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Large-scale manipulation of radial positioning does not affect most aspects of genome organization

ABSTRACT: Although the spatial organization of the genome is closely linked to biological function, genome structure is highly stochastic. Within this heterogeneity, specific function-associated structural elements must be maintained, even when the nucleus is deformed due to physiological physical constraints. The radial positioning of genomic loci - their distance from the nuclear periphery - has long been considered an important feature of genome organization which is correlated with both structural elements and genomic activity. In the current study, we developed an experimental system for manipulating the radial positioning of the genome, by expressing the sperm-specific protein Prm1 in somatic cells. By microscopy, we observe that initial Prm1 nuclear foci develop within 72 hours into a large Prm1 focus occupying most of the nuclear interior, while the entire genome is driven towards the nuclear periphery, resulting in a 3-5 fold reduction in the volume that the genome occupies. Noting that this system enables isolation of a pure population of cells with reorganized nuclei, we then used Hi-C to study the effects of this perturbation. Remarkably, we find that interaction patterns are largely robust to this major nuclear reorganization, with minor changes which mostly reflect a strengthening of heterochromatin self-interactions. Our experimental system provides means for manipulating nuclear organization in a reproducible manner, potentially allowing to examine radial positioning decoupled from other features of genome organization. Highlighting the complementary nature of microscopic and genomic methods, our work further suggests a remarkable resilience of genome structure such that large-scale nuclear changes, including chromosome compression and changes in radial positioning, can occur without extensive alteration of functional genome organization.

ORGANISM(S): Homo sapiens

PROVIDER: GSE276554 | GEO | 2024/09/06

REPOSITORIES: GEO

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Project description:The chromatin of individual chromosomes is organized into chromosome territories (CTs) in the interphase nucleus. The spatial arrangement of CTs is non-random and evolutionarily conserved. The gene-dense and gene-poor CTs are positioned in the nuclear center and periphery, respectively. As candidates for key molecules involved in nuclear organization, we have investigated the nuclear actin-related proteins (Arps), which include the evolutionarily conserved actin-family together with conventional actin. We used a conditional knockout system with chicken DT40 cells to analyze the functions of the actin-related protein Arp6. Consistent with a previous identification of Arp6 in the SRCAP chromatin remodeling complex, the histone variant H2AZ was significantly decreased in the chromatin of Arp6-deficient cells. Most importantly, Arp6-deficient cells had impaired radial positioning of both gene-poor macrochromosome and gene-rich microchromosome CTs. A transcription microarray analysis of the cells suggests that the radial positioning of CTs impacts only a limited number of genes and plays an active role in repression, rather than in induction. As far as we know, this report is the first observation that an inner nuclear protein is required for the radial distribution of CTs, and will provide new insight into the mechanisms and physical significance of the positioning of CTs in the nucleus. The total RNA was extracted with RNeasy Maxi Kit (QIAGEN) according to the manufacturer protocol. The poly-A mRNA was isolated from total RNA using Oligotex-dT30 Super (JSR corporation) and biotinylated cRNA was synthesized according to Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix). Shortly, 2 ug of poly-A mRNA was reverse transcribed to cDNA using 100 pmol of a T7-Oligo (dT) Primer (Invitrogen), and biotinylated cRNA was prepared by in vitro transcription amplification. For hybridization, 15 ug of biotinylated cRNA was fragmented and added into a hybridization cocktail containing four biotinylated hybridization controls (bioB, bioC, bioD, and cre) as recommended by the manufacturer. GeneChip Chicken genome Arrays (Affymetrix) were hybridized with the cocktail at 45ËC for 16 hours. After washing with Non-Stringent wash buffer (6xSSPE, 0.01% Tween20) and staining with SAPE in the Affymetrix GeneChip Fluidics Station 400, the GeneChips were read using the Affymetrix GeneChip scanner 3000 7G. These systems and data analyses were operated with the GeneChip Operating Software 1.3.

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Large-scale manipulation of radial positioning does not affect most aspects of genome organization

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