feat: Expose equation exports (#869)

* pin new docling-core and exploit it via assembler changes

Signed-off-by: Michele Dolfi <dol@zurich.ibm.com>

* update test results

Signed-off-by: Michele Dolfi <dol@zurich.ibm.com>

* update with docling-core release

Signed-off-by: Michele Dolfi <dol@zurich.ibm.com>

---------

Signed-off-by: Michele Dolfi <dol@zurich.ibm.com>

tests/data/groundtruth/docling_v2/elife-56337.xml.md

@@ -18,7 +18,7 @@ TEs, especially long terminal repeat (LTR) retrotransposons, also known as endog
 
 We analyzed the RNA expression profiles of mouse KRAB-ZFPs across a wide range of tissues to identify candidates active in early embryos/ES cells. While the majority of KRAB-ZFPs are expressed at low levels and uniformly across tissues, a group of KRAB-ZFPs are highly and almost exclusively expressed in ES cells (Figure 1—figure supplement 1A). About two thirds of these KRAB-ZFPs are physically linked in two clusters on chromosome 2 (Chr2-cl) and 4 (Chr4-cl) (Figure 1—figure supplement 1B). These two clusters encode 40 and 21 KRAB-ZFP annotated genes, respectively, which, with one exception on Chr4-cl, do not have orthologues in rat or any other sequenced mammals (Supplementary file 1). The KRAB-ZFPs within these two genomic clusters also group together phylogenetically (Figure 1—figure supplement 1C), indicating these gene clusters arose by a series of recent segmental gene duplications (Kauzlaric et al., 2017).
 
-To determine the binding sites of the KRAB-ZFPs within these and other gene clusters, we expressed epitope-tagged KRAB-ZFPs using stably integrating vectors in mouse embryonic carcinoma (EC) or ES cells (Table 1, Supplementary file 1) and performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). We then determined whether the identified binding sites are significantly enriched over annotated TEs and used the non-repetitive peak fraction to identify binding motifs. We discarded 7 of 68 ChIP-seq datasets because we could not obtain a binding motif or a target TE and manual inspection confirmed low signal to noise ratio. Of the remaining 61 KRAB-ZFPs, 51 significantly overlapped at least one TE subfamily (adjusted p-value<1e-5). Altogether, 81 LTR retrotransposon, 18 LINE, 10 SINE and one DNA transposon subfamilies were targeted by at least one of the 51 KRAB-ZFPs (Figure 1A and Supplementary file 1). Chr2-cl KRAB-ZFPs preferably bound IAPEz retrotransposons and L1-type LINEs, while Chr4-cl KRAB-ZFPs targeted various retrotransposons, including the closely related MMETn (hereafter referred to as ETn) and ETnERV (also known as MusD) elements (Figure 1A). ETn elements are non-autonomous LTR retrotransposons that require trans-complementation by the fully coding ETnERV elements that contain Gag, Pro and Pol genes (Ribet et al., 2004). These elements have accumulated to ~240 and~100 copies in the reference C57BL/6 genome, respectively, with ~550 solitary LTRs (Baust et al., 2003). Both ETn and ETnERVs are still active, generating polymorphisms and mutations in several mouse strains (Gagnier et al., 2019). The validity of our ChIP-seq screen was confirmed by the identification of binding motifs - which often resembled the computationally predicted motifs (Figure 1—figure supplement 2A) - for the majority of screened KRAB-ZFPs (Supplementary file 1). Moreover, predicted and experimentally determined motifs were found in targeted TEs in most cases (Supplementary file 1), and reporter repression assays confirmed KRAB-ZFP induced silencing for all the tested sequences (Figure 1—figure supplement 2B). Finally, we observed KAP1 and H3K9me3 enrichment at most of the targeted TEs in wild type ES cells, indicating that most of these KRAB-ZFPs are functionally active in the early embryo (Figure 1A).
+To determine the binding sites of the KRAB-ZFPs within these and other gene clusters, we expressed epitope-tagged KRAB-ZFPs using stably integrating vectors in mouse embryonic carcinoma (EC) or ES cells (Table 1, Supplementary file 1) and performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). We then determined whether the identified binding sites are significantly enriched over annotated TEs and used the non-repetitive peak fraction to identify binding motifs. We discarded 7 of 68 ChIP-seq datasets because we could not obtain a binding motif or a target TE and manual inspection confirmed low signal to noise ratio. Of the remaining 61 KRAB-ZFPs, 51 significantly overlapped at least one TE subfamily (adjusted p-value&lt;1e-5). Altogether, 81 LTR retrotransposon, 18 LINE, 10 SINE and one DNA transposon subfamilies were targeted by at least one of the 51 KRAB-ZFPs (Figure 1A and Supplementary file 1). Chr2-cl KRAB-ZFPs preferably bound IAPEz retrotransposons and L1-type LINEs, while Chr4-cl KRAB-ZFPs targeted various retrotransposons, including the closely related MMETn (hereafter referred to as ETn) and ETnERV (also known as MusD) elements (Figure 1A). ETn elements are non-autonomous LTR retrotransposons that require trans-complementation by the fully coding ETnERV elements that contain Gag, Pro and Pol genes (Ribet et al., 2004). These elements have accumulated to ~240 and~100 copies in the reference C57BL/6 genome, respectively, with ~550 solitary LTRs (Baust et al., 2003). Both ETn and ETnERVs are still active, generating polymorphisms and mutations in several mouse strains (Gagnier et al., 2019). The validity of our ChIP-seq screen was confirmed by the identification of binding motifs - which often resembled the computationally predicted motifs (Figure 1—figure supplement 2A) - for the majority of screened KRAB-ZFPs (Supplementary file 1). Moreover, predicted and experimentally determined motifs were found in targeted TEs in most cases (Supplementary file 1), and reporter repression assays confirmed KRAB-ZFP induced silencing for all the tested sequences (Figure 1—figure supplement 2B). Finally, we observed KAP1 and H3K9me3 enrichment at most of the targeted TEs in wild type ES cells, indicating that most of these KRAB-ZFPs are functionally active in the early embryo (Figure 1A).
 
