item-0 at level 0: unspecified: group _root_
  item-1 at level 1: title: KRAB-zinc finger protein gene ex ... retrotransposons in the murine lineage
    item-2 at level 2: paragraph: Gernot Wolf, Alberto de Iaco, Mi ...  Ralls, Didier Trono, Todd S Macfarlan
    item-3 at level 2: paragraph: The Eunice Kennedy Shriver Natio ... Lausanne (EPFL), Lausanne, Switzerland
    item-4 at level 2: section_header: Abstract
      item-5 at level 3: text: The Krüppel-associated box zinc  ... edundant role restricting TE activity.
    item-6 at level 2: section_header: Introduction
      item-7 at level 3: text: Nearly half of the human and mou ... s are active beyond early development.
      item-8 at level 3: text: TEs, especially long terminal re ... f evolutionarily young KRAB-ZFP genes.
    item-9 at level 2: section_header: Results
      item-10 at level 3: section_header: Mouse KRAB-ZFPs target retrotransposons
        item-11 at level 4: text: We analyzed the RNA expression p ... duplications (Kauzlaric et al., 2017).
        item-12 at level 4: text: To determine the binding sites o ... ctive in the early embryo (Figure 1A).
        item-13 at level 4: picture
          item-13 at level 5: caption: Figure 1. Genome-wide binding patterns of mouse KRAB-ZFPs. (A) Probability heatmap of KRAB-ZFP binding to TEs. Blue color intensity (main field) corresponds to -log10 (adjusted p-value) enrichment of ChIP-seq peak overlap with TE groups (Fisher’s exact test). The green/red color intensity (top panel) represents mean KAP1 (GEO accession: GSM1406445) and H3K9me3 (GEO accession: GSM1327148) enrichment (respectively) at peaks overlapping significantly targeted TEs (adjusted p-value<1e-5) in WT ES cells. (B) Summarized ChIP-seq signal for indicated KRAB-ZFPs and previously published KAP1 and H3K9me3 in WT ES cells across 127 intact ETn elements. (C) Heatmaps of KRAB-ZFP ChIP-seq signal at ChIP-seq peaks. For better comparison, peaks for all three KRAB-ZFPs were called with the same parameters (p<1e-10, peak enrichment >20). The top panel shows a schematic of the arrangement of the contact amino acid composition of each zinc finger. Zinc fingers are grouped and colored according to similarity, with amino acid differences relative to the five consensus fingers highlighted in white.
        item-14 at level 4: table with [9x5]
          item-14 at level 5: caption: Table 1. KRAB-ZFP genes clusters in the mouse genome that were investigated in this study. * Number of protein-coding KRAB-ZFP genes identified in a previously published screen (Imbeault et al., 2017) and the ChIP-seq data column indicates the number of KRAB-ZFPs for which ChIP-seq was performed in this study.
        item-15 at level 4: text: We generally observed that KRAB- ... responsible for this silencing effect.
        item-16 at level 4: text: To further test the hypothesis t ... t easily evade repression by mutation.
        item-17 at level 4: text: Our KRAB-ZFP ChIP-seq dataset al ... ntirely shift the mode of DNA binding.
      item-18 at level 3: section_header: Genetic deletion of KRAB-ZFP gen ...  leads to retrotransposon reactivation
        item-19 at level 4: text: The majority of KRAB-ZFP genes a ... ung et al., 2014; Deniz et al., 2018).
        item-20 at level 4: picture
          item-20 at level 5: caption: Figure 2. Retrotransposon reactivation in KRAB-ZFP cluster KO ES cells. (A) RNA-seq analysis of TE expression in five KRAB-ZFP cluster KO ES cells. Green and grey squares on top of the panel represent KRAB-ZFPs with or without ChIP-seq data, respectively, within each deleted gene cluster. Reactivated TEs that are bound by one or several KRAB-ZFPs are indicated by green squares in the panel. Significantly up- and downregulated elements (adjusted p-value<0.05) are highlighted in red and green, respectively. (B) Differential KAP1 binding and H3K9me3 enrichment at TE groups (summarized across all insertions) in Chr2-cl and Chr4-cl KO ES cells. TE groups targeted by one or several KRAB-ZFPs encoded within the deleted clusters are highlighted in blue (differential enrichment over the entire TE sequences) and red (differential enrichment at TE regions that overlap with KRAB-ZFP ChIP-seq peaks). (C) DNA methylation status of CpG sites at indicated TE groups in WT and Chr4-cl KO ES cells grown in serum containing media or in hypomethylation-inducing media (2i + Vitamin C). P-values were calculated using paired t-test.
