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. 2017 Feb 23;10:8.
doi: 10.1186/s13072-017-0115-7. eCollection 2017.

SMYD5 Regulates H4K20me3-marked Heterochromatin to Safeguard ES Cell Self-Renewal and Prevent Spurious Differentiation

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Free PMC article

SMYD5 Regulates H4K20me3-marked Heterochromatin to Safeguard ES Cell Self-Renewal and Prevent Spurious Differentiation

Benjamin L Kidder et al. Epigenetics Chromatin. .
Free PMC article

Abstract

Background: Epigenetic regulation of chromatin states is thought to control the self-renewal and differentiation of embryonic stem (ES) cells. However, the roles of repressive histone modifications such as trimethylated histone 4 lysine 20 (H4K20me3) in pluripotency and development are largely unknown.

Results: Here, we show that the histone lysine methyltransferase SMYD5 mediates H4K20me3 at heterochromatin regions. Depletion of SMYD5 leads to compromised self-renewal, including dysregulated expression of OCT4 targets, and perturbed differentiation. SMYD5-bound regions are enriched with repetitive DNA elements. Knockdown of SMYD5 results in a global decrease of H4K20me3 levels, a redistribution of heterochromatin constituents including H3K9me3/2, G9a, and HP1α, and de-repression of endogenous retroelements. A loss of SMYD5-dependent silencing of heterochromatin nearby genic regions leads to upregulated expression of lineage-specific genes, thus contributing to the decreased self-renewal and perturbed differentiation of SMYD5-depleted ES cells.

Conclusions: Altogether, these findings implicate a role for SMYD5 in regulating ES cell self-renewal and H4K20me3-marked heterochromatin.

Keywords: ChIP-Seq; Chromatin; Differentiation; Embryoid body; Embryonic stem cells; Epigenetics; Gene expression; Genomics; H4K20me3; Heterochromatin; Histone methyltransferase; LINE; LTR; Pluripotent; RNA-Seq; Repetitive DNA; SMYD5; Self-renewal.

