4.5.1 The interchromosome domain model
A functional importance of interchromatin space was postulated by Peter Lichter and colleagues (Zirbel et al., 1993). Accordingly interchromatin spaces would define a 3D network-like compartment, termed the interchromosome domain (ICD), which starts from nuclear pores and invades the nuclear interior in channels surrounding CTs (Zirbel et al., 1993, Fig. 1 G). The model was based on the finding that RNA transcripts and components of the splicing machinery are basically excluded from the interior of CTs, since CTs and RNA transcripts seemed to be spatially exclusive (Zirbel et al., 1993). The space of the ICD was postulated to define a structural and functional compartment, where nuclear components involved in transcription and mRNA processing are accumulated. The model implicates that
transcribed genes are located in the periphery of the corresponding CT. Investigations on the localization of a set of active and inactive genes with respect to their CT was performed in different cell lines and compared with the localization of non-coding sequences (Kurz et al., 1996). In support of the ICD model, genes were localized at the periphery of the respective CTs with no respect to their transcriptional status. Studies on the localization of highly expressed loci (see 4.1.4) are in accordance with the ICD model as well.
Additionally, the detection of a network of vimentin fibers in interchromatin areas (Bridger et al., 1998), which was aligning but never invading CTs, further supported the view of a CT surrounding channel system of functional importance.
The ICD model was challenged, however, when active loci were found within the very interior of a CT as well (Mahy et al., 2002b), and when it was demonstrated that a strict organization of active chromatin regions with respect to the CT periphery does not exist (Kupper et al., 2007).
4.5.2 The chromosome territory-interchromatin compartment model
Further subsequent studies also challenged the view of active processes exclusively localized at the periphery of CTs, since also ongoing transcription (Abranches et al., 1998; Cmarko et al., 1999; Verschure et al., 1999) was reported to localize in the deep interior of FISH painted CTs. The chromosome territory- interchromatin compartment model (CT- IC model) accounted for these findings and was introduced by Thomas and Christoph Cremer (Cremer and Cremer, 2001). It basically represents an advancement of the preceding ICD model (Cremer and Cremer, 2001; Cremer et al., 2004b). Now, CTs are considered to be built with a sponge like conformation with extensions and invaginations which invade the CTs interior (Fig. 1 H). This architecture of a CT would be mediated by a hierarchy of domains as described by the MLS model (Munkel et al., 1999; Sadoni et al., 2004): Several DNA loops with a length of 30-200 kb (~100 kb domains) build up sub- compartments termed according to their rough DNA content '~1 Mb chromatin domains', which correspond to foci detected by replication labeling (Jackson and Pombo, 1998, see 4.1.4). Computer modeling of CTs built up from such sphere-like ~1 Mb chromatin domains, yielded an interchromatin space of variable width expanding between the spherical domains (Kreth et al., 2004b). In such a porous architecture of a CT, with interchromatin channels pervading its entire volume, the functional machinery can access the interior of a CT. Accordingly, active processes are no longer restricted to locate at the CTs periphery (ICD model), but can be situated at the surface of a chromatin domain within a CT, which is in full agreement with the experimental data stated above. Still, a topological separation of chromatin and interchromatin spaces is postulated, since active processes are still postulated to locate at the border between chromatin and interchromatin space (Cremer andCremer, 2001). This proposed complex architecture of the interchromatin space was emphasized by terming it the interchromatin compartment (IC). The postulated restriction of functional processes to the border of chromatin domains is further consistent with the mentioned EM observations, which demonstrated that ongoing transcription, splicing and replication are exclusively observed at the perichromatin region (Cremer et al., 2004b, see 4.4.3)
4.5.2.1 In-/Accessibility of chromatin domains
The postulation that active processes are restricted to the surfaces of chromatin domains raised the question if the interior of these compact chromatin domains is indeed physically inaccessible for molecules in the size range of e.g. transcription factors or preassembled functional machineries. Several studies addressed that question by the use of micro-injected FITC-conjugated dextrans of varying size or FITC-poly-L-lysine in the nuclei of living cells. Accordingly, small dextrans (3-42 kDa) displayed homogeneous signal distributions with no preferential exclusion from chromatin regions (Gorisch et al., 2003; Verschure et al., 2003). With increasing masses (77- 2500 kDa), however, FITC dextrans were progressively excluded from chromatin regions (Verschure et al., 2003) and accumulated in interchromatin channels (Gorisch et al., 2003). However, 70-kDa dextrans were detected in some compact chromatin regions, demonstrating that the overall exclusion is not necessarily due to a strict size threshold at this level (Verschure et al., 2003). These results implied that different types of condensed chromatin domains may exist, distinguishable by their accessibility to 70-kDa dextrans. Anionic FITC-dextrans (500-kDa) were exclusively observed in interchromatin spaces, whereas positively charged FITC-poly-L-lysine was to some extent also detected within the chromatin regions (Gorisch et al., 2003). Görisch et al. therefore concluded that the nucleoplasmic accessibility for macromolecules not only depends on molecule size but additionally on electrical charge properties.
However, limitations in accessibility were only detected in size ranges greater than that of components of the transcription machinery. This result suggested that these molecules can diffuse freely also inside condensed chromatin domains. In accordance with this claim RNA-polymerase II as well as the transcription factor TFIIH was found homogenously distributed throughout the nucleoplasm (Verschure et al., 2003). Nevertheless there seems to be a size threshold in the range of 70 kDa dextrans, consistent with the observation that large hnRNP particles localized in interchromatin channels demonstrated when poly-(A) RNA molecules were tagged in living cells (Politz et al., 1999, see 4.4.1).
The claim of limited accessibility was further challenged by living cell studies and diffusion measurements obtained by the observation of transfected GFP-tagged nuclear proteins. Applying either FRAP (fluorescence recovery after photobleaching) or FCS (fluorescence
for the heterochromatin protein HP1 (Cheutin et al., 2003; Festenstein et al., 2003). It is important to note, however, that a decreased mobility of HP1 was observed in heterochromatic (compact) versus euchromatic (decondensed) chromatin regions in unstimulated T cells (Festenstein et al., 2003).
Taken together, simple steric exclusions are not sufficient to explain the restricted localization of active processes to chromatin domain surfaces. Considering these results two functional scenarios were put up (Cremer and Cremer, 2006b) to explain the observed topology of functional processes: [1] parts of functional machineries may pre-assemble in the IC forming macromolecules with limited access to the interior of domains according to a size threshold and/or [2] a cascade of protein binding interactions forming a functional machinery would start at promoters exposed at a domain surface, which is the closest region for proteins released from neighboring aggregates like speckles or bodies. The advantage of the second scenario would be a closer pathway for the interactions increasing the probability for binding partners to meet.