Research ArticleCANCER

An Mll4/COMPASS-Lsd1 epigenetic axis governs enhancer function and pluripotency transition in embryonic stem cells

See allHide authors and affiliations

Science Advances  31 Jan 2018:
Vol. 4, no. 1, eaap8747
DOI: 10.1126/sciadv.aap8747
  • Fig. 1 Mll4 depletion results in transcription deregulation but does not impair self-renewal of ESCs.

    (A) University of California at Santa Cruz (UCSC) genome browser view of RNA-seq signals of WT and Mll4KO ESCs showing successful deletion of the indicated exons (green arrows) at the Kmt2d gene. The green line indicates the deleted genomic region in Mll4KO ESCs. CPM, counts per million mapped reads. (B) Western blotting assay with antibodies against Mll4 and Rbbp5 using nuclear extracts of WT and Mll4KO ESCs. (C) Immunostaining of pluripotency factors Oct4 and Nanog in WT, Mll4KO, and Mll4ΔSET ESCs. Scale bars, 10 μm. (D) Correlation analysis of gene expression levels between WT and respective Mll4KO (top) and Mll4ΔSET (bottom) ESCs. Plots were generated with two biological replicates of RNA-seq experiments from two cell clones for each genotype. Significantly down-regulated genes (compared with WT, adjusted P < 0.01, logFC > |1|) are labeled green, and up-regulated ones are labeled purple. The major pluripotency marker genes are marked on the plot with significantly changed ones labeled in red. Other unchanged genes are labeled as gray dots. FPKM, fragments per kilobase of transcript per million mapped reads. (E) K-means clustering analysis of expression levels of the 1702 differentially expressed genes (DEGs) in Mll4KO cells comparing WT, Mll4KO, and Mll4ΔSET ESCs. Z scores were used to generate the heat map. Numbers below the heat map denote the two biological replicates of each genotype. (F) UCSC genome browser view of RNA-seq signals of WT, Mll4KO, and Mll4ΔSET ESCs at Ckb (top) and Serpinh1 (bottom) loci.

  • Fig. 2 Mll4 is responsible for enhancer activity independent of its catalytic function.

    (A) Donut diagram showing the genome-wide distribution of Mll4-enriched regions. (B) Top transcription factors motifs at Mll4-enriched regions. (C) UCSC genome browser view of Mll4 ChIP-seq signals of WT, Mll4KO, and Mll4ΔSET ESCs at Ckb (top) and Serpinh1 (bottom) loci. ChIP-seq experiments were performed using the CT antibody. (D) Western blotting assay of WT, Mll4KO, and Mll4ΔSET ESC lysates using antibodies against H3K4me1, H3K4me2, H3K4me3, and H3. (E) Heat maps showing the log2 fold change (LogFC) in the levels of H3K4me1, H3K27ac, and gene expression at Mll4-enriched regions between Mll4KO, or Mll4ΔSET, and WT cells. Clusters were generated by K-means clustering, and the nearest gene to each Mll4 peak was used to generate the RNA-seq heat map. Signals 5 kb up- and downstream of Mll4 peak regions were included. (F) UCSC genome browser view of H3K4me1 and H3K27ac ChIP-seq signals of WT, Mll4KO, and Mll4ΔSET ESCs at Ckb (top) and Serpinh1 (bottom) loci.

  • Fig. 3 Lsd1 is required for the full deactivation of enhancers caused by Mll4 loss.

