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

A. Top: Heatmaps of Xist coverage on autosomes and chromosome X genes in WT female ES cells at day 0, day 4, day 7, and day 14. Bottom: Average profiles shown as metagene maps of the data in the top panel. TSS, transcription start site. TES, transcription end site.

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

C. Representative site-specific binding of Xist on autosome locus (Kmt2e) in WT female ES cells at day 4 and day 7, WT male ES cells are used as control.

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

E. Xist CHART signal coverage on Xist-autosomal peak region (top 100 peaks) in WT female ES cells at day 4, day 7, and day 14. Sense and input are used as control. P-values are determined using the Wilcoxon rank sum test.

F. Average profile of CHART (subtracted input) signal on Xist-autosomal peak region (top 100 peaks) in WT female and male ES cells at day 4, day 7, and day 14. Sense and WT male ES are used as control.

G. Feature annotation of Xist binding loci on autosomes by ChIPseeker in WT female ES cells at day 4.

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

A. Top: Heatmaps of Xist coverage on autosomes and chromosome X genes in WT female ES cells at day 0, day 4, day 7, and day 14. Bottom: Average profiles shown as metagene maps of the data in the top panel.

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. Representative site-specific binding of Xist on autosome locus (Kmt2e) in WT and ΔRepB female ES cells at day 4 and day 7.

D. Feature annotation of Xist binding loci on autosomes by ChIPseeker in ΔRepB female ES cells at day 4.

E. Xist CHART signal coverage on Xist-autosomal peak region in WT and ΔRepB female ES cells at day 4, day 7, and day 14. P-values are determined using the Wilcoxon rank sum test.

F. Top: Heatmaps of Xist coverage on Xist-autosomal peak region (top 100 peaks) in WT and ΔRepB female ES cells at day4, day 7, and day 14. Bottom: Average profiles shown as metagene maps of the data in the top panel.

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. P-values are determined using the Wilcoxon rank sum test.

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

D. Evaluation of gene expression levels for XCI genes in WT and ΔRepB female ES cells at day4, day7, and day 14. P-values are determined using the Wilcoxon rank sum test.

E-F. Representative 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♀).

G. 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.

H. Gene expression levels for Xist targets on autosomes (identified in day4 and day7) in undifferentiated WT, ΔRepB, female and male ES cells (day 0) show no obvious changes. 2 biological replicates were used. 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 doxycycline responsive Xist overexpressed ES cells (129/Sv-Cast/Ei) of neuronal differentiation (Tg-1 and Tg-2 have different insertion sites), the control (Ctrl) was doxycycline-treated wildtype cell.

B. Analysis of gene expression levels for XCI genes in Xist Tg ES cells (129/Sv-Cast/Ei) at of neuronal differentiation. P-values are determined using the Wilcoxon rank sum test.

C. RNA-seq track illustrating expression alongside representative autosome genes with Xist binding (Rbm14) in Xist Tg ES cells (129/Sv-Cast/Ei) of neuronal differentiation. Change in coverage (Δ) is shown below (Tg - Ctrl).

D. Analysis of gene expression levels for Xist autosomal targets in Xist Tg ES cells (129/Sv-Cast/Ei) of neuronal differentiation. P-values are determined using the Wilcoxon rank sum test.

E. RNA-seq track illustrating expression level of autosome genes without Xist binding (Stau1) in Xist Tg ES cells (129/Sv-Cast/Ei) of neuronal differentiation. Change in coverage (Δ) is shown below (Tg - Ctrl).

F. Analysis of gene expression levels for Xist non-targets on autosomes in Xist Tg ES cells (129/Sv-Cast/Ei) of neuronal differentiation. 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 female ES cells at day 5 of differentiation. Change in coverage (Δ) is shown below (X1 - DMSO). The Xist peak on autosomes is from CHART at day 4.

E. Evaluation of gene expression levels for Xist autosomal targets in DMSO (control) and X1 treated WT female ES cells at day 5 of differentiation. The Xist peak on autosomes is from CHART at day 4. P-values are determined using the Wilcoxon rank sum test.

F. Evaluation of gene expression levels for Xist non-targets on autosomes in in DMSO (control) and X1 treated WT female ES cells at day 5 of differentiation. P-values are determined using the Wilcoxon rank sum test.

