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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Summary |
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Key words: Gene expression, Nuclear organization, Oligodendrocyte, Proteolipid protein, Splicing factors
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Introduction |
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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.,
1999). 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., 1996
;
Croft et al., 1999
).
Many nuclear proteins are found concentrated in discrete domains
(Lamond and Earnshaw, 1998).
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., 1997
;
Smith et al., 1999
;
Dundr and Misteli, 2001
).
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, 1998
; Wei et al.,
1999
). 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.,
1998
). SFCs may serve as storage sites from which splicing factors
are recruited to adjacent transcriptionally active genes
(Misteli et al., 1997
). 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.,
1996
; Grande et al.,
1997
; Jolly et al.,
1997
; Schul et al.,
1998
).
The organization of both nuclear proteins and chromatin changes during cell
differentiation (Antoniou et al.,
1993; Santama et al.,
1996
). 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., 1986); (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, 1990)
and the DNA-binding protein myelin transcription factor 1 (Myt1)
(Kim and Hudson, 1992
) to
determine the spatial relationship between myelin-specific genes and related
nuclear proteins as the cells undergo terminal differentiation.
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Materials and Methods |
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PLP mRNA in situ hybridization
In situ hybridization for PLP mRNA was performed as previously
described (Redwine and Armstrong,
1998). 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., 1987
), 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., 1991). 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, 1994
). 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., 1995) 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.
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Results |
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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, 1992;
Lewin, 1994
). 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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|
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.
|
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., 1986), 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 -amanitin to
inhibit RNA polymerase II activity. In the presence of
-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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|
The transcription factor Myt1 localizes to different nuclear domains
from splicing factors
Splicing can occur as a co-transcriptional event
(Misteli and Spector, 1999).
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., 1995
). SC35
and Myt1 immunoreactivities exhibited very different nuclear patterns
(Fig. 7A,B). As previously
described in other systems (Fu and
Maniatis, 1990
), 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.
|
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.
|
|
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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Discussion |
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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,
1999). 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, 1991
).
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., 1997
). 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., 1993
; Brown et al.,
1999
; Gerasimova et al.,
2000
).
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, 1998;
Wei et al., 1999
) and
identification of increased levels of acetylated chromatin
(Hendzel et al., 1998
). 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, 1999
). 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., 1992
;
Timsit et al., 1992
) and in
oligodendrocyte progenitors (Mallon et
al., 2002
). 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., 2000
). 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., 1999
). 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., 1996;
Jolly et al., 1997
).
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.,
2001). Relative to Myt1, gliomas exhibit variable subnuclear and
subcellular Myt1 immunoreactivity compared to normal oligodendrocyte lineage
cells (Armstrong et al., 1997
).
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., 1994
; Tomura et al.,
1995
). 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., 1995
).
Our data support a model in which discrete functional domains are regulated
by localized changes in protein distribution
(Carmo-Fonseca, 2002). 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, 2000
;
Carmo-Fonseca, 2002
). 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., 1994;
Armstrong et al., 1997
).
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Acknowledgments |
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