The inactive X chromosome adopts a unique three-dimensional conformation that is dependent on Xist RNA

Abstract

Three-dimensional topology of DNA in the cell nucleus provides a level of transcription regulation beyond the sequence of the linear DNA. To study the relationship between the transcriptional activity and the spatial environment of a gene, we used allele-specific chromosome conformation capture-on-chip (4C) technology to produce high-resolution topology maps of the active and inactive X chromosomes in female cells. We found that loci on the active X form multiple long-range interactions, with spatial segregation of active and inactive chromatin. On the inactive X, silenced loci lack preferred interactions, suggesting a unique random organization inside the inactive territory. However, escapees, among which is Xist, are engaged in long-range contacts with each other, enabling identification of novel escapees. Deletion of Xist results in partial refolding of the inactive X into a conformation resembling the active X without affecting gene silencing or DNA methylation. Our data point to a role for Xist RNA in shaping the conformation of the inactive X chromosome at least partially independent of transcription.

Characterization and dynamics of pericentromere-associated domains in mice

“…Distances were measured manually from the center of each BAC signal to the edge of the closest chromocenter (marked by satellite probe) using ImageJ software as previously described (Splinter et al 2011). Association was called when distances between the center of the BAC signal and the edge of the closest chromocenter were <0.3 μm, the maximum distance at which visual associations (touching signals) have been observed in our measurements.…”

“…For probe labeling, the lacO transgene and a bacterial artificial chromosome (BAC) across the Llgl2 locus (RP23-143F14) were fragmented with Sau3A, while major satellite repeats were PCR-amplified from diluted genomic DNA to obtain fragments corresponding to 1-3 repeats. Probes were labeled as previously described (Splinter et al 2011) with the BAC, lacO transgene, and satellites labeled in green, Cy3 and Cy5, respectively (Cat. #42845, 42501, and 42502, Enzo Life Sciences).…”

“…1A), we used the 234-base pair (bp) major satellite repeat unit as a viewpoint in a chromosome conformation capture approach followed by high-throughput sequencing (4C-seq) (Simonis et al 2006;Splinter et al 2011). Pericentromeric DNA is organized in tandem arrays of ∼200,000 major satellite repeats (Martens et al 2005), so we had to adapt the standard 4C strategy to prevent sequencing of the thousands of satellite-to-satellite ligation events (see Methods; Supplemental Fig.…”

Gene Silencing and Polycomb Group Proteins: An Overview of their Structure, Mechanisms and Phylogenetics

“…For a better understanding of histone modifications and chromatin, a number of excellent reviews (Grimaud et al, 2006;Ringrose and Paro, 2004;Schuettengruber et al, 2007) focus on these subjects. Moreover, Pc complexes contribute crucially during embryogenesis, tissue differentiation (Pietersen and van Lohuizen, 2008;Sparmann and van Lohuizen, 2006) and tumorigenesis (Schlesinger et al, 2007;Widschwendter et al, 2007) through X-chromosome inactivation (Casanova et al, 2011;Splinter et al, 2011) and regulation of imprinted genes ( Jullien et al, 2006;Makarevich et al, 2006;Schubert et al, 2006). Pc complexes are also associated with nuclear reprogramming and chromatin remodeling (for a review, see Golbabapour et al, 2011).…”

Linking dosage compensation and X chromosome nuclear organization inC. elegans

“…Xist spreads along the entire X chromosome and represses transcription by multiple mechanisms. 28 Xist binding changes the chromatin conformation of the X chromosome 29 and its spreading depends on the 3D organization of the X chromosome. Chromosome Conformation Capture techniques demonstrated that the first regions bound by Xist are in close proximity to the Xist locus in the nuclear space.…”