CHART-Seq reveals ∼100 discrete binding sites in autosomal genes.

A. Heatmap and profile of Xist coverage on autosomes and chromosome X genes in WT female ES cells at day 0, day 4, day 7, and day 14.

B. Representative consecutive binding signals of Xist on chromosome X (∼320 kb) in WT female ES cells on day 4 and day 7. WT male ES cells are used as control.

C-D. Two representative site-specific binding of Xist on autosome locus in WT female ES cells on day 4 and day 7, WT male ES cells are used as control.

E. Number of Xist peaks determined by MACS2 peak calling on autosomes in WT female ES cells at day 4, day 7, and day 14.

F. Distribution pattern of Xist peaks identified by MACS2 peak calling on autosomes in WT female ES cells.

G. 10 examples of Xist binding position information and related genes on autosomal chromosomes in WT female ES cells.

H. Feature annotation of Xist binding loci on autosomes in WT female ES cells.

Xist’s Repeat B is required for proper binding of Xist to autosomal genes.

A. Heatmap and profile of Xist coverage on autosomes and chromosome X genes in ΔRepB female ES cells at day 0, day 4, day 7, and day 14.

B. Number of Xist peaks determined by MACS2 peak calling on autosomes in ΔRepB female ES cells at day 4, day 7, and day 14.

C. Distribution pattern of Xist peaks identified by MACS2 peak calling on autosomes in ΔRepB female ES cells.

D. 10 examples of Xist binding position information and related genes on autosomal chromosomes in ΔRepB female ES cells.

E. Representative consecutive binding signals of Xist on chromosome X (∼320 kb) in WT and ΔRepB female ES cells on day 4 and day 7.

F-G. Two representative site-specific binding of Xist on autosome locus in WT and ΔRepB female ES cells on day 4 and day 7.

H. Feature annotation of Xist binding loci on autosomes in ΔRepB female ES cells.

Autosomal target genes are actively transcribed.

A. Xist preferentially targets actively expressed genes. The proportion of overall genes (label as “All”) and Xist targets on autosomes expression levels (label as “Autosomal”) in different expression categories. Genes are classified into distinct categories (Q1-Q5) based on RPKM values, representing various levels of expression. Category Q1 includes non-expressed genes (RPKM=0), while categories Q2, Q3, Q4, and Q5 represent 25%, 50%, 75%, and 100% expression, respectively.

B. Genes bound by Xist exhibit elevated expression levels compared to surrounding regions. The analysis of gene expression within the 10, 20, 50, and 100-kilobase binding regions of Xist is conducted in WT female ES cells at day 4.

C. The H2AK119ub and H3K27me3 (Chip-Seq) profile of genes within the 10 and 50-kilobase binding regions of Xist in WT female cells at day 4.

D-E. Exemplifying CHART-seq (D) and RNA-Seq (E) patterns of an X-linked gene (Med12) at day 7. Change in coverage (Δ1 and Δ2) is shown below (Δ1 for ΔRepB♀ -WT♀, and Δ2 for WT♂ -WT♀).

F-G. Representing CHART-seq (F) and RNA-Seq (G) patterns of an autosomal gene bound by Xist (Kmt2e) at day 4. Change in coverage (Δ1 and Δ2) is shown below (Δ1 for ΔRepB♀ -WT♀, and Δ2 for WT♂ -WT♀).

H. Assessing gene expression levels of Xist targets on autosomes (10 kb within the peak region) in WT, ΔRepB female ES cells, and male ES cells at different time points. P-values are determined using the Wilcoxon rank sum test.

Xist binding suppresses, but does not silence, autosomal gene expression.

A. RNA-seq track shows the Xist expression level in ectopic Xist overexpressed ES cell lines (Tg).

B. RNA-seq track shows the MED14 (X-linked gene) expression level in ectopic Xist overexpressed ES cell lines (Tg). Change in coverage (Δ) is shown below (Tg - Ctrl).

C-D. RNA-seq track illustrating expression alongside representative autosome genes with Xist binding (Bcl7B and Rbm14) in differentiated Xist Tg ES cells (neurons). Change in coverage (Δ) is shown below (Tg - Ctrl).

E. RNA-seq track illustrating expression level of autosome genes without Xist binding (Stau1) in differentiated Xist Tg ES cells (neurons). Change in coverage (Δ) is shown below (Tg - Ctrl).

F-G. Analysis of gene expression levels for Xist targets (F) or non-targets(G) on autosomes in differentiated Xist Tg ES cells (neurons). P-values are determined using the Wilcoxon rank sum test.

Treating cells with the X1 inhibitor of Xist RNA perturbs autosomal target genes.

A-D. RNA-seq track depicting Mecp2 (A) (an X-linked gene), Bcl7b (B) and Rbm14 (C) (Xist-regulated autosome target), and Cbx2 (D) (autosomal gene without Xist binding) expression in DMSO (control) and Xist inhibitor (X1) treated differentiated WT ES female cells at day 5 of differentiation. Change in coverage (Δ) is shown below (X1 - DMSO).