    (A) UCSC genome browser view of Mll4 and Lsd1 ChIP-seq signals at Ckb (top) and Serpinh1 (bottom) loci. (B) Metaplot showing Mll4 and Lsd1 ChIP-seq signals at 2.5 kb up- and downstream of Mll4 peaks. (C) Venn diagram showing the overlap between Mll4 and Lsd1 peak regions. (D) Western blotting of Lsd1 and tubulin in ESCs infected with control virus (nonTsh) and virus containing shRNA-targeting Lsd1 (Lsd1sh). (E) UCSC genome browser view of RNA-seq signals of control and Lsd1-depleted ESCs at Ckb (top) and Serpinh1 (bottom) loci. (F) Correlation analysis of gene expression levels between nonTsh-infected WT and Mll4KO ESCs. Significantly down-regulated genes are labeled green, and up-regulated ones are labeled purple. Unchanged genes are labeled as gray dots. (G) Correlation analysis of the expression levels of DEGs in (F) between nonTsh- and Lsd1sh-infected Mll4KO ESCs. Significantly down-regulated genes by Mll4 depletion in (F) are labeled green, and up-regulated ones are labeled purple. (H) UCSC genome browser view of H3K4me1 and H3K27ac ChIP-seq signals of nonTsh- and Lsd1sh-infected Mll4KO ESCs at Ckb (top) and Serpinh1 (bottom) loci. (I) Heat maps showing the log2 fold change in the levels of H3K4me1, H3K27ac, and gene expression at Mll4/Lsd1 co-bound regions for Mll4KO versus WT cells and nonTsh-infected versus Lsd1sh-infected Mll4KO cells. Clusters were generated by K-means clustering and the nearest gene to each peak was used to generate the RNA-seq heat map. Signals 5 kb up- and downstream of Lsd1/Mll4 co-bound peaks were included.

  • Fig. 4 Mll4 deletion impairs the exit of naive pluripotency.

    (A) Phase images of WT, Mll4KO, and Mll4ΔSET ESCs grown in FBS/LIF condition. Scale bar, 100 μm. (B) Correlation analysis of gene expression levels between ESCs grown in FBS/LIF and 2i/LIF conditions. Genes significantly up-regulated in FBS/LIF are labeled purple, whereas genes significantly increased in 2i/LIF are labeled green. The major naive pluripotency and primed pluripotency markers are marked, with red dots denoting significantly differentially expressed ones. (C) Correlation analysis of gene expression levels between WT and Mll4KO ESCs grown in FBS/LIF condition. Genes significantly down-regulated in Mll4KO cells are labeled green, whereas up-regulated ones are labeled purple. The major naive pluripotency and primed pluripotency markers are marked, with red dots denoting significantly changed ones. (D) Correlation analysis of the expression levels of DEGs in (B) between WT and Mll4KO ESCs grown in FBS/LIF condition. Genes significantly up-regulated in FBS/LIF are labeled purple, whereas genes significantly increased in 2i/LIF are labeled green. (E) UCSC genome browser view of RNA-seq signals of WT, Mll4KO, and Mll4ΔSET ESCs cultured in FBS/LIF condition at the Pou5f1 gene. (F) UCSC genome browser view of RNA-seq signals of WT, Mll4KO, and Mll4ΔSET ESCs cultured in FBS/LIF condition at Nr0b1, Tet2, Lefty1, and Myl9 genes. (G) Immunostaining of pluripotency factors Oct4 and Nanog in WT, Mll4KO, and Mll4ΔSET ESCs cultured in FBS/LIF condition. Scale bars, 10 μm.

  • Fig. 5 Lsd1 depletion rescues the pluripotency transition defects in Mll4KO ESCs.

    (A) UCSC genome browser view of RNA-seq signals of nonTsh- and Lsd1sh-infected Mll4KO ESCs cultured in FBS/LIF condition at Nr0b1, Tet2, Lefty1, and Myl9 genes. (B) Correlation analysis of gene expression levels between nonTsh-infected WT and Mll4KO ESCs grown in FBS/LIF condition. Genes significantly down-regulated in Mll4KO cells are labeled green, whereas up-regulated ones are labeled purple. (C) Correlation analysis of the expression levels of DEGs in (B) between nonTsh- and Lsd1sh-infected Mll4KO ESCs cultured in FBS/LIF condition. Significantly down-regulated genes by Mll4 depletion in (B) are labeled green, and up-regulated ones are labeled purple. (D) Immunostaining of pluripotency factors Oct4 and Nanog in nonTsh- and Lsd1sh-infected Mll4KO ESCs cultured in FBS/LIF condition. Scale bars, 10 μm.

