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. 2021 Apr 15;4(1):485.
doi: 10.1038/s42003-021-01945-1.

Chd8 regulates X chromosome inactivation in mouse through fine-tuning control of Xist expression

Andrea Cerase  1   2 , Alexander N Young  3   4 , Nerea Blanes Ruiz  4 , Andreas Buness  3   5 , Gabrielle M Sant  3   6 , Mirjam Arnold  3   7 , Monica Di Giacomo  3 , Michela Ascolani  3 , Manish Kumar  3   8 , Andreas Hierholzer  3   9 , Giuseppe Trigiante  4 , Sarah J Marzi  10 , Philip Avner  11
Affiliations

Chd8 regulates X chromosome inactivation in mouse through fine-tuning control of Xist expression

Andrea Cerase et al. Commun Biol. .

Abstract

Female mammals achieve dosage compensation by inactivating one of their two X chromosomes during development, a process entirely dependent on Xist, an X-linked long non-coding RNA (lncRNA). At the onset of X chromosome inactivation (XCI), Xist is up-regulated and spreads along the future inactive X chromosome. Contextually, it recruits repressive histone and DNA modifiers that transcriptionally silence the X chromosome. Xist regulation is tightly coupled to differentiation and its expression is under the control of both pluripotency and epigenetic factors. Recent evidence has suggested that chromatin remodelers accumulate at the X Inactivation Center (XIC) and here we demonstrate a new role for Chd8 in Xist regulation in differentiating ES cells, linked to its control and prevention of spurious transcription factor interactions occurring within Xist regulatory regions. Our findings have a broader relevance, in the context of complex, developmentally-regulated gene expression.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Characterization of the genomic distribution of Chd8 and H3K4me3 peaks.
a Overlap between Chd8 and H3K4me3 peaks; top undifferentiated (Und) and bottom differentiated conditions (Dif) from two biological experiments. b Heatmaps showing the distribution of Chd8 and H3K4me3 peaks in the genome (left). Association of Chd8 peaks with transcriptional start sites (TSS); top undifferentiated, bottom differentiated conditions (right). Data from two biological replicas is shown. c Genomic features distribution of Chd8 consensus peak sets; top undifferentiated, bottom differentiated conditions. Selected-features are shown (e.g. Promoters, Immediate downstream region (ImDown), etc.). Data from two biological experiments is shown. d Chd8 distribution at aligned sequence reads at the Xist and Tsix promoter regions. Black arrows indicate Chd8 peaks at the Xist promoter. A single representative set of ChIP-seq profiles is shown. Red: Chd8, green: H3K4me3, grey: IgG, black: input. Samples and antibodies used are indicated in the figure.
Fig. 2
Fig. 2. Chd8 KD leads to Xist de-regulation.
a CTRL-normalised Chd8 qRT-PCR at 3-day differentiated cells in CTRL and Chd8 KD cells is shown. CRTL: scrambled siRNA control (black bars), Chd8KD: specific siRNA pool to Chd8 (red bars). Data from three independent experiments is shown. b CTRL-normalised Xist and Tsix qPCR at 3-day differentiated cells in CTRL and Chd8KD cells is shown. CRTL: scrambled siRNA control (black bars), Chd8KD: specific siRNA pool to Chd8 (red bars). Data from three independent experiments is shown. c Selected differentiation markers used for qRT-PCR analysis are shown. Data is normalised for the undifferentiated condition (Und). Data from two independent experiments is shown. d Xist-mediated gene silencing is not affected in Chd8 KD (two randomly selected X-linked genes are showed). Data is normalised for the undifferentiated condition (Und). Data from two independent experiments is shown. Und (undifferentiated cells), Dif (3 day differentiated cells). Error bars represent standard error of the mean (SEM). Statistical significance was tested by means of two-tailed unpaired t-test (*p ≤ 0.05; **p ≤ 0.01). p-values, Xist = 0.00141, Chd8 = 0.00390. Single points represent independent biological samples. Gapdh was used as internal normalization control.
Fig. 3
Fig. 3. Chd8 KO affects XCI initiation.