      item-21 at level 3: section_header: KRAB-ZFP cluster deletions license TE-borne enhancers
        item-22 at level 4: text: We next used our RNA-seq dataset ... vating effects of TEs on nearby genes.
        item-23 at level 4: picture
          item-23 at level 5: caption: Figure 3. TE-dependent gene activation in KRAB-ZFP cluster KO ES cells. (A) Differential gene expression in Chr2-cl and Chr4-cl KO ES cells. Significantly up- and downregulated genes (adjusted p-value<0.05) are highlighted in red and green, respectively, KRAB-ZFP genes within the deleted clusters are shown in blue. (B) Correlation of TEs and gene deregulation. Plots show enrichment of TE groups within 100 kb of up- and downregulated genes relative to all genes. Significantly overrepresented LTR and LINE groups (adjusted p-value<0.1) are highlighted in blue and red, respectively. (C) Schematic view of the downstream region of Chst1 where a 5’ truncated ETn insertion is located. ChIP-seq (Input subtracted from ChIP) data for overexpressed epitope-tagged Gm13051 (a Chr4-cl KRAB-ZFP) in F9 EC cells, and re-mapped KAP1 (GEO accession: GSM1406445) and H3K9me3 (GEO accession: GSM1327148) in WT ES cells are shown together with RNA-seq data from Chr4-cl WT and KO ES cells (mapped using Bowtie (-a -m 1 --strata -v 2) to exclude reads that cannot be uniquely mapped). (D) RT-qPCR analysis of Chst1 mRNA expression in Chr4-cl WT and KO ES cells with or without the CRISPR/Cas9 deleted ETn insertion near Chst1. Values represent mean expression (normalized to Gapdh) from three biological replicates per sample (each performed in three technical replicates) in arbitrary units. Error bars represent standard deviation and asterisks indicate significance (p<0.01, Student’s t-test). n.s.: not significant. (E) Mean coverage of ChIP-seq data (Input subtracted from ChIP) in Chr4-cl WT and KO ES cells over 127 full-length ETn insertions. The binding sites of the Chr4-cl KRAB-ZFPs Rex2 and Gm13051 are indicated by dashed lines.
        item-24 at level 4: text: While we generally observed that ... he internal region and not on the LTR.
      item-25 at level 3: section_header: ETn retrotransposition in Chr4-cl KO and WT mice
        item-26 at level 4: text: IAP, ETn/ETnERV and MuLV/RLTR4 r ... s may contribute to reduced viability.
        item-27 at level 4: text: We reasoned that retrotransposon ... Tn insertions at a high recovery rate.
        item-28 at level 4: text: Using this dataset, we first con ... nsertions in our pedigree (Figure 4A).
        item-29 at level 4: picture
          item-29 at level 5: caption: Figure 4. ETn retrotransposition in Chr4-cl KO mice. (A) Pedigree of mice used for transposon insertion screening by capture-seq in mice of different strain backgrounds. The number of novel ETn insertions (only present in one animal) are indicated. For animals whose direct ancestors have not been screened, the ETn insertions are shown in parentheses since parental inheritance cannot be excluded in that case. Germ line insertions are indicated by asterisks. All DNA samples were prepared from tail tissues unless noted (-S: spleen, -E: ear, -B:Blood) (B) Statistical analysis of ETn insertion frequency in tail tissue from 30 Chr4-cl KO, KO/WT and WT mice that were derived from one Chr4-c KO x KO/WT and two Chr4-cl KO/WT x KO/WT matings. Only DNA samples that were collected from juvenile tails were considered for this analysis. P-values were calculated using one-sided Wilcoxon Rank Sum Test. In the last panel, KO, WT and KO/WT mice derived from all matings were combined for the statistical analysis.
        item-30 at level 4: text: To validate some of the novel ET ... ess might have truncated this element.
        item-31 at level 4: text: Besides novel ETn insertions tha ... tions (Figure 4—figure supplement 3D).
        item-32 at level 4: text: Finally, we asked whether there  ... s clearly also play an important role.
    item-33 at level 2: section_header: Discussion
      item-34 at level 3: text: C2H2 zinc finger proteins, about ... ) depending upon their insertion site.
      item-35 at level 3: text: Despite a lack of widespread ETn ... ion of the majority of KRAB-ZFP genes.
    item-36 at level 2: section_header: Materials and methods
      item-37 at level 3: table with [31x5]
        item-37 at level 4: caption: Key resources table
      item-38 at level 3: section_header: Cell lines and transgenic mice
        item-39 at level 4: text: Mouse ES cells and F9 EC cells w ... KO/KO and KO/WT (B6/129 F2) offspring.