  • Fig. 6 Deletion of Mll4 NT leads to enhancer decommissioning and pluripotency transition defects.

    (A) UCSC genome browser view of RNA-seq signals of WT and Mll4ΔNT ESCs showing successful deletion of the indicated exons (red arrows) at Kmt2d gene. The red line indicates the deleted genomic region in Mll4ΔNT ESCs. (B) A representative phase image of Mll4ΔNT ESCs grown in 2i/LIF condition. Scale bar, 100 μm. (C) Correlation analysis of gene expression levels between WT and Mll4ΔNT ESCs cultured in 2i/LIF. Significantly down-regulated genes by Mll4 NT deletion are labeled green, and up-regulated ones are labeled purple. The major pluripotency marker genes are marked on the plot with significantly changed ones labeled in red. Other unchanged genes are labeled as gray dots. (D) K-means clustering analysis of expression levels of the 1702 DEGs in Mll4KO cells in 2i/LIF culture conditions. Z scores of WT, Mll4KO, and Mll4ΔNT ESCs of the genes are shown. Numbers below the heat map denote the two biological replicates of each genotype. (E) Heat map showing Mll4 occupancy at the 11,071 Mll4 peaks and the surrounding 5-kb regions in WT and Mll4ΔNT ESCs. The profiles are sorted in a descending order of Mll4 occupancy in WT ESCs. (F) Heat maps showing the log2 fold change in the levels of H3K4me1, H3K27ac, and gene expression at Mll4 peaks and 10-kb surrounding regions between Mll4KO, or Mll4ΔNT, and WT cells. Cluster division and the order within the clusters are the same as Fig. 2E. (G) Correlation analysis of gene expression levels between WT and Mll4ΔNT ESCs grown in FBS/LIF condition. Genes significantly down-regulated in Mll4ΔNT cells are labeled green, whereas up-regulated ones are labeled purple. The major naive pluripotency and primed pluripotency markers are marked, with red dots denoting significantly changed ones. (H) Venn diagrams showing the overlap of DEGs in Mll4KO and Mll4ΔNT ESCs cultured in FBS/LIF. Down-regulated genes (versus WT) are plotted on the left, and up-regulated genes are plotted on the right.

  • Fig. 7 A working model for the regulation of enhancer function by Mll4/COMPASS and Lsd1.

    Mll4/COMPASS and Lsd1 co-occupy a large cohort of enhancers in WT ESCs. In Mll4ΔSET ESCs, the levels of H3K4me1 are reduced at enhancers without affecting transcription. Upon the deletion (Mll4KO) or loss of recruitment of Mll4 (Mll4ΔNT), Lsd1 and its associated co-repressors decommission enhancers, which results in repression of transcription and failures in pluripotency transition.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/1/eaap8747/DC1

    fig. S1. Characterization of Mll4KO ESCs.

    fig. S2. Enhancer inactivation by Mll4 depletion.

    fig. S3. Epigenetic balance between Lsd1 and Mll4/COMPASS.

    fig. S4. Mll4 deletion impairs enhancer activation during pluripotency transition.

    fig. S5. Lsd1 is indispensable for enhancer decommissioning in FBS/LIF-cultured Mll4KO cells.

    fig. S6. Characterization of Mll4ΔNT ESCs.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Characterization of Mll4KO ESCs.
    • fig. S2. Enhancer inactivation by Mll4 depletion.
    • fig. S3. Epigenetic balance between Lsd1 and Mll4/COMPASS.
    • fig. S4. Mll4 deletion impairs enhancer activation during pluripotency transition.
    • fig. S5. Lsd1 is indispensable for enhancer decommissioning in FBS/LIF-cultured Mll4KO cells.
    • fig. S6. Characterization of Mll4ΔNT ESCs.

    Download PDF

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article