a Schematic representation of Chd8 protein domains; red lines indicate the position of the CRISPR/Cas9 guides used (not in scale). b Western Blot analysis of Chd8 KO.1 (clone C4.1) and parental ES cells (CTRL, Fa2L-S4) are shown. c qRT-PCR results showing expression levels of Xist, Tsix, Nestin, Sox2. In differentiating ESCs Xist is strongly up-regulated in Chd8 KO cells whilst cell differentiation is not affected (Nestin, Nanog); CTRL, parental cell line: Fa2L-S4; KO, sub-clone C4.1. Data is normalised for the undifferentiated condition (Und). Data from three independent experiments is shown. p-values, Xist = 0.0367. d Xist qRT-PCR data from parental (CTRL) and KO.1 cells in undifferentiated state is shown. Und (undifferentiated cells), Dif (3 day differentiated cells). Data from three independent experiments is shown. Error bars represent standard error of the mean (SEM). Statistical significance was tested by means of two-tailed unpaired t-test (*p ≤ 0.05; **p ≤ 0.01). p-values, Xist = 0.00362. Single points represent independent biological sample. Gapdh was used as internal normalization control. e Representative images of H3K27me3 in CTRL vs. Chd8 KO cells (left) and the normalised scoring of H3K27me3 domains (IF) is shown (right), n = 658. Data from three independent experiments is shown. Error bars represent standard error of the mean (SEM). Statistical significance was tested by means of two-tailed unpaired t-test (*p ≤ 0.05; **p ≤ 0.01). CTRL vs. KO.1, p-value = 0.0463. Single points represent independent biological samples.
Fig. 4
Fig. 4. ATAC-seq analysis zoom at the Xist locus.
Zoom-in on the XIC around the Xist and Tsix genes. In black the parental cell line and in red two representative KO cell lines (C4.1/C4.3 sub-clones). Undifferentiated and differentiating ES cells are shown (Und/Dif). Light-blue arrows indicate changes in chromatin structure at the Xist promoter in differentiating cells. A black box surrounds the Tsix and Jpx main regulatory regions, indicates no change of chromatin structure. Green arrows indicate chromatin changes around the Tsix promoter/Tsix intron and Xist promoter, (CTRL, parental cell line: Fa2L-S4; Chd8 KO.1, sub-clone C4.1; Chd8 KO.2, sub-clone C4.3).
Fig. 5
Fig. 5. Complementation of Full-length (FL) Chd8 into Chd8 KO cells.
a Schematic representation of the Chd8 expression construct. Protein domains are indicated. CAG promoter is shown (CAG P) in the red box. b Western Blot analysis of the Chd8 KO line (C4.1) and complemented cells. Top CDH8 and bottom loading control (GAPDH) blots are shown. c H3K27me3 IF analysis of Chd8 KO and complemented cells. Right representative images, left CTRL-normalized quantification of the number of cells having an Xi at 3 days of differentiation. To test for statistical significance, our data was fitted using Poisson regression method and multiple correction testing (*p ≤ 0.05). Data from two and three experiments are shown, n = 827. Single points represent independent biological samples. Standard error of the mean (SEM) is shown. d Top, schematic of Xist promoter and primers used for analysis (peaks 1–6). Bottom, qPCR analysis of YY1 Cut&Run qPCR analysis at the Xist promoter. Data from two experiments are shown. Single points represent independent biological samples. Matched input samples were used as normalization control. Sample names are shown in the legend. e Top, CTRL-normalised qRT-PCR analysis of scrambled siRNA (CTRL) and YY1 siRNA (YY1KD) in Chd8 KO.1 (KO.1 line). Tested genes are indicated. Statistical significance was tested by means of two-tailed unpaired t-test (*p ≤ 0.05). Data from three experiments are shown. Bottom, representative images of H3K27me3 in CTRL vs. YY1KD cells (left) and the CTRL-normalised scoring of H3K27me3 domains (IF) is shown (right), n = 744. White arrows indicate examples of H3K27me3 domains. Statistical significance was tested by means of two-tailed unpaired t-test (*p ≤ 0.05). CTRL vs. YY1KD, p-value = 0.00196. Data from three experiments is shown. Gapdh was used as internal normalization control. Single points represent independent biological samples.
Fig. 6
Fig. 6. Proposed model of action of Chd8 on the Xist promoter.
a Schematic representation of the Xist (blue) and Tsix genes (green). b Chd8 is crucial for correct Xist expression in undifferentiated and differentiating ESCs. CHD8 is shown in light-blue; Tsix-specific TF/remodelers are shown in green. Top, CHD8 localization at the Xist promoter is shown (light-blue peak). In undifferentiated conditions (Und), in the absence of CHD8, there is reduction of the basal transcription level of Xist (blue wavy lines), while Tsix transcription level is unaffected (green wavy lines). c Differentiating conditions in Chd8 KD and Chd8 KO background. Right, a mild Chd8 KD leads to a small reduction of Xist steady levels (KD). Complete and severe CHD8 depletions instead, lead to increased Xist expression and a further Tsix downregulation (blue wavy lines Xist, green wavy lines Tsix). This condition also leads to a more-open chromatin state of the Xist regulatory regions, but not at Tsix’s, during differentiation (light-brown peaks). We suggest that YY1 (green circle) and/or known (red circle) and yet-to-be-identified protein (purple circle with question mark) may bind to the Xist promoter in the absence of the CHD8 protein (red arrow) and strongly activate it. Left, WT situation is depicted.
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