 We generally observed that KRAB-ZFPs present exclusively in mouse target TEs that are restricted to the mouse genome, indicating KRAB-ZFPs and their targets emerged together. For example, several mouse-specific KRAB-ZFPs in Chr2-cl and Chr4-cl target IAP and ETn elements which are only found in the mouse genome and are highly active. This is the strongest data to date supporting that recent KRAB-ZFP expansions in these young clusters is a response to recent TE activity. Likewise, ZFP599 and ZFP617, both conserved in Muroidea, bind to various ORR1-type LTRs which are present in the rat genome (Supplementary file 1). However, ZFP961, a KRAB-ZFP encoded on a small gene cluster on chromosome 8 that is conserved in Muroidea targets TEs that are only found in the mouse genome (e.g. ETn), a paradox we have previously observed with ZFP809, which also targets TEs that are evolutionarily younger than itself (Wolf et al., 2015b). The ZFP961 binding site is located at the 5’ end of the internal region of ETn and ETnERV elements, a sequence that usually contains the primer binding site (PBS), which is required to prime retroviral reverse transcription. Indeed, the ZFP961 motif closely resembles the PBSLys1,2 (Figure 1—figure supplement 3A), which had been previously identified as a KAP1-dependent target of retroviral repression (Yamauchi et al., 1995; Wolf et al., 2008). Repression of the PBSLys1,2 by ZFP961 was also confirmed in reporter assays (Figure 1—figure supplement 2B), indicating that ZFP961 is likely responsible for this silencing effect.
 