E. Evaluation of gene expression levels for Xist targets on autosomes in WT and X1 treated ES cells. P-values are determined using the Wilcoxon rank sum test.

F. Evaluation of gene expression levels for Xist non-targets on autosomes in WT and X1 treated ES cells.

G. ChIP-seq track for H3K27me3 on Bcl7b (within a 160 kb window) in WT and X1 treated ES cells. Change in coverage (Δ) is shown below (X1 - DMSO).

H-I. Profile plot displaying H3K27me3 levels for Xist-flanking genes (H) (within a 100 kb window) or Xist non-targets (I) in WT and X1 treated ES cells.

Xist RNA also binds autosomal genes in post-XCI MEF cells.

A. Coverage of CHART-Seq reads (input, Xist, and sense control) on Chromosome X in WT, ΔRepB, and ΔRepE female MEF cells.

B. Xist peak number (MACS2 peak calling) on autosomes in WT, ΔRepB, and ΔRepE female MEF cells.

C. Xist peak patterns (MACS2 peak calling) on autosomes in WT, ΔRepB, and ΔRepE female MEF cells.

D. Feature annotation of Xist binding loci on autosomes in WT female ES cells.

E. Representative CHART-seq and RNA-seq track patterns of Xist in WT, ΔRepB, and ΔRepE female MEF cells.

F-G. Heatmap (F) and profile (G) depicting Xist coverage on autosome targets and chromosome X in WT, ΔRepB, and ΔRepE female MEF cells.

Xist autosomal binding does not alter gene expression in MEF Cells.

A-C. Representative CHART-seq and RNA-seq track patterns of Xist autosomal binding genes such as Mecp2 (A) and Rbm14 (B), and autosome genes without Xist binding such as kmt2a (C) in WT, ΔRepB, and ΔRepE female MEF cells. Change in coverage (Δ1 and Δ2) is shown below (Δ1 for ΔRepB -WT, and Δ2 for ΔRepE -WT♀).

D-E. Gene expression levels for Xist targets (D) or non-targets (E) on autosomes in WT, ΔRepB, and ΔRepE female MEF cells show no obvious changes.

F-G. Gene expression levels for Xist targets on autosomes in male and female MEF cells show no obvious changes.

H. Schematic of the Xist autosome binding pattern influences the gene expression. During the differentiation process of female ES cells, the Xist is specifically expressed from the Xist loci of one X chromosome (Xi). It then binds to the Xist loci of the Xi in cis, spreading across this chromosome and effectively silencing the expression of most of its genes. Additionally, some Xist complex will also bind to hundreds of loci on autosomes, thereby inhibiting the expression levels of target genes located there.

Xist binds select autosomal genes in trans.

A. Xist CHART-seq reads percent of autosomes and Chromosome X in WT and ΔRepB female ES cells. ES day 7 exhibits the highest Xist coverage. Sense probe and male ES cells are used as a control. B-E. Representative binding signals of Xist RNA on Xist locus (B), genes such to XCI such as Cdkl5 (C), Mecp2 (D), and escapee genes (Kdm6a) (E) in WT female ES cells on day 4 and day 7. WT male ES cells and sense probe are used as control.

Genes bound by Xist exhibit higher expression levels and lower H3K27me3 and H2AK119ub binding levels.

A. Xist peak pattern (MACS2 peak calling) on autosomes in WT and ΔRepB female ES cells.

B-C. The analysis of gene expression within the 10, 20, 50, and 100-kilobase binding regions of Xist is performed in WT female ES cells at day 7 (B), and day 14 (C), respectively.

D. Profile plots showing H3K27me3 and H2AK119ub coverage over genes within the 10 and 50-kilobase binding regions of Xist in WT female ES cells.

Example of Xist binding on autosomal genes and influence on gene expression.

This figure illustrates CHART-seq and RNA-seq patterns of autosomal genes, including Srp9, Brf1, Thra, Cand2, and Kmt2c, which exhibit Xist binding on different days. Ces1l, which lacks Xist binding, is used as a control. Change in coverage (Δ1 and Δ2) is shown below (Δ1 for ΔRepB♀ - WT♀, and Δ2 for WT♂ -WT♀).

Genes not bound by Xist exhibit no changes in gene expression or differences in H3K27me3 and H2AK119ub signals.

A-C. The analysis of gene expression within the 10, 20, 50, and 100-kilobase randomly selected regions is performed in WT female ES cells at day 4 (A), day 7 (B), and day 14 (C), respectively.

D. Profile plots showing H3K27me3 and H2AK119ub coverage over genes within the 10 and 50-kilobase randomly selected regions in WT female ES cells at different time points.

E. Assessing gene expression levels of Xist non-targets on autosomes in WT, ΔRepB female ES cells, and male ES cells at different time points.