High-salt–recovered sequences are associated with the active chromosomal compartment and with large ribonucleoprotein complexes including nuclear bodies

Abstract

The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the high-salt–recovered sequences-sequencing (HRS-seq) method, a straightforward genome-wide approach whereby we isolated and sequenced genomic regions associated with large high-salt insoluble RNP complexes. By using mouse embryonic stem cells (ESCs), we showed that these regions essentially correspond to the most highly expressed genes, and to cis-regulatory sequences like super-enhancers, that belong to the active A chromosomal compartment. They include both cell-type–specific genes, such as pluripotency genes in ESCs, and housekeeping genes associated with nuclear bodies, such as histone and snRNA genes that are central components of Histone Locus Bodies and Cajal bodies. We conclude that HRSs are associated with the active chromosomal compartment and with large RNP complexes including nuclear bodies. Association of such chromosomal regions with nuclear bodies is in agreement with the recently proposed phase separation model for transcription control and might thus play a central role in organizing the active chromosomal compartment in mammals.

Exploring Mammalian Genome within Phase-Separated Nuclear Bodies: Implications for the Regulation of Gene Expression

“…Indeed, our recent work in mouse embryonic stem cells showed that HRS include sequences associated with nuclear bodies (like the Cajal bodies, the HLBs, the speckles and paraspeckles). Moreover, transcriptional condensates formed around super-enhancers are also retained in our assay [52]. In full agreement with the first prediction, we found that two classes of genes can be defined according to the criterion of their association (or lack thereof) with large high-salt insoluble RNP complexes [52].…”

“…Moreover, transcriptional condensates formed around super-enhancers are also retained in our assay [52]. In full agreement with the first prediction, we found that two classes of genes can be defined according to the criterion of their association (or lack thereof) with large high-salt insoluble RNP complexes [52]. Our work showed that HRS-located genes are highly expressed and associated with the active chromosomal compartment and active super-enhancers in a cell-type specific manner, while genes that do not lie in HRS are moderately or weakly expressed.…”

“…So far, these sequences have been difficult to analyse because no efficient and reliable method was available to purify nuclear bodies, presumably due to their membrane-less phase-separated nature. We have recently shown that performing a liquid (or liquid-like) to solid phase transition through high-salt treatments of transcriptionally active nuclei makes large RNP complexes, including nuclear bodies, insoluble [52]. This traps the genomic DNA associated with these complexes.…”

“…Figure 2. Principle of the HRS-seq method allowing the high-throughput identification of genomic sequences significantly associated with large RNP complexes and nuclear bodies (adapted from [52]).…”

Early Polycomb-target deregulations in Hutchinson-Gilford Progeria Syndrome revealed by heterochromatin analysis

“…1h, i, Supplementary Table 2). Of note, other chromatin fractionation-based NGS methods have been previously described 41,42 , however they are not directly comparable to our technology as they adopt different conditions, enzymes and cellular models.…”