differentiation
To examine whether gene localization may contribute to coordinate transcriptional regulation, double genomic in situ hybridization was performed to compare the relative nuclear localization of two myelin-specific genes during oligodendrocyte differentiation. The PLP and the MBP genes were chosen for analysis because expression of each gene is upregulated at a very similar time during oligodendrocyte differentiation. There was no co-localization of the PLP gene with either of the MBP alleles in almost every cell examined (Fig. 10). The same lack of co-localization was observed in mature oligodendrocytes (31 of 31 cells), progenitors (53 of 53 cells), and astrocytes/microglia (28 of 29). In most cells, the PLP gene and the MBP gene were found in disparate regions of the nucleus. The autosomal MBP alleles were nearly always found clearly separated from each other (Fig. 10), and were evenly distributed relative to the peripheral or the central regions of nuclei (data not shown). These data argue against a hypothesis involving gene co-localization to coordinate transcription of a set of tissue-specific genes associated with terminal differentiation.



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Fig. 10. Double genomic in situ hybridization for MBP and PLP genes. Genomic in situ hybridization with the X-linked PLP gene being detected with FITC-labeled PLP probe (green) and the autosomal MBP gene being simultaneously detected with a digoxigenin-labeled MBP probe (red). In progenitors (Fig. 10A, phase-contrast 10C) and in oligodendrocytes (10B, phase contrast 10D), the PLP and the MBP genes were spatially separated. Bar, 10 µm.

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study extends previous work on gene expression and nuclear organization into a primary cell culture model that undergoes differentiation to analyze changes associated with the corresponding upregulation of tissue-specific gene expression. As a model system, we focus on the PLP gene in the oligodendrocyte lineage. We show that PLP transcriptional activity is associated with localized changes in specific nuclear protein domains. However, the PLP gene does not undergo large-scale translocation relative to the nuclear periphery or another myelin-specific gene, MBP.

The position of a gene in the three-dimensional space of the nucleus may be an important transcriptional regulatory mechanism. Ribosomal genes located on different chromosomes are segregated into the nucleolus presumably to facilitate efficient transcription, modification, and assembly of ribosomal gene products (Scheer and Hock, 1999Go). In contrast, when expression of the immediate early gene c-fos is induced in NIH-3T3 cells, the two alleles are transcriptionally active but are not located adjacent to one another (Huang and Spector, 1991Go). Thus, different organizing principles appear to be applied to different classes of genes. Few studies have addressed the question of whether sets of tissue-specific genes that are coordinately regulated and share similar regulatory factors also exhibit regulated spatial localization within the nucleus. One example that is available for tissue-specific genes showed that immunoglobulin genes are non-randomly and differentially positioned in the nucleus in two mature B-cell lines (Parreira et al., 1997Go). These immunoglobulin genes each maintained a different topography relative to each other and to peripheral versus central regions of the nuclear volume, regardless of transcriptional activity. However, several studies have reported large scale movements of genes within the nucleus, particularly between the peripheral and central regions (Palladino et al., 1993Go; Brown et al., 1999Go; Gerasimova et al., 2000Go).

Our data extend these findings to developmentally regulated tissue-specific genes in primary cultures of oligodendrocyte lineage cells undergoing terminal differentiation. Our data clearly show that the PLP gene was not spatially associated with either MBP allele. This result was observed in progenitors as well as after differentiation and upregulation of PLP and MBP transcription in oligodendrocytes. In addition, we show that the PLP gene is consistently associated with a peripheral nuclear localization in oligodendrocyte progenitors, differentiated oligodendrocytes, and astrocytes.

The periphery of SFCs have been demonstrated to be transcriptionally active sites based upon labeling to reveal nascent RNA transcripts (Misteli and Spector, 1998Go; Wei et al., 1999Go) and identification of increased levels of acetylated chromatin (Hendzel et al., 1998Go). In preliminary studies, we compared the PLP gene localization to regions in the nucleus enriched in acetylated chromatin, but did not find a marked association of acetylated chromatin with the PLP gene (data not shown). Previous studies in cell lines demonstrate splicing occurring at the site of transcription (Misteli and Spector, 1999Go). Importantly, our analysis allowed SFCs and the PLP gene to be compared at multiple stages of regulation of the PLP gene locus. SC35 splicing factors accumulated in discrete nuclear compartments adjacent to sites of transcriptionally active PLP genes in differentiated oligodendrocytes. Isoforms of the PLP gene have been reported to be expressed embryonically (Ikenaka et al., 1992Go; Timsit et al., 1992Go) and in oligodendrocyte progenitors (Mallon et al., 2002Go). However, the abundance of PLP mRNA transcripts in progenitors is dramatically lower than in mature oligodendrocytes (Fig. 1B). In addition, astrocytes (Fig. 1F) do not express PLP isoforms (Fuss et al., 2000Go). Therefore, the detectable accumulation of splicing factors adjacent to the PLP gene only in oligodendrocytes is likely to be related to the active production of PLP transcripts. Interestingly, accumulation of SC35 splicing factors relative to active genes may be a gene-specific process (Smith et al., 1999Go). Thus, our results characterize the PLP gene locus within the class of genes that demonstrate SC35 accumulation with transcriptional activity.