Figures

Fig. 1
Fig. 1
SMYD5 regulates ES cell self-renewal. a Bright-field microscopy of ES cells infected with shLuc or shSmyd5 lentiviral particles and wild-type (WT) SMYD5 or an enzymatically mutant (mut) version of SMYD5 (H315L and C317A) lentiviral particles and stably selected with puromycin and G418. b ES cell colonies were scored by morphology. The percentage of colonies with an ES-like morphology (compact and round vs. flattened) are represented as mean ± SEM. P values were calculated using a t test. c Alkaline phosphatase (AP) staining of ES cells. d ES cells were scored by AP staining. The percentage of AP positive colonies is represented as mean ± SEM. p values were calculated using a t test. e Quantitative RT-PCR (Q-RT-PCR) expression of SMYD5 using primers for three different regions of the SMYD5 coding region. f Scatter plot of RNA sequencing (RNA-Seq) gene expression analysis between shLuc and shSmyd5 ES cells. Log2 adjusted differentially expressed genes are plotted. Genes whose expression is greater than twofold (shLuc vs shSmyd5) and with an RPKM > 1 (reads per kilo bases of exon model per million reads) and FDR < 0.001 are shown in black. g UCSC genome browser view of differential expression of self-renewal genes in shSmyd5 and shLuc control ES cells. h Q-RT-PCR analysis of expression of Smyd5 and self-renewal genes in shLuc and shSmyd5 ES cells. i Gene set enrichment analysis (GSEA) [22] of differentially expressed genes in Smyd5 knockdown ES cells relative to undifferentiated and differentiated embryoid bodies (EBs). j Gene ontology (GO) functional annotation of differentially expressed genes analyzed using DAVID [23]. k Mouse gene atlas expression analysis evaluated using Network2Canvas [66] demonstrates that lineage and ES cell genes are misexpressed in shSmyd5 ES cells. Each node (square) represents a gene list (shLuc vs shSmyd5 DE genes) associated with a gene-set library (mouse gene atlas). The brightness (white) of each node is determined by its p value
Fig. 2
Fig. 2
Altered differentiation of SMYD5-depleted ES cells. a Venn diagrams showing overlap between differentially expressed genes in shSmyd5 and shLuc ES cells and genes bound by OCT4, OCT4, and NANOG, or OCT4, SOX2, and NANOG. b Embryoid body (EB) formation shows abnormal differentiation of shSmyd5 ES cells. c Hematoxylin and eosin (H&E) histological sections of shLuc and shSmyd5 day 9 EBs. The arrowheads depict altered and advanced differentiation of shSmyd5 EBs relative to control (shLuc) EBs. The bottom left panel shows an EB with an atypical internal epithelial-like structure; the bottom right panel shows a thick epithelial-like layer. d H&E histological sections of teratomas generated from shLuc and shSmyd5 ES cells injected into SCID–beige mice. Tumors were harvested 4–6 weeks post-injection and evaluated using standard H&E histological methods. Transmitted white-light microscopy of sectioned teratomas. Heterogeneous differentiation of shLuc and shSmyd5 ES cells into ectoderm (keratinized epidermal cells), mesoderm (muscle and mesenchymal cells, adipocytes), and endoderm (glandular structures)
Fig. 3
Fig. 3
Transcriptome analysis reveals altered differentiation of SMYD5-depleted ES cells. a K-means clustering analysis of RNA-Seq data from shLuc and shSmyd5 ES cells differentiated without LIF for 14 days. The experimental design is shown on top. Differentially expressed genes (>twofold; RPKM > 1) clustered according to k-means. b Custom tracks of RNA-Seq data in the UCSC genome browser for undifferentiated and differentiated shLuc control and shSmyd5 ES cells. c Principal component analysis (PCA) of differentially expressed genes during EB differentiation of shSmyd5 and shLuc ES cells. d Prediction of differentially expressed genes due to chance or altered differentiation. The percentage of genes that lag behind during differentiation of shSmyd5 ES cells is less than expected. Top each bar represents a group of genes upregulated by at least alpha-fold (X axis) from ESC (0 h) to EB day 6 in the control cells. The percentage of genes with expression values that follow the order: EB day 6 (shLuc) > EB day 6 (shSmyd5) > ES cell is calculated (observed; red bars); error bars are generated by bootstrapping. The expression values of all genes are randomly shuffled independently for EB day 6 (shLuc), EB day 6 (shSmyd5), and ES cells and are repeated many times to give the means and standard deviations for the expectations (expected; blue bars). The red bars represent observed data. Bottom each bar represents a group of genes upregulated by at least alpha-fold (X axis) from ESC (0 h) to EB day 10 in the control cells. The percentage of genes with expression values that follow the order: EB day 10 (shLuc) > EB day 10 (shSmyd5) > ES cell is calculated (observed; red bars); error bars are generated by bootstrapping. The expression values of all genes are randomly shuffled independently for EB day 10 (shLuc), EB day 10 (shSmyd5), and ES cells and are repeated many times to give the means and standard deviations