Identification of Developmentally Specific Enhancers for Tsix in the Regulation of X Chromosome Inactivation

Abstract

X chromosome inactivation silences one of two X chromosomes in the mammalian female cell and is controlled by a binary switch that involves interactions between Xist and Tsix, a sense-antisense pair of noncoding genes. On the future active X chromosome, Tsix expression suppresses Xist upregulation, while on the future inactive X chromosome, Tsix repression is required for Xist-mediated chromosome silencing. Thus, understanding the binary switch mechanism depends on ascertaining how Tsix expression is regulated. Here we have taken an unbiased approach toward identifying Tsix regulatory elements within the X chromosome inactivation center. First, we defined the major Tsix promoter and found that it cannot fully recapitulate the developmental dynamics of Tsix expression, indicating a requirement for additional regulatory elements. We then delineated two enhancers, one classical enhancer mapping upstream of Tsix and a bipartite enhancer that flanks the major Tsix promoter. These experiments revealed the intergenic transcription element Xite as an enhancer of Tsix and the repeat element DXPas34 as a component of the bipartite enhancer. Each enhancer contains DNase I-hypersensitive sites and appears to confer developmental specificity to Tsix expression. Characterization of these enhancers will facilitate the identification of trans-acting regulatory factors for X chromosome counting and choice.In mammals, the expression of X-linked genes is equalized in males and females by the transcriptional inactivation of one X chromosome in the female (2, 25). During embryogenesis, random X chromosome inactivation (XCI) occurs as the epiblast lineage differentiates and is regulated by counting and choice mechanisms, such that one randomly selected X chromosome remains active in each cell (33). Appropriate developmental regulation of XCI requires the X chromosome inactivation center (Xic), a cis-acting locus on the X chromosome that functions as a binary switch between the on and off transcriptional states (34). Molecular and genetic analyses of the Xic have focused on two genes, Xist and Tsix, that are integral to its function. Xist produces a large noncoding RNA that triggers X chromosome inactivation and coats the inactive X chromosome (6,7,27,31), while Tsix, the antisense partner of Xist, antagonizes Xist expression and negatively regulates X chromosome inactivation (11,21,22,35). Thus, Tsix works genetically upstream of Xist, and the opposing interplay between these two genes determines the pattern of XCI in the female cell.Many aspects of Tsix-Xist dynamics remain poorly understood. Because the suppression and induction of Xist are regulated by Tsix, understanding how XCI is regulated in turn requires an understanding of how Tsix expression is controlled. During cell differentiation, Tsix expression is silenced asynchronously on the active and inactive X chromosomes (X a and X i , respectively) (21, 22). On the X i chromosome, the loss of Tsix expression permits the upregulation of Xist and chromosome inactivation in cis. ...

IGF2: Epigenetic regulation and role in development and disease

“…(B) Model of allele-specific repression in X chromosome inactivation by CTCF. Adapted from [26][27][28]. DMR: differentially methylated region.…”

“…In a region implicated in controlling both random and imprinted X chromosome inactivation, functional methylation-sensitive CTCF binding sites were identified ( Figure 1B). This region was later found to contain developmentally specific enhancers [28] and to be differentially methylated in vivo [27]. CTCF has since been demonstrated to control imprinting at several other gene domains, and putative binding sites have been discovered in several other imprinted loci [32].…”

Natural and artificial routes to pluripotency

“…Xic functions both in trans, through transient pairing of the two X chromosomes that marks the onset of XCI (Xu et al, 2006), and in cis through the non-coding RNAs Xite, Tsix, and Xist located within it. Allele-specific upregulation of Xist on the future inactive X chromosome (Xi) is preceded by the down-regulation of the antisense transcript Tsix and the enhancer-like Xite (Ogawa and Lee, 2003;Stavropoulos et al, 2005). Xist coats the future Xi, initiating a cascade of epigenetic events that result in transcriptional silencing of the future Xi along its entire length.…”

Expression Profile and Localization of Mouse Calcitonin (CT) Sense/Antisense Transcripts in Pre- and Postnatal Tissue Development

“…Co-localization of the sense and antisense transcripts of the CT gene suggests that they may operate in regulatory networks during development; this is consistent with a previous study [18] indicating that the treatment of antisense oligonucleotide of CT mRNA caused severe impairment of embryo implantation. Taken together, these findings suggest that, like Xist/Tsix gene [8,13], CT antisense transcripts might be involved in control of CT gene expression.…”