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. 2020 May 19;48(11):6001–6018. doi: 10.1093/nar/gkaa425

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — ZKSCAN3 regulates heterochromatin architecture and represses repetitive sequence expression in hMSCs. (A) Schematic representation of DamID-seq strategy. (B) Reduced DamID signals [log₂ (Dam-EMD vs. Dam)] at LAD regions in ZKSCAN3^-/- hMSCs (P10) compared to ZKSCAN3^+/+ hMSCs (P10), shown by both violin plot (left) and heatmaps (right). (C) Line plots showing DamID signals [log₂ (Dam-EMD vs. Dam)] at LAD-localized repetitive sequence loci in ZKSCAN3^+/+ and ZKSCAN3^-/- hMSCs (P10). (D) Sketch map showing the loss of ‘H3K9me3 mountain’ distribution over 23 chromosomes in ZKSCAN3^-/- hMSCs (P10) compared to ZKSCAN3^+/+ hMSCs (P10). (E) Violin plots showing the loss of H3K9me3 signals in H3K9me3 mountains, LAD and heterochromatin (HC) regions in ZKSCAN3^-/- hMSCs (P10) compared to ZKSCAN3^+/+ hMSCs (P10). (F) Violin plots showing the loss of H3K9me3 signals at LAD-localized repetitive sequence regions (LINE, SINE, Satellite family) in ZKSCAN3^-/- hMSCs (P10) compared to ZKSCAN3^+/+ hMSCs (P10). (G) Enrichment of H3K9me3 within the regions of repetitive sequences in ZKSCAN3^+/+ and ZKSCAN3^-/- hMSCs (P10) as measured by ChIP-qPCR. Data are presented as the mean ± SEMs, n = 4. *P < 0.05, ***P < 0.001 (two tailed t-test). (H) Violin plot showing the increase of ATAC signals at ATAC-seq peaks within LADs in ZKSCAN3^-/- hMSCs (P10) compared to ZKSCAN3^+/+ hMSCs (P10). (I) The distribution feature of ATAC-seq peaks annotated to different repetitive sequence regions in ZKSCAN3^+/+ and ZKSCAN3^-/- hMSCs (P10). (J) Violin plots showing ATAC signals of LAD-localized repetitive sequence regions in ZKSCAN3^+/+ and ZKSCAN3^-/- hMSCs (P10). (K) Representative tracks of DamID-seq, H3K9me3 ChIP-seq and ATAC-seq analyses showing decreased DamID signal, reduced H3K9me3 signal coupled with increased ATAC signal in ZKSCAN3^-/- hMSCs (P10) compared to ZKSCAN3^+/+ hMSCs (P10). (L) RNA-seq analysis showing the increased mRNA expression levels of repetitive sequences in ZKSCAN3^-/- hMSCs (P10) compared to ZKSCAN3^+/+ hMSCs (P10). (M) Heatmap showing the mRNA expression levels of repetitive sequences in the ZKSCAN3^+/+ and ZKSCAN3^-/- hMSCs (P10) by RT-qPCR.