The Chromosome Organization in the Cell Nuclei: History
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The spatial organization of the genome into the cell nucleus plays a central role in controlling several genome functions, such as gene expression and DNA replication timing during the S-phase of the cell cycle. Here we show how chromosomes are organized in the cell nucleus according to the gene density and to the GC-level of the various chromosomal bands, allowing a corrected and coordinated gene expression during cell life. The human genome, such as the genome of the other mammals, is composed by two very different part: one very gene-dense, replicated at the onset of the S-phase, very GC-rich and the other endowed by opposite features. These two genomic compartments are localized in the chromosome distant one to each other, with regions having intermediate properties located between them. This determine a zig-zag organization of the larger chromosomes, to position the gene-poorest genome compartment at the nuclear periphery and the gene-richest one at the nuclear interior.

  • cancer
  • genome organization
  • cell nucleus
  • chromosomal bands

1. Radial nuclear organization of the chromosome territories

Using DNA probes to “paint” individual chromosomes with the fluorescence in situ hybridization (FISH) technique, it has been possible to visualize whole chromosome territories in the interphase nuclei allowing precise analyses to understand the organization of each chromosome in the nucleus[1]. Indeed, it was determined that individual chromosomes occupy specific radial positions according to their average gene density in proliferating cells[2]. This was demonstrated, for the first time, for two small human chromosomes, chromosomes 18 and 19, endowed with opposing properties taking into consideration the GC level of the genomic sequence and gene density. The authors found that the chromosome territory of the gene-poor/GC-poor chromosome 18 was located at the nuclear periphery, whereas the territory of the gene-rich/GC-rich chromosome 19 was located more internally in the nucleus[2][3]. The spatial organization in the cell nuclei of these two chromosomes has also been observed in other organisms such as mice[4] and primates[5], where the syntenic chromosomes show similar nuclear distribution.
Chromosomes 18 and 19 occupy different intranuclear positions (the former more peripheral and the latter more internal) because they are small and relatively homogeneous in their nucleotide composition and gene density, as previously shown[6][7][8][9]. Thus, these two chromosomes can occupy different nuclear locations due to their small size and different genomic features (Figure 1). On the other hand, larger chromosomes, being highly heterogeneous in terms of nucleotide composition and gene distribution, cannot occupy a specific position close to the nuclear periphery or more internally in the nucleus[10]. In this case, because the genomic regions with opposite properties are not contiguous but distant from each other, large and heterogeneous chromosomes can organize their different chromosomal regions using a “zig-zag” distribution to position all the GC-poorest DNA at the nuclear periphery and all the GC-rich DNA toward the inner part of the nucleus[10]. Genomic DNA with intermediate properties connects the above regions (Figure 1). Confirmation of this chromosomal organization in the nucleus was also obtained by localization in the cell nucleus of the compositionally fractionated DNA used as a probe in FISH experiments: the GC-rich DNA was located in the inner part of the nucleus. At the same time, the GC-poor was observed only in the nuclear periphery. This means that all GC-rich chromosomal bands reside in the nuclear interior, even if they belong to different chromosomes and even if they occupy different positions along each chromosome.
Figure 1. Chromosome organization in the cell nucleus. The left panel displays ideograms of four representative human chromosomes. Red and blue bands indicate the gene/GC-richest and the gene/GC-poorest bands, respectively. The other bands are endowed with intermediate properties. The right panel displays a schematic representation of the chromosomal band sequence distribution through interphase nuclei according to their genomic properties with respect to gene/GC content. Small chromosomes can be located at the nuclear edge or in the interior, with gene-poor chromosomes being located at the nuclear edge and gene-rich in the interior, in proliferating cells. Large chromosomes, generally heterogeneous in their genomic properties (such as the human chromosome 7), assume a “zig-zag” conformation and are positioned with the GC-richest bands exposed towards the interior of nuclei. Nucleolus organizer regions (NOR) containing chromosomes (such as the human chromosome 21) have associations with internal nuclear structure with the GC-poorest bands located close to the nucleoli.
Conversely, all the GC-poor bands, along with the centromeric sequences, are located at the nuclear periphery or close to the nucleolar periphery[2]. Indeed, in this latter case, chromosomes containing the rRNA gene clusters (Nucleolus organizer regions (NOR)), namely chromosomes 13, 14, 15, 21, and 22, reside close to the nucleolus, thus in these cases, the entire chromosomes are located in the inner part of the nucleus. However, their gene/GC-poorest bands maintain the same functional position in the peripheral part of the nucleolus, composed primarily by the centromeric and pericentromeric heterochromatin of the NOR-containing chromosomes. The rDNA clusters located in the short arm of these chromosomes are organized in a rosette mode, thus determining the nucleolus formation.
These results demonstrate that chromosome territories are endowed with a GC-level gradient increasing from the periphery to the inner nuclear compartment (Figure 1). This result was confirmed by further studies [11], showing the active gene sequences located in chromatin loops stretching into the nuclear interior and the gene-poor sequences located at the nuclear periphery.

