Abstract. Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of welldefined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis.1991. EMBO (Eur. Mol. Biol. Organ.)J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that cotranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.T HE cell nucleus comprises all factors required for faithful replication of the genome and regulated synthesis, processing and transport of RNA. In recent years much information on nuclear organization has become available. It is clear now that the nucleus is highly organized (reviewed by Jackson, 1991; van Driel et al., 1991). The most conspicuous subnuclear domain is the nucleolus in which ribosomal genes from different chromosomes are clustered and ribosome assembly takes place (reviewed by Scheer and Benavente, 1990; HernandezVerdun, 1991). Other examples of a domainlike organization in the nucleus are: replication clusters during S-phase (reviewed by Berezney, 1991), clustered splicing components (Spector, 1990;Fu and Maniatis, 1990; CarmoFonseca et al., 1992), hnRNP proteins (Pifiol-Roma et al., 1989; Ghetti et al., 1992), and tracks and foci of specific RNAs (Lawrence and Singer, 1991; Huang and Spector, 1991). In addition, a number of structures have been visualized of which the function is still unknown (Ascoli and Maul, 1991;Saunders et al., 1991; Ra~ka et al., 1991; al., 1992). The structural basis of the occurrence of nuclear activities in domains and the functional significance of this organizing principle for the regulation of gene expression and DNA replication are not understood.Essential for understandi...

Heterochromatin proteins are thought to play key roles in chromatin structure and gene regulation, yet very few genes have been identified that are regulated by these proteins. We performed large-scale mapping and analysis of in vivo target loci of the proteins HP1, HP1c, and Su(var)3-9 in Drosophila Kc cells, which are of embryonic origin. For each protein, we identified ∼100-200 target genes among >6000 probed loci. We found that HP1 and Su(var)3-9 bind together to transposable elements and genes that are predominantly pericentric. In addition, Su(var)3-9 binds without HP1 to a distinct set of nonpericentric genes. On chromosome 4, HP1 binds to many genes, mostly independent of Su(var)3-9. The binding pattern of HP1c is largely different from those of HP1 and Su(var)3-9. Target genes of HP1 and Su(var)3-9 show lower expression levels in Kc cells than do nontarget genes, but not if they are located in pericentric regions. Strikingly, in pericentric regions, target genes of Su(var)3-9 and HP1 are predominantly embryo-specific genes, whereas on the chromosome arms Su(var)3-9 is preferentially associated with a set of male-specific genes. These results demonstrate that, depending on chromosomal location, the HP1 and Su(var)3-9 proteins form different complexes that associate with specific sets of developmentally coexpressed genes.[Keywords: Heterochromatin; Drosophila; target genes; gene expression; genome organization] Supplemental material is available at http://www.genesdev.org.

We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300–450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) ␣ (HP1␣) and HP1␤ on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1␣ and HP1␤ in vivo targeting. HP1␣ targeting causes the recruitment of endogenous HP1␤ to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1␣ and HP1␤ targeting is sufficient to induce heterochromatin formation.Packing of the eukaryotic genome into higher-order chromatin structures is tightly related to gene expression (reviewed in references 17 and 37). Changes in chromatin structure are a key element in epigenetic gene control. Transcriptional activation is associated with large-scale chromatin decondensation, whereas silencing is related to condensation (2, 31, 36, 51, 52). The molecular mechanisms that establish and maintain functionally distinct higher-order chromatin states in the interphase nucleus are poorly understood (8,42). Posttranslational modifications of histones have been directly linked with transcriptional regulation and with changes in chromatin structure (reviewed in references 22 and 24). Such modifications alter the interactions between histones and DNA and between chromatin-associated proteins. The "histone code" apparently defines functionally distinct chromatin domains of different transcriptional states (15,21,35,49). Many key questions concerning the role of histone modifications regulating chromatin structure and gene expression are unsolved.A number of heterochromatin-associated proteins, including heterochromatin protein 1 (HP1) and polycomb group (PcG) proteins, which all are involved in epigenetic silencing, share a common evolutionarily conserved domain, the chromodomain (CD), which is responsible for the association with chromatin (4,9,19,24,48). HP1 also contains a chromoshadow domain (CSD), which mediates protein-protein interactions, including homodimerization (1). Moreover, HP1 also binds to DNA and linker histones through its hinge domain (HD), positioned between the CD and the CSD. This HD may also be involved in targeting HP1 to heterochromatin through an RNA binding activity (30). A link between histone modifications and the formation of a repres...

PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components Grande, M.A.; van der Kraan, K.; van Steensel, B.; Schul, W.; de The, H.; van der Voort, H.T.M.; de Jong, L.; van Driel, R. Link to publicationCitation for published version (APA): Grande, M. A., van der Kraan, K., van Steensel, B., Schul, W., de The, H., van der Voort, H. T. M., ... van Driel, R. (1996). PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components. Journal of Cellular Biochemistry, 63, 280-291. 3.0.CO;2-T" class="link">https://doi.org/10.1002/(SICI)1097-4644(19961201)63:33.0.CO;2-T General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D-distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual-labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2-snRNP, or transcription factors (glucocorticoid receptor, 'TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle-late S-phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RARa gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML-RAR fusion protcin, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non-APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bod...