      item-40 at level 3: section_header: Generation of KRAB-ZFP expressing cell lines
        item-41 at level 4: text: KRAB-ZFP ORFs were PCR-amplified ... led and further expanded for ChIP-seq.
      item-42 at level 3: section_header: CRISPR/Cas9 mediated deletion of KRAB-ZFP clusters and an MMETn insertion
        item-43 at level 4: text: All gRNAs were expressed from th ... PCR genotyping (Supplementary file 3).
      item-44 at level 3: section_header: ChIP-seq analysis
        item-45 at level 4: text: For ChIP-seq analysis of KRAB-ZF ... 010 or Khil et al., 2012 respectively.
        item-46 at level 4: text: ChIP-seq libraries were construc ...  were re-mapped using Bowtie (--best).
      item-47 at level 3: section_header: Luciferase reporter assays
        item-48 at level 4: text: For KRAB-ZFP repression assays,  ... after transfection as described above.
      item-49 at level 3: section_header: RNA-seq analysis
        item-50 at level 4: text: Whole RNA was purified using RNe ... lemented in the R function p.adjust().
      item-51 at level 3: section_header: Reduced representation bisulfite sequencing (RRBS-seq)
        item-52 at level 4: text: For RRBS-seq analysis, Chr4-cl W ... h sample were considered for analysis.
      item-53 at level 3: section_header: Retrotransposition assay
        item-54 at level 4: text: The retrotransposition vectors p ... were stained with Amido Black (Sigma).
      item-55 at level 3: section_header: Capture-seq screen
        item-56 at level 4: text: To identify novel retrotransposo ... assembly using the Unicycler software.
    item-57 at level 2: section_header: Funding Information
      item-58 at level 3: text: This paper was supported by the following grants:
      item-59 at level 3: list: group list
        item-60 at level 4: list_item: http://dx.doi.org/10.13039/10000 ... ment 1ZIAHD008933 to Todd S Macfarlan.
        item-61 at level 4: list_item: http://dx.doi.org/10.13039/50110 ... ndation 310030_152879 to Didier Trono.
        item-62 at level 4: list_item: http://dx.doi.org/10.13039/50110 ... dation 310030B_173337 to Didier Trono.
        item-63 at level 4: list_item: http://dx.doi.org/10.13039/50110 ... ch Council No. 268721 to Didier Trono.
        item-64 at level 4: list_item: http://dx.doi.org/10.13039/50110 ... rch Council No 694658 to Didier Trono.
    item-65 at level 2: section_header: Acknowledgements
      item-66 at level 3: text: We thank Alex Grinberg, Jeanne Y ...  268721; Transpos-X, No. 694658) (DT).
    item-67 at level 2: section_header: Additional information
    item-68 at level 2: section_header: Additional files
    item-69 at level 2: section_header: Data availability
      item-70 at level 3: text: All NGS data has been deposited  ... GenBank database (MH449667- MH449669).
      item-71 at level 3: text: The following datasets were generated:
      item-72 at level 3: text: Wolf G. Retrotransposon reactiva ... ession Omnibus (2019). NCBI: GSE115291
      item-73 at level 3: text: Wolf G. Mus musculus musculus st ... e. NCBI GenBank (2019). NCBI: MH449667
      item-74 at level 3: text: Wolf G. Mus musculus musculus st ... e. NCBI GenBank (2019). NCBI: MH449668
      item-75 at level 3: text: Wolf G. Mus musculus musculus st ... e. NCBI GenBank (2019). NCBI: MH449669
      item-76 at level 3: text: The following previously published datasets were used:
      item-77 at level 3: text: Castro-Diaz N, Ecco G, Coluccio  ... ssion Omnibus (2014). NCBI: GSM1406445
      item-78 at level 3: text: Andrew ZX. H3K9me3_ChIPSeq (Ctrl ... ssion Omnibus (2014). NCBI: GSM1327148
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  item-137 at level 1: caption: Figure 1. Genome-wide binding pa ... onsensus fingers highlighted in white.
  item-138 at level 1: caption: Table 1. KRAB-ZFP genes clusters ...  ChIP-seq was performed in this study.
  item-139 at level 1: caption: Figure 2. Retrotransposon reacti ... s were calculated using paired t-test.
  item-140 at level 1: caption: Figure 3. TE-dependent gene acti ... Gm13051 are indicated by dashed lines.
  item-141 at level 1: caption: Figure 4. ETn retrotransposition ... combined for the statistical analysis.
  item-142 at level 1: caption: Key resources table