Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation

Abstract

X-chromosome inactivation (XCI) is highly dynamic during early mouse embryogenesis and strictly depends on the Xist noncoding RNA. The regulation of Xist and its antisense partner Tsix remains however poorly understood. We provide here the first evidence of transcriptional control of Xist expression. We show that RNA polymerase II (RNAPolII) preinitiation complex recruitment and H3 Lys 4 (H3-K4) methylation at the Xist promoter form the basis of the Xist expression profiles that drives both imprinted and random XCI. In embryonic stem (ES) cells, which are derived from the inner cell mass where imprinted XCI is reversed and both Xs are active, we show that Xist is repressed at the level of preinitiation complex (PIC) recruitment. We further demonstrate that Tsix, although highly transcribed in ES cells, is not itself responsible for the transcriptional down-regulation of Xist. Rather, Tsix induces efficient H3-K4 methylation over the entire Xist/Tsix unit. We suggest that chromatin remodeling of the Xist locus induced by biallelic Tsix transcription renders both Xist loci epigenetically equivalent and equally competent for transcription. In this model, Tsix, by resetting the epigenetic state of the Xist/Tsix locus, mediates the transition from imprinted to random XCI.

Figure 1.

ChIP analysis of histone modifications within the Xist/Tsix region in female TS cells and in MEFs. (A) Schematic representation of the Xist/Tsix locus. Xist exons are represented by black boxes, and Tsix exons are represented by white boxes. Dotted arrows indicate the positions of the primer pairs used in the ChIP analysis. (B-D) Analysis of H3-K4 dimethylation (B), H3-K9 dimethylation (C), and H3-K27 trimethylation (D) in female TS cells and MEFs. In TS cells, two allelic primers (X1, black bars, and X11, gray bars) allowed us to distinguish the paternal allele (Xi) from the maternal (Xa). White bars (XEx5) correspond to a primer pair detecting both the paternal and maternal alleles. As both X chromosomes in female MEFs are of 129/Sv origin, they cannot be distinguished using allelic primers. All graphs were obtained after normalization with position XEx5 located in the body of Xist.

Figure 2.

Binding of the transcription machinery to the Xist/Tsix region in female TS cells and in MEFs. (A,B) Binding of RNA polymerase II (A) and TFIIB (B) in female TS cells. As in Figure 1, the positions corresponding to the Xist P1 promoter (X1) and the Tsix promoter (X11) are in black and gray, respectively. Allelic primers for X1 and X11 allowed us to distinguish the maternal and paternal alleles. All the other positions (in white) detect both paternal and maternal alleles. Male TS cells exhibit a profile similar to that of the maternal X chromosome of female TS cells (data not shown). (C,D) Similar analysis in female and male MEFs. The graphs show the fold-enrichment value of each position compared with position X3.

Figure 3.

Xist down-regulation in undifferentiated ES cells is regulated by repression of PIC recruitment to the P1 Xist promoter. (A,B) Binding of RNA polymerase II (A) and TFIIB (B) in female ES cells (LF2). Identical results were obtained in male ES cells (data not shown). Graphs show the fold-enrichment value of each position compared with position X3. (C) Comparative analysis of the Xist/Tsix transcripts ratio (black bars) with the ratio of TFIIB binding to Xist P1 promoter versus Tsix promoter (gray bars) in female TS cells, ES cells, and MEFs. The RNA levels of Xist and Tsix were determined by real-time PCR using primers XcR1 for Xist and X6 for Tsix, and standardized against the mRNA levels of Arpo P0.

Figure 4.

Transcriptional and chromatin analyses of Tsix-truncated undifferentiated male ES cells. Ma2L cells (white bars) carry a transcriptional stop signal 4 kb downstream of the Tsix promoter. Ma1L (black bars) is the corresponding control cell line. (A) ChIP analysis of RNA polymerase II distribution within the Xist/Tsix region in Ma1L and Ma2L cell lines. The position of the stop signal is indicated in the graph. (B) ChIP analysis of TFIIB binding to the Xist P1 and P2 promoters and the Tsix promoter in Ma1L and Ma2L. (C) Extensive ChIP analysis of H3-K4 dimethylation within the Xist/Tsix locus in Ma1L and Ma2L. The graphs show the fold-enrichment value of each position compared with position X3. The schematic map below the graph shows the position of the primers used and of the stop signal.

Figure 5.

A model of the regulation of the Xist/Tsix region during early mouse development. Developmental stages are shown on the left, and the status of the Xist/Tsix region in terms of transcription and chromatin at each stage is shown on the right. (A) In the preimplantation embryo, the segregation of PIC together with H3-K4 dimethylation over the paternal and maternal Xist and Tsix promoters ensures paternal Xist and maternal Tsix expression, respectively. Bold arrows represent high levels of expression, dotted arrows represent low levels of expression. (B) At the implantation stage, an unknown ICM-specific repressive mechanism of PIC recruitment at P1 Xist promoter avoids biallelic X-inactivation (represented by a question mark and a transparent PIC at the Xist promoter). In the meantime, biallelic Tsix activation erases the histone methylation pattern by inducing high levels of H3-K4 dimethylation across the region (pink arrows), rendering both Xist alleles epigenetically equivalent. (C) At the onset of random X-inactivation, monoallelic down-regulation of Tsix results in an increase of H3-K4 dimethylation over the corresponding Xist promoter region. The absence of ICM-specific Xist repression allows stable binding of the transcriptional apparatus, inducing high levels of monoallelic Xist expression and X-inactivation. (D) In the female epiblast, other epigenetic marks lock in the expression profile of Xist and the silencing of Tsix transcription.