G. ChIP-seq track for H3K27me3 on Bcl7b (an Xist autosomal target, within a 160 kb window) in DMSO (control) and X1 treated WT female ES cells at day 5 of differentiation. Change in coverage (Δ) is shown below (X1 - DMSO).

H-I. Profile plot displaying H3K27me3 levels for Xist-autosomal targets flanking genes (H) (within a 100 kb window) or Xist non-targets (I) in DMSO (control) and X1 treated WT female ES cells at day 5 of differentiation. The Xist autosomal target is from CHART at day 4.

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

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

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

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

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

E-F. Heatmaps (E) and average profiles (F) 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. P-values are determined using the Wilcoxon rank sum test.

F-G. Gene expression levels for Xist targets on autosomes in male and female MEF cells show no obvious changes. P-values are determined using the Wilcoxon rank sum test.

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 autosomal genes in trans.

A. Spearman (rank-based relationship) and Pearson (linear relationship) correlation analysis show that a strong positive correlation between the two biological CHART sequencing.

B. Xist CHART-seq reads percent of autosomes and Chromosome X in WT and ΔRepB female ES cells at day 0, day 4, day 7, and day 14. ES day 7 exhibits the highest Xist coverage. Sense probe and male ES cells are used as a control.

C. Principal Component Analysis (PCA) of CHART-seq reads include Xist, sense, and input in WT and ΔRepB female, and male ES cells at day 4. Samples clustering closer together share similar genome-wide coverage patterns.

Xist binds X chromosome during female ES cell differentiation.

A-D. Representative binding signals of Xist RNA on Xist locus (A), genes subjective to XCI such as Cdkl5 (B), Mecp2 (C), and escapee genes (Kdm6a) (D) in WT female ES cells at day 4 and day 7. WT male ES cells and sense probe are used as control.

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

F. Representative site-specific binding of Xist on autosome locus (Stau2) in WT female ES cells at day 4 and day 7, WT male ES cells are used as control.

Xist binds XCI genes during differentiation.

A. Xist CHART signal coverage on X chromosome genes (XCI-active/inactive, and escapee) in WT female ES cells at day 4. Sense and male ES cells are used as control. P-values are determined using the Wilcoxon rank sum test.

B. Average profile of Xist CHART signal on XCI escapee genes (day 0, 4, 7, and 14) shows that Xist accumulated in the upstream of the promoter but depleted in the escapee gene body.

Xist binds select autosomal genes in trans.

A. IDR (Irreproducible Discovery Rate) analysis to assess the reproducibility of the peaks detected across biological replicates (day 4). The results showed a strong correlation between the replicates, with an IDR threshold of 0.05 (red point > 0.05).

B. Xist peak pattern (MACS2 peak calling) on autosomes in WT and ΔRepB female ES cells at day 4, day 7, and day 14.

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

D. Representative site-specific binding of Xist on autosome locus (Stau2) in WT and ΔRepB female ES cells at day 4 and day 7.

E. The Venn diagram illustrates the overlap of peak sites identified in WT and ΔRepB female ES cells at day 4, day 7, and day 14.

Xist binds X-linked genes is Xist’s RepB dependent.

A. RNA-seq track patterns of Xist in WT, ΔRepB female, and male ES cells at day 0, day 4, day 7, and day 14.

B. Exemplifying CHART-seq and RNA-Seq 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♀).

C. Evaluation of gene expression levels for X-linked genes in WT and ΔRepB female, and male ES cells at day 0. P-values are determined using the Wilcoxon rank sum test.

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

A-B. 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. P-values are determined using the Wilcoxon rank sum test.

C. Average 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. P-values are determined using the Wilcoxon rank sum test.

D. Average 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. P-values are determined using the Wilcoxon rank sum test.

Xist overexpression inhibits select autosomal genes and X-linked genes.

RNA-seq track shows the Med14 (X-linked gene) (A) and Bcl7b (an Xist autosomal target gene) (B) expression levels in differentiated ectopic Xist overexpressed ES cell lines (Tg), the control (Ctrl) was doxycycline-treated wildtype cell. Change in coverage (Δ) is shown below (Tg - Ctrl).

Xist has overlapped autosomal binding peaks in differentiated ES and 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. The Venn diagram illustrates the overlap of peak sites identified in WT female ES cells at day 4, day 7 and MEF cells.