XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells

Abstract

Background: The establishment of facultative heterochromatin by X-chromosome inactivation requires the long non-coding RNA XIST/Xist. However, the molecular mechanism by which the RNA achieves chromosome-wide gene silencing remains unknown. Mouse Xist has been shown to have redundant domains for cis-localization, and requires a series of well-conserved tandem 'A' repeats for silencing. We previously described a human inducible XIST transgene that is capable of cis-localization and suppressing a downstream reporter gene in somatic cells, and have now leveraged these cells to dissect the sequences critical for XIST-dependent gene silencing in humans.

Results: We demonstrated that expression of the inducible full-length XIST cDNA was able to suppress expression of two nearby reporter genes as well as endogenous genes up to 3 MB from the integration site. An inducible construct containing the repeat A region of XIST alone could silence the flanking reporter genes but not the more distal endogenous genes. Reporter gene silencing could also be accomplished by a synthetic construct consisting of nine copies of a consensus repeat A sequence, consistent with previous studies in mice. Progressively shorter constructs showed a linear relationship between the repeat number and the silencing capacity of the RNA. Constructs containing only two repeat A units were still able to partially silence the reporter genes and could thus be used for site-directed mutagenesis to demonstrate that sequences within the two palindromic cores of the repeat are essential for silencing, and that it is likely the first palindrome sequence folds to form a hairpin, consistent with compensatory mutations observed in eutherian sequences.

Conclusions: Silencing of adjacent reporter genes can be effected by as little as 94 bp of XIST, including two 'monomers' of the A repeat. This region includes a pair of essential palindromic sequences that are evolutionarily well-conserved and the first of these is likely to form an intra-repeat hairpin structure. Additional sequences are required for the spread of silencing to endogenous genes on the chromosome.

Figure 1

The repeat A region of XIST is necessary and sufficient for silencing of flanking reporter genes. (A) Approximate location of genes analyzed on chromosome 3 relative to the schematic of full-length XISTcDNA construct showing regions included in shorter XIST constructs and location of qRT-PCR primer pairs p1 to p4 and p5 (vector primer pair used to amplify all XIST constructs). (B) Enhanced Green Fluorescent Protein gene (EGFP) expression following one to five days (d1 to d5) induction of full-length XIST or 5’A, measured by flow cytometry and shown relative to d0. (C) qRT-PCR analysis of expression within full length XIST transgene (p2) and upstream (p1) and downstream (p3, p4) of XIST sequence. Genomic DNA was used to normalize for amplification efficiency. Location of qPCR amplicon positions is shown in Figure 1A. (D) Expression of the reporter genes (Hygromycin gene (Hyg) and EGFP) and endogenous genes CLDN16 and IL1RAP following five days of transgene induction measured by qRT-PCR, relative to expression in uninduced cells (d0) and normalized to ACTB expression. Transgene constructs were full XIST, 5’A only, full XIST lacking the 5’A region or vector with no XIST as indicated. Error bars indicate ± 1 S.D. of four to six biological replicates. Significance (P-value <0.05) was calculated using a Mann–Whitney test comparing each transgene construct with the vector alone construct. (E) Allele-specific silencing of flanking endogenous genes following five days of transgene induction. The percent change in allelic ratio upon DOX induction relative to the ratio without DOX was measured by pyrosequencing for expressed polymorphisms in five genes up to 20 Mb from the integration site (see A). Transgene constructs were full XIST, 5’A only, full XIST lacking the 5’A region or vector with no XIST as indicated. Two technical replicates of three biological replicates were averaged for each datapoint.

Figure 2

Repeat A monomers additively contribute to silencing. (A) Human repeat A sequence consists of 8.5 copies of a well-conserved CG-rich core and T-rich spacer sequences. Palindromic sequences hypothesized to form a secondary structure are underlined. Artificial repeat A was constructed as a 9-mer repetition of consensus monomer sequence and restriction enzyme sites were introduced to allow for the creation of shorter constructs. (B) Enhanced Green Fluorescent Protein gene (EGFP) expression following five days of transgene induction as measured by qRT-PCR, relative to d0 and normalized to changes in expression caused by induction of the vector alone and to ACTB expression for two biological replicates. (C)EGFP expression was measured by flow cytometry every 2 days for 16 days following induction of repeat A 2-mer. Data are normalized to EGFP expression in cells that were not induced with DOX.

Figure 3

Mutation of the core repeat A sequences abrogates its silencing ability. (A) Sequence of the canonical repeat A monomer and four mutant constructs that were created to target the hypothesized repeat A hairpins. Underlined sequences correspond to stem 1 and stem 2. Dashes indicate no sequence change. (B) Mean Enhanced Green Fluorescent Protein gene (EGFP) expression following five days of transgene induction, measured by flow cytometry, relative to d0 (two-tailed paired t-test). Error bars indicate ± 1 S.D. of two single-cell clones. (C)EGFP and hygromycin (Hyg) gene expression following five days of transgene induction, measured by qRT-PCR, relative to d0 and normalized to ACTB expression for two independent single-cell clones.

Figure 4

Stem-loop 1 structure is required to maintain repeat A silencing ability. Silencing ability of 2-mer repeat A construct is retained when forced to form stem-loop 1 structure, but abrogated when pairing between the monomers is enforced. (A) Sequence of the canonical repeat A 2-mer and four mutant constructs that either enforce formation of stem-loop 1 (A1, A2) or an alternative folding (B1, B2) of repeat A sequences, as indicated by schematics. Dashes indicate no change in sequence. (B) Mean Enhanced Green Fluorescent Protein gene (EGFP) expression following five days of transgene induction, measured by flow cytometry, relative to d0 (two-tailed paired t-test). Error bars indicate ± 1 S.D. of two independent single-cell clones and a total of seven biological replicates.

Figure 5

Compensatory changes in putative stems of repeat A hairpin sequences of 27 mammals. (A) All bona fide repeat A core sequences that deviated in sequence from the canonical stem 1 sequence were categorized by their potential to form a base pair with a reciprocally-mutated base within the same repeat A unit or within another unit. (B) As in (A), but stem 2 is analyzed.