Volume 55, Issue 2, Pages 277-290 (July 2014) Suv39h-Dependent H3K9me3 Marks Intact Retrotransposons and Silences LINE Elements in Mouse Embryonic Stem Cells  Aydan Bulut-Karslioglu, Inti A. De La Rosa-Velázquez, Fidel Ramirez, Maxim Barenboim, Megumi Onishi-Seebacher, Julia Arand, Carmen Galán, Georg E. Winter, Bettina Engist, Borbala Gerle, Roderick J. O’Sullivan, Joost H.A. Martens, Jörn Walter, Thomas Manke, Monika Lachner, Thomas Jenuwein  Molecular Cell  Volume 55, Issue 2, Pages 277-290 (July 2014) DOI: 10.1016/j.molcel.2014.05.029 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Genome-wide Definition of Suv39h-Dependent H3K9me3 (A) Schematic representation of domains of knockin Suv39h1-HA/Flag and Suv39h2-HA/Flag gene products. (B) Genomic distribution of Suv39h-dependent H3K9me3 (WT/Suv39h dn), Suv39h1- and Suv39h2-HA/Flag peaks. Indicated percentages for intergenic regions include promoters. The overall number of called peaks is listed above the pie charts, and overlaps between peaks are shown in the accompanying table. (C) Example of a 2.4 Mbp region that shows Suv39h-dependent H3K9me3 at ERV and LINE-rich domains. Normalized ChIP-seq reads and peaks are indicated. Repeat data were retrieved from Repeat Masker via UCSC genome browser. Two representative regions comprising ERV (purple) or LINE (green) elements are shown in the two bottom panels. See also Figures S1–S3. Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Bioinformatic Workflow and Hierarchical Clustering (A) Schematic representation of the hierarchical clustering. H3K9me3 peaks were clustered based on their Suv39h-HA/Flag coverage levels. This first grouping resulted in the identification of three major H3K9me3 decorated chromatin domains, which are displayed in (B). Suv39h-dependent H3K9me3 clusters were then associated with the presence or absence of ERV and LINE-rich regions and sorted based on combined conservation score for repeats. This resulted in two types of repeat-rich, Suv39h-decorated heterochromatin, which are displayed in Figures 3 and 4. (B) Heatmaps showing the three major H3K9me3-enriched chromatin domains. Each row represents one scaled H3K9me3 peak that includes ±4 kb of flanking regions. + or − strand of LINEs are indicated in blue or red; other features (e.g., exon, intron, etc.) are also indicated. The right panel illustrates a comparison of random versus unique mapping for H3K9me3 reads. Mappability of these regions is indicated in blue. Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Intact ERVs Are Marked by Eset- and Suv39h-Dependent H3K9me3 (A) Heatmap showing unscaled peaks (±6 kb of flanking regions) of H3K9me3, Suv39h1-HA/Flag and Suv39h2-HA/Flag, Eset, and HP1α-EGFP at intact, degenerate, and truncated ERVs. Additionally, LINE and ERV content and repeat conservation score are plotted. (B) Meta-analysis of mean H3K9me3 coverage (WT and Suv39h dn) versus ERV repeat length. A schematic diagram of an intact ERV element is shown above. (C) Two representative genomic regions comprising ERV repeats that accumulate H3K9me3 and Suv39h-HA/Flag are shown. Strand-specific nuclear RNA-seq profiles (blue and red) and coverage of RNA output (black) are also displayed. See also Figures S4–S6 and Table S1, Table S2, and Table S3. Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Suv39h-Dependent H3K9me3 Represses Intact LINE Elements (A) Heatmap showing unscaled peaks (±6 kb of flanking regions) of H3K9me3, Suv39h1-HA/Flag and Suv39h2-HA/Flag, Eset, and HP1α-EGFP at LINE repeats (− and + strand). Other features are as described in Figure 3A. (B) Meta-analysis of mean H3K9me3 coverage (WT and Suv39h dn) versus LINE repeat length. A schematic diagram of an intact LINE element is shown above. (C) Two representative genomic regions comprising LINE repeats that accumulate H3K9me3 and Suv39h-HA/Flag are shown. Strand-specific nuclear RNA-seq profiles (blue and red) and coverage of RNA output (black) are also displayed. See also Figures S4–S6 and Table S1, Table S2, and Table S3. Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Retrotransposon Silencing via H3K9me3 is an ESC-Specific Pathway (A) Heatmaps showing scaled (±4 kb of flanking region) ESC-H3K9me3 peaks with the corresponding H3K9me3 profiles in ESCs, NPCs, and iMEFs. (B) Total number of H3K9me3 reads that were mapped to the consensus major satellite sequence (GSAT_MM) in WT and Suv39h dn ESCs, NPCs, and MEFs are displayed. (C) Two representative genomic regions comprising either an ERV-rich or an LINE-rich region together with their H3K9me3 and RNA coverage in WT and Suv39h dn ESCs, NPCs, and iMEFs are shown. RNA-seq data are represented as coverage without strand-specific reads. See also Figure S7. Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 DNA Methylation Governs Suv39h-Targeted Repeats in Committed Cells Hairpin bisulfite analysis of top Suv39h targets. Each column corresponds to a cytosine in a CpG dinucleotide. CpG positions in the different repeats are schematically indicated in the bottom diagram. Full or hemimethylation is displayed in different colors. Percentages indicate fully methylated CpG sites relative to all analyzed reads. Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Model for Suv39h-Dependent H3K9me3 Decoration at Intact Retrotransposons (A) In ESCs, intact ERVs are characterized by a broad domain of H3K9me3, which is initiated by Eset/HP1α and then extended by the Suv39h KMTs. Transcriptional silencing is primarily mediated by Eset, as there is only modest upregulation (dashed line) of ERV transcripts in Suv39h dn ESCs. (Β) By contrast, Suv39h KMTs are the chief enzymes to silence intact LINE elements through focal H3K9me3 enrichment at the 5′ UTR. In Suv39h dn ESCs, LINE transcripts are significantly derepressed (bold line). (A and B) Committed cells (NPCs, iMEFs) progressively lose Suv39h-dependent H3K9me3 over ERV and LINE repeats, and this decline in H3K9me3 levels appears to be compensated by an increase in DNA methylation (black bars indicate 5meC). Molecular Cell 2014 55, 277-290DOI: (10.1016/j.molcel.2014.05.029) Copyright © 2014 Elsevier Inc. Terms and Conditions