Dynamics of the Two Heterochromatin Types during Imprinted X Chromosome Inactivation in Vole Microtus levis

Abstract

In rodent female mammals, there are two forms of X-inactivation – imprinted and random which take place in extraembryonic and embryonic tissues, respectively. The inactive X-chromosome during random X-inactivation was shown to contain two types of facultative heterochromatin that alternate and do not overlap. However, chromatin structure of the inactive X-chromosome during imprinted X-inactivation, especially at early stages, is still not well understood. In this work, we studied chromatin modifications associated with the inactive X-chromosome at different stages of imprinted X-inactivation in a rodent, Microtus levis. It has been found that imprinted X-inactivation in vole occurs in a species-specific manner in two steps. The inactive X-chromosome at early stages of imprinted X-inactivation is characterized by accumulation of H3K9me3, HP1, H4K20me3, and uH2A, resembling to some extent the pattern of repressive chromatin modifications of meiotic sex chromatin. Later, the inactive X-chromosome recruits trimethylated H3K27 and acquires the two types of heterochromatin associated with random X-inactivation.

Transcriptome Characteristics and X-Chromosome Inactivation Status in Cultured Rat Pluripotent Stem Cells

“…S8). This alternating pattern was previously found on the inactive X chromosome of other placental mammals and seems to reflect two different types of facultative heterochromatin acting in XCI [15,16,[38][39][40][41]. However, no H3K9me3 enrichment (excluding the centromeric region) was detected on the inactive X chromosome in the female rEFs used for the NF13 line generation.…”

“…Nuclei and metaphase spread preparation, immunofluorescence staining (IF), IF-RNA-DNA FISH, IF-DNA FISH, as well as BrdU incorporation, and detection were performed as described [15,16]. Xist RNA was detected using BAC PR23 31G17 as a probe.…”

“…5B-D and Supplementary Fig.S8). Using a combination of IF, RNA, and DNA FISH, we tested X chromosomes for loss of active chromatin marks and enrichment of repressive histone modifications that had been found on the inactive X chromosome in other mammals [15,16,[38][39][40][41]. In all the three cell lines, shortly after Xist RNA coating, X chromosome undergoing XCI was depleted in H3K4me2, an active chromatin marker, and enriched with H3K27me3 and ubiquitylated H2A (uH2A), repressive chromatin modifications.…”

The making of a Barr body: the mosaic of factors that eXIST on the mammalian inactive X chromosome

“…This domain becomes enriched with XIST, H3K27me3, ubH2A, H4K20me1, H3K9me2 and MacroH2A during the course of XCI. The second domain is enriched with H3K9me3, HP1 and H4K20me3, marks generally associated with constitutive heterochromatin, and roughly coincides with the gene poor and LINE-rich G-dark chromosome regions (Brinkman et al 2006;Chadwick 2007;Chadwick and Willard 2004;Csankovszki et al 2001;Vaskova et al 2014). The functional and component similarity with constitutive and facultative heterochromatin is also reflected in the domains' replication timing, with the former domains replicating two hours prior to the latter domains during late S phase (Chadwick and Willard 2004).…”

“…The second heterochromatin domain of the Xi is characterized by the heterochromatin marks H3K9me3, H4K20me3 and HP1 ( (Brinkman et al 2006;Chadwick 2007;Chadwick and Willard 2004;Csankovszki et al 2001;Vaskova et al 2014). Initially, H3K9me2 and H4K20me1 marks are established across both heterochromatin domains, however only in Domain 2 are the enzymes SETDB1 and SUV420H2 recruited to further modify these methylated lysine residues (Chaumeil et al 2011;Koina et al 2009).…”