@@ -38,7 +38,7 @@ While we generally observed that TE-associated gene reactivation is not caused b
 
 ### ETn retrotransposition in Chr4-cl KO and WT mice
 
-IAP, ETn/ETnERV and MuLV/RLTR4 retrotransposons are highly polymorphic in inbred mouse strains (Nellåker et al., 2012), indicating that these elements are able to mobilize in the germ line. Since these retrotransposons are upregulated in Chr2-cl and Chr4-cl KO ES cells, we speculated that these KRAB-ZFP clusters evolved to minimize the risks of insertional mutagenesis by retrotransposition. To test this, we generated Chr2-cl and Chr4-cl KO mice via ES cell injection into blastocysts, and after germ line transmission we genotyped the offspring of heterozygous breeding pairs. While the offspring of Chr4-cl KO/WT parents were born close to Mendelian ratios in pure C57BL/6 and mixed C57BL/6 129Sv matings, one Chr4-cl KO/WT breeding pair gave birth to significantly fewer KO mice than expected (p-value=0.022) (Figure 4—figure supplement 1A). Likewise, two out of four Chr2-cl KO breeding pairs on mixed C57BL/6 129Sv matings failed to give birth to a single KO offspring (p-value<0.01) while the two other mating pairs produced KO offspring at near Mendelian ratios (Figure 4—figure supplement 1A). Altogether, these data indicate that KRAB-ZFP clusters are not absolutely essential in mice, but that genetic and/or epigenetic factors may contribute to reduced viability.
+IAP, ETn/ETnERV and MuLV/RLTR4 retrotransposons are highly polymorphic in inbred mouse strains (Nellåker et al., 2012), indicating that these elements are able to mobilize in the germ line. Since these retrotransposons are upregulated in Chr2-cl and Chr4-cl KO ES cells, we speculated that these KRAB-ZFP clusters evolved to minimize the risks of insertional mutagenesis by retrotransposition. To test this, we generated Chr2-cl and Chr4-cl KO mice via ES cell injection into blastocysts, and after germ line transmission we genotyped the offspring of heterozygous breeding pairs. While the offspring of Chr4-cl KO/WT parents were born close to Mendelian ratios in pure C57BL/6 and mixed C57BL/6 129Sv matings, one Chr4-cl KO/WT breeding pair gave birth to significantly fewer KO mice than expected (p-value=0.022) (Figure 4—figure supplement 1A). Likewise, two out of four Chr2-cl KO breeding pairs on mixed C57BL/6 129Sv matings failed to give birth to a single KO offspring (p-value&lt;0.01) while the two other mating pairs produced KO offspring at near Mendelian ratios (Figure 4—figure supplement 1A). Altogether, these data indicate that KRAB-ZFP clusters are not absolutely essential in mice, but that genetic and/or epigenetic factors may contribute to reduced viability.
 
 We reasoned that retrotransposon activation could account for the reduced viability of Chr2-cl and Chr4-cl KO mice in some matings. However, since only rare matings produced non-viable KO embryos, we instead turned to the viable KO mice to assay for increased transposon activity. RNA-seq in blood, brain and testis revealed that, with a few exceptions, retrotransposons upregulated in Chr2 and Chr4 KRAB-ZFP cluster KO ES cells are not expressed at higher levels in adult tissues (Figure 4—figure supplement 1B). Likewise, no strong transcriptional TE reactivation phenotype was observed in liver and kidney of Chr4-cl KO mice (data not shown) and ChIP-seq with antibodies against H3K4me1, H3K4me3 and H3K27ac in testis of Chr4-cl WT and KO mice revealed no increase of active histone marks at ETn elements or other TEs (data not shown). This indicates that Chr2-cl and Chr4-cl KRAB-ZFPs are primarily required for TE repression during early development. This is consistent with the high expression of these KRAB-ZFPs uniquely in ES cells (Figure 1—figure supplement 1A). To determine whether retrotransposition occurs at a higher frequency in Chr4-cl KO mice during development, we screened for novel ETn (ETn/ETnERV) and MuLV (MuLV/RLTR4\_MM) insertions in viable Chr4-cl KO mice. For this purpose, we developed a capture-sequencing approach to enrich for ETn/MuLV DNA and flanking sequences from genomic DNA using probes that hybridize with the 5’ and 3’ ends of ETn and MuLV LTRs prior to deep sequencing. We screened genomic DNA samples from a total of 76 mice, including 54 mice from ancestry-controlled Chr4-cl KO matings in various strain backgrounds, the two ES cell lines the Chr4-cl KO mice were generated from, and eight mice from a Chr2-cl KO mating which served as a control (since ETn and MuLVs are not activated in Chr2-cl KO ES cells) (Supplementary file 4). Using this approach, we were able to enrich reads mapping to ETn/MuLV LTRs about 2,000-fold compared to genome sequencing without capture. ETn/MuLV insertions were determined by counting uniquely mapped reads that were paired with reads mapping to ETn/MuLV elements (see materials and methods for details). To assess the efficiency of the capture approach, we determined what proportion of a set of 309 largely intact (two LTRs flanking an internal sequence) reference ETn elements could be identified using our sequencing data. 95% of these insertions were called with high confidence in the majority of our samples (data not shown), indicating that we are able to identify ETn insertions at a high recovery rate.
 
