A regulatory potential of the Xist gene promoter in vole M. rossiaemeridionalis

Abstract

X chromosome inactivation takes place in the early development of female mammals and depends on the Xist gene expression. The mechanisms of Xist expression regulation have not been well understood so far. In this work, we compared Xist promoter region of vole Microtus rossiaemeridionalis and other mammalian species. We observed three conserved regions which were characterized by computational analysis, DNaseI in vitro footprinting, and reporter construct assay. Regulatory factors potentially involved in Xist activation and repression in voles were determined. The role of CpG methylation in vole Xist expression regulation was established. A CTCF binding site was found in the 5' flanking region of the Xist promoter on the active X chromosome in both males and females. We suggest that CTCF acts as an insulator which defines an inactive Xist domain on the active X chromosome in voles.

Figure 1. mVista plot of Xist 5′ end and upstream sequences based on LAGAN alignment with reference sequence Microtus rossiaemeridionalis.

The horizontal and vertical axes represent the position in the sequences (in base-pairs) and the percent identity of the two sequences (50–100%), respectively. Parameters: Calc Window – 20 bp, Min Cons Width – 20 bp. Regions in which the identity is higher than or equal to 70% are colored in pink (non-coding regions), or blue (coding region).

Figure 2

(A) Multiple alignment of CNS3 sequences of Xist 5′ regulatory regions in different mammalian lineages. (Bt) Bos taurus; (Cf) Canis familiaris; (Dn) Dasypus novemcinctus (armadillo); (Ec) Equus caballus (horse); (Hs) Homo sapiens, (La) Loxodonta africana (elephant); (Oc) Oryctolagus cuniculus (rabbit); (Sa) Sorex araneus (shrew); (Ss) Sus scrofa (pig); (Cp) Cavia porcellus (guinea pig); (Mm) Mus musculus; (Rn) Rattus norvegicus; (Mr) M. rossiaemeridionalis (vole). Putative CTCF binding sites are shown with yellow frames. Consensus of CTCF binding site is present . (B) Alignment of Xist minimal promoter. Human CTCF is shown with red frames . Vole AP2 binding site is shown with blue frame.

Figure 3. DNAse I in vitro footprinting analysis of the 5′-region of the vole Xist gene.

(A) and (B) footprinting of the «+» strand of the minimal promoter. (C) «−» strand. Lane C is a control digestion without nuclear extract. Nuclear extracts were used in binding reactions: 5 µg (lane 1) and 10 µg (lane 2). (D) Footprinting with the recombinant transcription factor SP1 (1 µg of the protein). The binding reactions were performed with 1 µl (lane 1) and 2 µl (lane 2) of the template; G, G+A, C+T, and C are the corresponding Maxam–Gilbert sequencing reactions. Vertical bars and numbers indicate footprints. (E) footprinting analysis of the second conserved region and the adjacent regions.

Figure 4. Analysis of activity of reporter constructs.

Scheme of the Xist 5′ region is shown above; CNS1 and CNS2 are shown with green and red rectangles, respectively. pCx1–pCx14-Me are the variants of reporter constructs in the vector pGL4.10[luc2]; nucleotide positions relative to the Xist transcription start site are shown to the left. Diagram shows the relative luciferase activity of the constructs in female vole fibroblast culture (Sd10). Arrow and black dots indicate the Xist transcription start site and methylated CpG dinucleotides, respectively; R.U., relative luciferase activity units. Significant differences *** P≥0.999, ** P≥0.99, and * P≥0.95.

Figure 5. Analysis of the effect of −43G/A substitution on the activity of reporter constructs.

Schemes of the constructs pCx14G/A and pCx5G/A are shown to the left; green and red rectangles denote the conserved regions CNS1 and CNS2, respectively. Relative luciferase activity of constructs in the fibroblast culture Sd10 is shown to the right. Arrow shows the Xist transcription start site; R.U., relative luciferase activity units.

Figure 6. CTCF interacts with the Xist allele on the active X chromosome in voles.

Sequencing of the vole Xist minimal promoter using as a template Sad4 or Sa006 genomic DNA (A), CTCF-bound fraction from line Sad4 (B), CTCF-bound fraction from line Sa006 (C).

Figure 7. Methylation profile of the Xist 5′ regulatory region in the Sa006 and Sad4 vole fibroblast lines.

White circles denote unmethylated CpG sites; black, methylated; the Xist transcription start site is indicated by arrow; Xa, active X chromosome; Xi, inactive X chromosome; M. r., Microtus rossiaemeridionalis.

Figure 8. CTCF interaction with the vole Xist regulatory region.

(A) ChIP performed using anti-CTCF monoclonal antibodies (CTCF Ab) followed by PCR analysis. The fourth Xist exon and Igf2/H19 ICR were used as negative and positive controls of CTCF binding, respectively. (B) Real-time PCR analysis of ChIP experiments. Each bar indicates the average of two independent PCR analyses with the standard deviation. Percent of input is calculated as described in Materials and Methods.