Many transcription factors are found in discrete nuclear domains, and an unresolved question is whether these domains correspond with regulation of target gene transcription. Our data for the DNA-binding protein Myt1 suggests that Myt1 domains are not strictly associated with a particular state of PLP gene activation. We have not attempted the extensive quantitative analysis of the nuclear volume occupied by the Myt1 domains to formally compare a random occurrence with the 50% association of Myt1 domains relative to the active versus inactive states of the PLP gene. However, several other studies have not found an association of transcription factor domains relative to their presumed genomic targets (Elefanty et al., 1996Go; Jolly et al., 1997Go). Presumably, the number of transcription factor molecules required to bind to the promoter of a target gene to regulate transcription is likely to be relatively few, which may explain why transcription factor domains are not clearly associated with sites of active transcription.

Accumulations of transcription factors into domains may still be functionally important even if detectable domains are not preferentially localized adjacent to target gene transcription sites. For example, the subnuclear localization of Runx2/CBFA1/AML3 transcription factors in discrete domains appears to be critical for tissue-specific gene expression and differentiation (Choi et al., 2001Go). Relative to Myt1, gliomas exhibit variable subnuclear and subcellular Myt1 immunoreactivity compared to normal oligodendrocyte lineage cells (Armstrong et al., 1997Go). In preliminary studies (data not shown), we determined that the Myt1 nuclear pattern was distinct from that of several other nuclear proteins, including thyroid hormone receptor ß1, which is known to bind to the promoters of both PLP and MBP (Bogazzi et al., 1994Go; Tomura et al., 1995Go). Therefore, Myt1 domains exhibit a specific pattern that is not likely to reflect a general pattern of accumulated transcription factors in oligodendrocyte lineage cells. In addition, the nuclear domains of Myt1 appeared larger and less abundant than domains associated with BrUTP-labeled nascent RNA transcripts (data not shown). We predict that domains of Myt1 might serve as a mechanism to sequester and thereby regulate the concentration of available protein. This concept of regulated Myt1 availability would be consistent with our previous observation that Myt1 immunoreactivity shifts from the nucleus to the cytoplasm as mature oligodendrocytes accumulate PLP protein, and Myt1 is subsequently down-regulated (Armstrong et al., 1995Go).

Our data support a model in which discrete functional domains are regulated by localized changes in protein distribution (Carmo-Fonseca, 2002Go). Recent work suggests that the nucleus is an extremely dynamic environment with many nuclear proteins showing high rates of mobility throughout the nucleus (Phair and Misteli, 2000Go; Carmo-Fonseca, 2002Go). SFCs adjacent to active genes may reflect the relative accumulation of molecules required to process multiple copies of RNA transcripts as they are generated. In contrast, DNA-binding proteins may not accumulate to detectable levels near active genes because fewer molecules are likely to be required to regulate transcription from a single copy genomic DNA.

Coordinate transcriptional control of tissue-specific genes may then be accomplished through binding interactions that regulate the local concentrations of available DNA-binding proteins, which are relevant for a given set of target genes. These protein-protein and protein-nucleic acid interactions would be expected to establish an appropriate nuclear environment for regulated transcription. Future studies will be required to test this model using methods that assess these functional interactions without dramatically disrupting the balance of concentrations and spatial relationships among nuclear elements. This model has implications for understanding the regulation of cell differentiation and tissue-specific gene expression during normal development. These regulatory mechanisms may also provide insight for pathological observations, as in the example of aberrant protein expression in tumors and dysplasia (Weis et al., 1994Go; Armstrong et al., 1997Go).


    Acknowledgments
 
We thank Tuan Le for laboratory support and Diane Borst for the IRBP probe, technical advice, and critical reading of the manuscript. We thank George Cox, Tom Misteli and Aviva Symes for technical advice. We thank Emma Frost, Josh Murtie and Adam Vana for critical reading of the manuscript. We thank Franca Cambi for the rat PLP promoter plasmid. This work was supported by Uniformed Services University of the Health Sciences grant RO70IE.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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