@@ -74,7 +74,7 @@ All gRNAs were expressed from the pX330-U6-Chimeric\_BB-CBh-hSpCas9 vector (RRID
 
 For ChIP-seq analysis of KRAB-ZFP expressing cells, 5–10 × 107 cells were crosslinked and immunoprecipitated with anti-FLAG (Sigma-Aldrich Cat# F1804, RRID:AB\_262044) or anti-HA (Abcam Cat# ab9110, RRID:AB\_307019 or Covance Cat# MMS-101P-200, RRID:AB\_10064068) antibody using one of two previously described protocols (O'Geen et al., 2010; Imbeault et al., 2017) as indicated in Supplementary file 1. H3K9me3 distribution in Chr4-cl, Chr10-cl, Chr13.1-cl and Chr13.2-cl KO ES cells was determined by native ChIP-seq with anti-H3K9me3 serum (Active Motif Cat# 39161, RRID:AB\_2532132) as described previously (Karimi et al., 2011). In Chr2-cl KO ES cells, H3K9me3 and KAP1 ChIP-seq was performed as previously described (Ecco et al., 2016). In Chr4-cl KO and WT ES cells KAP1 binding was determined by endogenous tagging of KAP1 with C-terminal GFP (Supplementary file 3), followed by FACS to enrich for GFP-positive cells and ChIP with anti-GFP (Thermo Fisher Scientific Cat# A-11122, RRID:AB\_221569) using a previously described protocol (O'Geen et al., 2010). For ChIP-seq analysis of active histone marks, cross-linked chromatin from ES cells or testis (from two-week old mice) was immunoprecipitated with antibodies against H3K4me3 (Abcam Cat# ab8580, RRID:AB\_306649), H3K4me1 (Abcam Cat# ab8895, RRID:AB\_306847) and H3K27ac (Abcam Cat# ab4729, RRID:AB\_2118291) following the protocol developed by O'Geen et al., 2010 or Khil et al., 2012 respectively.
 