2. The GC-richest and the GC-poorest nuclear compartments

More recent techniques were developed to study the organization of the genome in 3D within the cell nucleus. These are derived from the chromosome conformation capture (3C) method [12], namely the variants 4C (chromosome conformation capture-on-chip) [13], 5C (chromosome conformation capture carbon copy) [14], and Hi-C (high-throughput chromosome conformation capture) [15]. This latter variant, Hi-C, has greatly enhanced our knowledge of the nuclear chromatin architecture. Indeed, these methodologies have revealed the closeness of individual genomic sequences to other sequences by cross-linking of chromatin regions that are joined or very close to one another. In this way, intra- and inter-chromosomal contacts can be determined, and thus a 3D model of chromosome and gene interactions was developed. Hi-C has given us the most extensive view of the 3D organization of the genome as all interactions between chromosomal regions are sequenced and considered when the computer-generated map of the genome is constructed [16].
The analysis of the human genome’s spatial organization through Hi-C confirmed what was initially found by FISH, namely that gene-rich chromosomal regions are spatially linked, as demonstrated by the high number of contacts observed [15]. Interestingly, gene-poor chromosomes 13 and 18 make very few contacts with other gene-rich chromosomes such as 17, 19, and 22, confirming that chromosomes, and more precisely chromosomal bands, share similar or different positions based on their gene density [17].
The Hi-C data continued to enhance our knowledge on chromatin organization in the cell nuclei, revealing the presence of Topologically Associated Domains (TADs), a structural organization of the chromatin endowed by a loop structure where the zinc finger CCCTC-binding factor (CTCF) sites play an essential role [18][19]. CTCF sites are critical in TAD organization, with these sites being located at the base of the TAD loops, aiding in the insulation of the sequence present inside each loop. Thus, a TAD loop could be considered not only a structural but also a functional region of the genome isolated from the other neighbor TADs [20], and dysfunction of TADs is involved in human diseases [21] and leukemogenesis [22].
These biochemical–molecular approaches have disclosed the presence in interphase of two genomic compartments A and B [15], organized into TADs of various sizes, with the former compartment located more internally in the nucleus and the latter more towards the nuclear periphery and surrounding nucleoli [23][24], where heterochromatin is located. These studies clearly showed the correspondence of compartment A and B with the GC-richest and GC-poorest chromosomal band DNA, respectively [20]. Thus, information obtained with different methodologies (biochemical and cytogenetic), joining data from the high-resolution molecular Hi-C method (at Kb level resolution) to that involving molecular cytogenetic techniques (with a lower level of resolution), demonstrated a unified model of the organization of chromosomal DNA in the interphase nucleus. This has highlighted the correspondence of the GC-rich isochores (and the GC-rich chromosomal bands) with the TADs located more internally in the cell nucleus. On the other hand, the GC-poorest isochores, corresponding to the GC-poor chromosomal bands, correspond to the genomic regions with the highest number of TADs specifically located close to the nuclear envelope and thus also defined as Lamina Associated Domains (LADs) [20][25].

3. Gene location in the cell nucleus, and transcriptional activity

Since genes are part of chromosomes it also matters where those genes sit in relation to the body of their home interphase chromosome territory, i.e. intra-chromosomal organization. Indeed, gene loci can be located deep within chromosome territories, more towards the surface of the chromosome territories, or even at some distance from the main body of the chromosome territory, out on a chromatin loop. There is evidence that even within interphase chromosomes there is spatial organization of gene-rich and gene-poor areas, which has been revealed by FISH, with inactive genes more likely to be located in the interior of chromosome territories [23][26][27][28][29][30]. When chromosomes are located at the nuclear periphery, their active genes are generally pointed towards the nuclear interior and not at the nuclear envelope side [10][31].

The arrangement of genes within a chromosome territory would permit the active regions of the genome to be closer and exposed to the components, machinery and structures required for transcription and processing[32]. We know that active transcription can take place at the surfaces and around channels that inveigle their way into chromosomes[33]. However, some gene loci are located away from the main body of their chromosome territory out on loops to be transcribed at a distance from the chromosome[10][29][34][35], at transcription factories or splicing speckles[36][37], becoming colocalized with other genes[38][39]. This positioning of chromatin loops belonging to different chromosomes in the same nuclear compartments can determine chromosomal rearrangements such as translocations.

Several studies have explored the link between chromosomal abnormalities and their effect on gene expression [reviewed in [40]]. Due to the precise nature of chromosome positioning within the nucleus, according to the GC-level of the genomic regions composing each band, the gross changes created by chromosomal rearrangements lead to major spatial disorders that go beyond local and in-cis effects. Chromosomal anomalies determine a change in the nuclear location of the chromosomal bands located around the breakpoints, and this spatial repositioning can cause changes in the accessibility status of the chromatin involved in the rearrangement, with repercussions on the transcriptional regulation of genomic loci not directly altered by the rearrangements[41].

4. Conclusion

Many different experimental procedures have demonstrated the presence of two very different nuclear compartments, one endowed with a high GC level, a high gene density, a very early replication during the S-phase of the cell cycle, and a position in the inner part of the nucleus. Another compartment, generally located more peripherally in the nucleus, is endowed with opposite properties. Rearrangements between loci belonging to these two different compartments determines the repositioning of genes in a compartment with different environmental properties, this determining the ectopic activation or inactivation of the relocated genes.

This entry is adapted from the peer-reviewed paper 10.3390/cancers13225860

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