ARTICLE |
Correspondence to: Pernette J. Verschure, Swammerdam Instit. for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, PO Box 94062, 1090 GB Amsterdam, The Netherlands. E-mail: pjversch@science.uva.nl
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Summary |
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Compartmentalization of the interphase nucleus is an important element in the regulation of gene expression. Here we investigated the functional organization of the interphase nucleus of HeLa cells and primary human fibroblasts. The spatial distribution of proteins involved in transcription (TFIIH and RNA polymerase II) and RNA processing and packaging (hnRNP-U) were analyzed in relation to chromosome territories and large-scale chromatin organization. We present evidence that these proteins are present predominantly in the interchromatin space, inside and between chromosome territories, and are largely excluded by domains of condensed chromatin. We show that they are present throughout the active and inactive X-chromosome territories in primary female fibroblasts, indicating that these proteins can freely diffuse throughout the interchromatin compartment in the interphase nucleus. Furthermore, we established that the in vivo spatial distribution of condensed chromatin in the interphase nucleus does not depend on ongoing transcription. Our data support a conceptually simple model for the functional organization of interphase nuclei. (J Histochem Cytochem 50:13031312, 2002)
Key Words: chromatin structure, nuclear organization, subnuclear domains, gene expression
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Introduction |
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There is rapidly growing evidence that compartmentalization of the interphase nucleus is an important element in the regulation of gene expression (
Second, a direct relationship exists between large-scale chromatin organization and transcriptional activity. In chromosome territories, chromatin is folded in such a way that transcriptionally active sites are located near the surface of condensed chromatin domains (
Finally, there is a growing list of subnuclear domains (nuclear bodies) that contain little or no DNA. These domains are enriched in specific sets of nuclear factors involved in RNA synthesis and/or RNA processing. Examples are nucleoli (
Here we investigate the functional organization of the interphase nucleus of HeLa cells and primary human female fibroblasts. The spatial distribution in the nucleus of proteins involved in transcription and RNA processing and packaging is analyzed. We present evidence that these proteins are largely excluded from condensed chromatin and occur predominantly in the interchromatin compartment. Strikingly, we find that proteins involved in transcription and RNA processing are present in the interchromatin space of both the active and the inactive X-chromosome territories in female fibroblasts. This is in line with the notion that most nuclear proteins freely diffuse through the nucleus (
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Materials and Methods |
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Cell Culture
Human primary fibroblasts (kindly provided by Dr. L.H.F. Mullenders; University of Leiden, The Netherlands) with a normal female karyotype (46, XX) were grown at 37C in a 2.5% CO2 atmosphere in Ham's F-10 (Gibco; Breda, The Netherlands), supplemented with 15% (w/v) heat-inactivated fetal calf serum (FCS; Boehringer, Mannheim, Germany), 2 mM L-glutamine (Gibco), 100 IU/ml penicillin, and 100 mg/ml streptomycin (Gibco). Cells were cultured on Alcian Blue-coated coverslips (
A HeLa cell line (cell line 212-HeLa) that stably expresses H2B-GFP was used (
To inhibit RNA synthesis, cells were cultured either with 50 µM 5,6-dichloro--D-ribofuranosylbenzimidazole (DRB) for 4 hr or with 100 µg/ml
-amanitin for 7 hr. The
-amanitin inhibitor of transcription by RNA polymerase II can at low concentration (1 µg/ml) also reduce transcription by RNA polymerase I, but usually it does not completely block preribosomal synthesis. However, at high concentration (100 µg/ml)
-amanitin also inhibits transcription by RNA polymerase III. DRB inhibits transcription by RNA polymerase II only.
Immunofluorescent Labeling
Immunofluorescent labeling was performed as described previously ( (
Chromosome-specific Probes
Labeling of the human X chromosome was achieved using chromosome-specific DNA library probes directly conjugated to either Cy3 or FITC (Cambio; Cambridge, UK). Probes were prepared as described previously (
ISH Procedure
The fluorescence ISH procedure has been described previously (
Cells were rinsed with PBS at RT and DNA staining was performed with 0.4 µg/ml Hoechst 33258 (Sigma) in PBS. Slides were mounted in Vectashield. Slides were kept at 4C until evaluation and were analyzed within 24 hr.
Confocal Laser Scanning Microscopy
All experiments were performed at least three times in duplicate. For each experiment, 10 nuclei were visualized. Images were recorded with a Zeiss LSM 510 (Zeiss; Jena, Germany) confocal laser scanning microscope equipped with a x100/1.23 NA oil-immersion objective. We used an argon laser at 488 nm in combination with a helium neon laser at 543 nm to excite green and red fluorochromes simultaneously. Emitted fluorescence was detected with a 505530-nm bandpass filter for the green signal and a 560-nm longpass filter for the red signal. Pairs of images were collected simultaneously in the green and red channels. 3D images were scanned as 512 x 512 x 32 voxel images (sampling distance 49 nm lateral and 208 nm axial).
Image Processing
Images were corrected for optical crosstalk (
Analysis of the Images
All experiments were repeated at least two times. For all experiments we analyzed at least 10 cells. To compare the localization of the double-labeled images, we made line scans measuring the local intensity distribution of the two labels. Of all the analyzed cells we evaluated at least 20 line scans. In the figures we show only one of these evaluated line scans.
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Results |
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Condensed Chromatin Domains Are Accessible for Antibodies
Histone H2B-GFP expressing HeLa cells (). HP-1
is confined to highly compacted pericentric heterochromatin (
-immunolabeled domains in HeLa cells were compared after dual-color 3D imaging. Fig 1A shows that there is a close match between the intensely GFP-labeled compact heterochromatin domains and the HP-1
signal, showing that the anti-HP-1
antibody had access to condensed chromatin domains. We conclude that under our labeling conditions antibodies are able to recognize antigens in condensed chromatin domains.