-ChIP-seq libraries were constructed and sequenced as indicated in Supplementary file 4. Reads were mapped to the mm9 genome using Bowtie (RRID:SCR\_005476; settings: --best) or Bowtie2 (Langmead and Salzberg, 2012) as indicated in Supplementary file 4. Under these settings, reads that map to multiple genomic regions are assigned to the top-scored match and, if a set of equally good choices is encountered, a pseudo-random number is used to choose one location. Peaks were called using MACS14 (RRID:SCR\_013291) under high stringency settings (p<1e-10, peak enrichment >20) (Zhang et al., 2008). Peaks were called both over the Input control and a FLAG or HA control ChIP (unless otherwise stated in Supplementary file 4) and only peaks that were called in both settings were kept for further analysis. In cases when the stringency settings did not result in at least 50 peaks, the settings were changed to medium (p<1e-10, peak enrichment >10) or low (p<1e-5, peak enrichment >10) stringency (Supplementary file 4). For further analysis, all peaks were scaled to 200 bp regions centered around the peak summits. The overlap of the scaled peaks to each repeat element in UCSC Genome Browser (RRID:SCR\_005780) were calculated by using the bedfisher function (settings: -f 0.25) from BEDTools (RRID:SCR\_006646). The right-tailed p-values between pair-wise comparison of each ChIP-seq peak and repeat element were extracted, and then adjusted using the Benjamini-Hochberg approach implemented in the R function p.adjust(). Binding motifs were determined using only nonrepetitive (<10% repeat content) peaks with MEME (Bailey et al., 2009). MEME motifs were compared with in silico predicted motifs (Najafabadi et al., 2015) using Tomtom (Bailey et al., 2009) and considered as significantly overlapping with a False Discovery Rate (FDR) below 0.1. To find MEME and predicted motifs in repetitive peaks, we used FIMO (Bailey et al., 2009). Differential H3K9me3 and KAP1 distribution in WT and Chr2-cl or Chr4-cl KO ES cells at TEs was determined by counting ChIP-seq reads overlapping annotated insertions of each TE group using BEDTools (MultiCovBed). Additionally, ChIP-seq reads were counted at the TE fraction that was bound by Chr2-cl or Chr4-cl KRAB-ZFPs (overlapping with 200 bp peaks). Count tables were concatenated and analyzed using DESeq2 (Love et al., 2014). The previously published ChIP-seq datasets for KAP1 (Castro-Diaz et al., 2014) and H3K9me3 (Dan et al., 2014) were re-mapped using Bowtie (--best).
+ChIP-seq libraries were constructed and sequenced as indicated in Supplementary file 4. Reads were mapped to the mm9 genome using Bowtie (RRID:SCR\_005476; settings: --best) or Bowtie2 (Langmead and Salzberg, 2012) as indicated in Supplementary file 4. Under these settings, reads that map to multiple genomic regions are assigned to the top-scored match and, if a set of equally good choices is encountered, a pseudo-random number is used to choose one location. Peaks were called using MACS14 (RRID:SCR\_013291) under high stringency settings (p&lt;1e-10, peak enrichment &gt;20) (Zhang et al., 2008). Peaks were called both over the Input control and a FLAG or HA control ChIP (unless otherwise stated in Supplementary file 4) and only peaks that were called in both settings were kept for further analysis. In cases when the stringency settings did not result in at least 50 peaks, the settings were changed to medium (p&lt;1e-10, peak enrichment &gt;10) or low (p&lt;1e-5, peak enrichment &gt;10) stringency (Supplementary file 4). For further analysis, all peaks were scaled to 200 bp regions centered around the peak summits. The overlap of the scaled peaks to each repeat element in UCSC Genome Browser (RRID:SCR\_005780) were calculated by using the bedfisher function (settings: -f 0.25) from BEDTools (RRID:SCR\_006646). The right-tailed p-values between pair-wise comparison of each ChIP-seq peak and repeat element were extracted, and then adjusted using the Benjamini-Hochberg approach implemented in the R function p.adjust(). Binding motifs were determined using only nonrepetitive (&lt;10% repeat content) peaks with MEME (Bailey et al., 2009). MEME motifs were compared with in silico predicted motifs (Najafabadi et al., 2015) using Tomtom (Bailey et al., 2009) and considered as significantly overlapping with a False Discovery Rate (FDR) below 0.1. To find MEME and predicted motifs in repetitive peaks, we used FIMO (Bailey et al., 2009). Differential H3K9me3 and KAP1 distribution in WT and Chr2-cl or Chr4-cl KO ES cells at TEs was determined by counting ChIP-seq reads overlapping annotated insertions of each TE group using BEDTools (MultiCovBed). Additionally, ChIP-seq reads were counted at the TE fraction that was bound by Chr2-cl or Chr4-cl KRAB-ZFPs (overlapping with 200 bp peaks). Count tables were concatenated and analyzed using DESeq2 (Love et al., 2014). The previously published ChIP-seq datasets for KAP1 (Castro-Diaz et al., 2014) and H3K9me3 (Dan et al., 2014) were re-mapped using Bowtie (--best).
 