for the expectations (expected; blue bars). The red bars represent observed data. e Gene set enrichment analysis (GSEA) of differentially expressed genes during differentiation of shSmyd5 ES cells relative to ES cells and day 14 EBs. f DAVID gene ontology analysis of differentially expressed genes between shLuc and shSmyd5 ES cells and during EB differentiation. The hierarchical clustering heat map on the right shows enrichment of developmental GO terms. g Correlation matrix of differentially expressed (DE) genes during shSmyd5 ES cell differentiation with promoter binding of transcription factors and epigenetic modifiers. Heat map generated by evaluating pair-wise affinities between differentially expressed (DE) genes during shLuc and shSmyd5 EB differentiation using RNA-Seq datasets generated from this study (0, 24 h, 6, 10, 14 days) and published ChIP-Seq data [, , –29, 67]. AutoSOME [68] was used to generate pair-wise affinity values
Fig. 4
Fig. 4
SMYD5 and trimethylated histone co-occupy genomic regions in ES cells. a Comparison of SMYD5-bioChIP and SMYD5-FLAG ChIP-Seq peaks. Scatter plot of log2 SMYD5 density at ChIP-enriched regions. b Heat map of SMYD5 ChIP-Seq densities. c Average profiles of SMYD5-bioChIP and SMYD5-FLAG density at SMYD5-FLAG enriched regions. d Western blot of H4K20me3, H4K20me2, and H4K20me1 in shLuc and shSmyd5 ES cells (top), and H4K20me3 in shLuc, shSmyd5, shLuc + WT, and shSmyd5 + WT (bottom). The bar graph (bottom right) shows H4K20me3 levels normalized to actin using ImageJ software (https://imagej.nih.gov/ij/). e Comparison of SMYD5 and H4K20me3 ChIP-Seq peaks. f Empirical cumulative distribution function (ECDF) for SMYD5-FLAG and SMYD5-bioChIP density at H4K20me3-enriched regions in ES cells
Fig. 5
Fig. 5
SMYD5 and H4K20me3 occupy repetitive DNA elements in ES cells. Comparison of H4K20me3, H3K9me3, and SMYD5 enriched sequences and annotated repetitive sequences (http://www.repeatmasker.org). Empirical cumulative distribution (ECDF) for the percent coverage of a a LINE or LTR repeat class or b family member (L1 or ERVK) across all H4K20me3 (top) or H3K9me3 (middle) islands, or SMYD5 regions (bottom) relative to random genomic regions (black). Y-axis shows the percentage of genes that exhibit a percent repeat length less than the value specified by the x-axis. A line shifted to the right means a systematic increase in the percent coverage of a repeat element in ChIP-Seq peaks relative to random genomic sequences. p value for all <2.2e−16 (Kolmogorov–Smirnov test)
Fig. 6
Fig. 6
Depletion of SMYD5 leads to decreased H4K20me3, H3K9me3/2, and HP1α binding. a Change in the global distribution of H4K20me3 in shSmyd5 ES cells. b Average profile and boxplot of H4K20me3 ChIP-Seq tag density in shSmyd5 ES cells. c Average profiles of H4K20me3 at SMYD5-enriched regions in shLuc and shSmyd5 ES cells. d, e Changes in global distributions of d H3K9me3 (e) and H3K9me2 in shSmyd5 ES cells relative to shLuc ES cells. fh Average profiles and boxplot of f H3K9me3, g H3K9me2, and h HP1α densities in shLuc and shSmyd5 ES cells. i Boxplot depicting density of H4K20me3, H3K9me3, and H3K9me2 at H4K20me3 islands. j Empirical cumulative distribution (ECDF) for the fold-change in density of HP1α in shSmyd5 ES cells. The red line shifted to the left of the input (gray) shows a systematic decrease in enrichment in shSmyd5 ES cells. Boxplot below shows HP1α density (log2 fold-change vs. input) in shLuc and shSmyd5 ES cells. k H4K20me3 density at regions at regions with decreased or unaltered H3K9me3 levels. l SMYD5 associates with HP1α and G9a. FLBIO-SMYD5 (biotinylated SMYD5 + BirA) or BirA (control) ES cells were used to immunoprecipitate SMYD5 protein with avidin-agarose beads. Immunoprecipitates were analyzed by immunoblotting with anti-HP1α, anti-G9a, and anti-SMYD5 antibodies. m Changes in the global distribution of G9a in shSmyd5 ES cells relative to shLuc ES cells. n Altered profiles of H4K20me3 at SMYD5-enriched regions (H2-Q1, H2-Q7) in shLuc and shSmyd5 ES cells
Fig. 7
Fig. 7
Elevated expression of repetitive DNA elements in shSmyd5 ES cells. a Fold-change expression of LINE/LTR repetitive DNA sequences in shSmyd5 ES cells relative to shLuc ES cells. p value for all <2.2e−16 (Kolmogorov–Smirnov test). b Heat map showing expression of a subset of LINE and LTR regions in shLuc and shSmyd5 ES cells. c Q-RT-PCR expression analysis of two LINE elements (p value <0.05). d Fold-change expression of LINE (left) and LTR (right) repeat subfamilies in shLuc and shSmyd5 ES cells. e De novo search for LTR retrotransposons/ERVs in the mouse genome (mm9) using LTRharvest software, and annotated using LTRdigest software. A representative full-length region with internal features is shown. f Fold-change expression of LTR internal features and LTR UTR regions between shLuc and shSmyd5 ES cells. g Browser view of RNA-Seq expression and H4K20me3, and H3K9me3 in shLuc and shSmyd5 ES cells, and SMYD5-FLAG and SMYD5-bioChIP in ES cells

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