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The General Transcription Factor TFIIH, RNA Polymerase II and the hnRNP-U Protein Are Predominantly Localized in the Interchromatin Compartment
We have analyzed the spatial distribution of the general transcription factor TFIIH (using an antibody recognizing the p62-subunit) and that of RNA polymerase II (using antibodies H14 and 8 WG16, which recognize all phosphorylation states of RNA polymerase
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In primary human female fibroblasts we analyzed the distribution of RNA polymerase II and of acetylated histone H4 with respect to both X chromosomes, of which one is transcriptionally active and the other is inactive (Fig 2A). For the labeling of acetylated histone H4 we used antibodies that recognize histone H4, which is acetylated at lysine 8 (antibody R232) or at lysine 16 (antibody R252). These histone H4 isoforms are associated with transcriptionally competent chromatin (
In contrast to acetylated histone H4, we find that RNA polymerase II is present throughout both X chromosome territories (Fig 2B), indicating that this protein has access to the interchromatin space in the inactive X-chromosome territory equally well as to that of its transcriptionally active counterpart. A similar result was obtained for hnRNP-U, which is a member of the large group of hnRNP proteins that are involved in RNA packaging, processing, and transport (
The Chromatin Distribution in the Interphase Nucleus Does Not Require Ongoing Transcription
Results thus far are consistent with the idea that the interchromatin space is a depot for factors required for transcription, RNA processing and transport. Does this mean that RNA synthesis and processing are essential for maintaining the spatial distribution of chromatin in transcriptionally active nuclei? To address this question, we compared the distribution of chromatin in HeLa cells that express GFP-tagged histone H2B before and after inhibition of transcription with 100 µg/ml -amanitin, which blocks RNA polymerases I and II but does not affect RNA polymerase III. Alternatively, we inhibited transcription by exposing cells to 50 µM 5,6-dichloro-
-D-ribofuranosyl-benzimidazole (DRB), an adenosine analogue. Fig 3A and Fig 3B compare the spatial distribution of chromatin in living cells before and 4 hr after addition of the transcription inhibitor. Results show that after inhibition the distribution of chromatin remained unchanged. The line scans shown in Fig 3 underscore these observations.
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In addition, we analyzed whether inhibition of RNA polymerases changes the amount and the distribution of acetylated histone H4 (
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Discussion |
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Two major compartments can be distinguished in mammalian interphase nuclei. One is composed of apparently interconnected condensed chromatin domains and the other is the interchromatin compartment, i.e., the space between the compact chromatin domains, which is largely devoid of chromatin (
In the present study we focused on the spatial distribution of nuclear components involved in RNA synthesis and RNA processing in relation to the large-scale organization of chromatin in the nucleus. We systematically analyzed the spatial distribution of the following nuclear components involved in transcription and RNA processing and transport in relation to condensed chromatin domains and the interchromatin compartment: (a) the general transcription factor TFIIH, (b) RNA polymerase II, and (c) hnRNP-U, which is involved in RNA packaging and processing. Chromatin was visualized in HeLa cells that express GFP-tagged histone H2B (
Our results are in line with recent results from
Several groups have presented evidence that large-scale chromatin organization in the nucleus depends on transcriptional activity. -amanitin or DRB has no detectable effect on large-scale chromatin organization in HeLa cell nuclei. Comparison of the chromatin distribution before and after inhibition of transcription in one and the same living cell gave no evidence for a change in large-scale chromatin structure. In addition, we found that in HeLa cell nuclei the amount and the distribution pattern of acetylated histone H4 did not change after inhibition of transcription. We suspect that the chromatin expansion and condensation after inhibition of RNA synthesis that was observed by others (
What is the reason that factors involved in transcription and in RNA processing do not enter condensed chromatin domains? One obvious answer is that they are excluded by steric hindrance due to the tight packing of nucleosomes. This argument is often taken as the explanation for the inactivity of genes that are present in a heterochromatin environment. However, heterochromatin-specific genes have been described that are actively transcribed inside heterochromatin domains (
Summarizing, our results support a simple model for the functional organization of the interphase nucleus. Transcriptionally active genes are localized at the surface of relatively condensed chromosomal domains in the perichromatin compartment in contact with the interchromatin compartment. In this way, components of the transcriptional machinery that are present in the interchromatin space have unhindered access to regulatory sequences of chromatin. Moreover, newly synthesized RNA can be deposited directly into the interchromatin space, where factors that are required for processing, packaging, and transport through the interchromatin channels are readily available. Thus, large-scale folding of chromatin and compartmentalization of nuclear components create a functional nuclear organization that supports efficient gene expression.
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Acknowledgments |
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Supported by the EU Biomed II program (project number BMH4-CT95-1139) and by a grant to PJV from the Biological branch (ALW) of the Dutch Research Council (NWO) (project number 805-48011).
We thank Dr B.M. Turner (University of Birmingham, UK) for kindly providing us with antibodies recognizing acetylated histone H4 and Prof Dr P. Chambon (Université Louis Pasteur, Strassbourg, France) for the generous gift of the anti-HP1 antibodies. We are grateful to Dr G. Wahl (Salk Institute for Biological Studies, La Jolla, CA) for kindly providing us with HeLa cells transfected with histone H2B-GFP fusion protein. We thank Prof Dr C.J.F. van Noorden (University of Amsterdam, The Netherlands) for critical reading of the manuscript.
Received for publication February 20, 2002; accepted May 1, 2002.
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