 ### Luciferase reporter assays
 
@@ -149,7 +149,7 @@ Key resources table:
 ## Figures
 
 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.
+(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&lt;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&lt;1e-10, peak enrichment &gt;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.
 Figure 1—source data 1.KRAB-ZFP expression in 40 mouse tissues and cell lines (ENCODE).Mean values of replicates are shown as log2 transcripts per million.
 Figure 1—source data 2.Probability heatmap of KRAB-ZFP binding to TEs.Values corresponds to -log10 (adjusted p-value) enrichment of ChIP-seq peak overlap with TE groups (Fisher’s exact test).
 
@@ -161,7 +161,7 @@ Figure 1—figure supplement 1.: ES cell-specific expression of KRAB-ZFP gene cl
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 Figure 1—figure supplement 2.: KRAB-ZFP binding motifs and their repression activity.
-(A) Comparison of computationally predicted (bottom) and experimentally determined (top) KRAB-ZFP binding motifs. Only significant pairs are shown (FDR < 0.1). (B) Luciferase reporter assays to confirm KRAB-ZFP repression of the identified target sites. Bars show the luciferase activity (normalized to Renilla luciferase) of reporter plasmids containing the indicated target sites cloned upstream of the SV40 promoter. Reporter plasmids were co-transfected into 293 T cells with a Renilla luciferase plasmid for normalization and plasmids expressing the targeting KRAB-ZFP. Normalized mean luciferase activity (from three replicates) is shown relative to luciferase activity of the reporter plasmid co-transfected with an empty pcDNA3.1 vector.
+(A) Comparison of computationally predicted (bottom) and experimentally determined (top) KRAB-ZFP binding motifs. Only significant pairs are shown (FDR &lt; 0.1). (B) Luciferase reporter assays to confirm KRAB-ZFP repression of the identified target sites. Bars show the luciferase activity (normalized to Renilla luciferase) of reporter plasmids containing the indicated target sites cloned upstream of the SV40 promoter. Reporter plasmids were co-transfected into 293 T cells with a Renilla luciferase plasmid for normalization and plasmids expressing the targeting KRAB-ZFP. Normalized mean luciferase activity (from three replicates) is shown relative to luciferase activity of the reporter plasmid co-transfected with an empty pcDNA3.1 vector.
 
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@@ -171,7 +171,7 @@ Figure 1—figure supplement 3.: KRAB-ZFP binding to ETn retrotransposons.
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 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.
+(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&lt;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.
 Figure 2—source data 1.Differential H3K9me3 and KAP1 distribution in WT and KRAB-ZFP cluster KO ES cells at TE families and KRAB-ZFP bound TE insertions.Differential read counts and statistical testing were determined by DESeq2.
 
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@@ -182,7 +182,7 @@ Figure 2—figure supplement 1.: Epigenetic changes at TEs and TE-borne enhancer
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 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.
+(A) Differential gene expression in Chr2-cl and Chr4-cl KO ES cells. Significantly up- and downregulated genes (adjusted p-value&lt;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&lt;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&lt;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.
 
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@@ -194,7 +194,7 @@ Figure 4—source data 2.Sequences of capture-seq probes used to enrich genomic
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 Figure 4—figure supplement 1.: Birth statistics of KRAB-ZFP cluster KO mice and TE reactivation in adult tissues.
-(A) Birth statistics of Chr4- and Chr2-cl mice derived from KO/WT x KO/WT matings in different strain backgrounds. (B) RNA-seq analysis of TE expression in Chr2- (left) and Chr4-cl (right) KO tissues. TE groups with the highest reactivation phenotype in ES cells are shown separately. Significantly up- and downregulated elements (adjusted p-value<0.05) are highlighted in red and green, respectively. Experiments were performed in at least two biological replicates.
+(A) Birth statistics of Chr4- and Chr2-cl mice derived from KO/WT x KO/WT matings in different strain backgrounds. (B) RNA-seq analysis of TE expression in Chr2- (left) and Chr4-cl (right) KO tissues. TE groups with the highest reactivation phenotype in ES cells are shown separately. Significantly up- and downregulated elements (adjusted p-value&lt;0.05) are highlighted in red and green, respectively. Experiments were performed in at least two biological replicates.
 
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@@ -214,7 +214,7 @@ Figure 4—figure supplement 3.: Confirmation of novel ETn insertions identified
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