The MMSAT4 satellite repeat corresponds to KZFP genes and KZFP-like entities

The repository of murine repetitive elements lists as a member of the Satellite family a simple repeat named ‘MMSAT4’, the consensus sequence of which was derived from arrays of triplets of C2H2 ZNFs located on chromosome 4 (Fig 1A, http://www.girinst.org/repbase/ [32]). However, unlike other Satellite repeats, MMSAT4s are not restricted to specific chromosomal positions, as we found 715 of these units spread all over the genome, often concentrated in regions also containing high densities of bona fide C2H2 ZNF-coding sequences (Fig 1B). Of the annotated C2H2-protein coding units containing an MMSAT4, the large majority (82%) are canonical KZFP genes while a small fraction spans other types of poly-zinc finger protein genes. Conversely, of the currently annotated KZFP genes, 87% overlap with MMSAT4 sequences. Additional MMSAT4 elements, making up slightly more than half of the group, fall into previously unannotated sequences (Fig 1B), yet we found them to harbor an upstream KRAB-coding sequence and to coincide with the 3’ end of the cognate transcripts, comparably to canonical KZFP-overlapping MMSAT4s (Fig 1C and S1A Fig). Therefore, MMSAT4s correspond to almost the totality of KZFP genes and to additional KZFP-like entities. For convenience, we coined the sum of these elements KZFP/rGUs, for KZFP-related genetic units.

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Fig 1. Genomic features and evolutionary origins of murine KZFP/rGUs clusters.

(A) (Top) Schematic representation of a canonical C2H2 zinc finger (ZF) structure, coupled to its 7-amino acids linker, 6 of which (TGEKPY) are highly conserved. The 21–24 amino acid-long ZNF (C X_2-4 C X₁₂ H X_3-4 H) is depicted, with zinc-coordinating cysteines and histidines in blue and putative DNA-contacting residues located at positions -1, 3 and 6 of the alpha helix in red. (Bottom) Consensus sequence of MMSAT4 Satellite Repeat as reported in Repbase, spanning three consecutive but incomplete C2H2 ZNF motifs, each reported on a single line. Highlighted are the canonical cysteine and histidine residues (in blue) and the conserved part of the linker (in grey). (B) Circular genomic map displaying the distribution of MMSAT4 elements. Inside the circular plot, stacked bar plot of MMSAT4 elements belonging to C2H2 ZNF protein genes not encoding a KRAB domain, to KRAB C2H2 ZNF protein genes, or to genomic locations not annotated as any type of C2H2 encoding protein genes. (C) Positional correlation between MMSAT4 consensus sequences and KRAB-encoding sequences, distinguishing MMSAT4 elements belonging to C2H2 ZNF protein genes not encoding a KRAB domain, to KRAB C2H2 ZNF protein genes, or to genomic locations not annotated as any type of C2H2 encoding protein genes. Genes’ coordinates were extended by 1kb at the 3’ end, and the correlation was calculated over a symmetrical window of 15 kb. (D) Raw counts of 518 murine KZFP/rGUs shared between phylogenetic branches, belonging to annotated KZFP genes or other genetic entities (“other”), with the x-axis representing the evolutionary distances separating the branches. 517/518 of these KZFP/rGUs bear a KRAB-encoding sequence within 30 kb upstream of the element itself. (E) Age-distribution of KZFP/rGUs within clusters. The estimated time of emergence during evolution is color-coded as indicated by the legend. (F) Phylogenetic tree of consensus sequences derived per KZFP/rGUs cluster, with indication of the chromosome where the relative cluster is located.

https://doi.org/10.1371/journal.pone.0173746.g001

Evolutionary relationships between KZFP/rGUs clusters

Through sequence and synteny analysis of murine KZFP/rGUs and their flanking sequences [4], we could assign a putative age to about three quarters of these elements (518/715). We found the vast majority to be mouse-restricted, whether KZFP genes or pseudogenes and non-annotated entities, and very few to have human orthologs, corresponding to an evolutionary distance of 90.9 million years ago (mya) (Fig 1D). By setting a cutoff of at least 8 MMSAT4 elements less than 500 kilobases (kb) apart, we could further define 18 clusters distributed amongst 11 chromosomes (S1 Table). Some clusters contained both mouse-specific and evolutionarily older KZFP/rGUs (e.g. clusters 14 and 16 on chromosome 7), yet none was entirely constituted of highly conserved members, and 5 of the 18 predefined clusters contained exclusively mouse-restricted KZFP/rGUs (Fig 1E).

Consistent with previously proposed mechanisms of KZFP genes amplification [7], we observed that murine KZFP/rGUs shared higher sequence homology within a cluster than amongst spatially unrelated elements (S1B Fig). We thus derived a consensus sequence for each cluster and built a tree based on their degree of similarity. This confirmed that clusters located on a same chromosome were usually more closely related. However, there were exceptions to this trend. For instance, clusters 1 and 12, respectively on chromosomes 1 and 6, were highly homologous, as were clusters 2 and 17, on chromosomes 10 and 7. This strongly suggests that large segment duplications, followed by chromosomal rearrangements, have contributed to the expansion of KZFP/rGUs, as previously postulated for canonical KZFP genes [5]. Interestingly, clusters partially composed of conserved KZFP/rGUs, such as number 14, 16 and 18, segregated away from the others (Fig 1F). Careful examination revealed age-independent clustering of KZFP/rGUs within these clusters (S1C Fig).

When further studying KZFP/rGUs clusters, we very rarely found genes other than KZFPs. The murine genome harbors several million loci derived from transposable elements (TEs), and when examining the density of these units within clusters of KZFP/rGUs we found it to be no different than elsewhere in the genome (S1D Fig). However, in KZFP/rGUs clusters, we found a marked enrichment in members of two subgroups of ERVs, ERVK and ERV1 (S1E Fig). Similar to KZFPs, olfactory and vomeronasal receptor genes (OLFR and VMNR genes, respectively) have been amplified through rounds of segmental duplications while undergoing positive selection [33, 34]. However, we found neither 14 clusters of OLFR genes nor a cluster of VMNR genes, each defined as a group of at least 10 such genes situated less than 500 kb apart, to display similarly biased TE distribution (S2 Table, S1F Fig). KZFP/rGUs clusters thus exhibit a particular TE content when compared to the rest of the genome or to other gene super-families amplified by related mechanisms.

When examining KZFP/rGUs not situated in clusters, we identified several instances where they most likely resulted from retrotransposition, as indicated by the absence of introns, the presence of a poly-A-coding sequence at their 3’ end and their homology with intron-containing cluster-located elements (Fig 2A, left panel). Although they were situated on other chromosomes than their source element, such retrotransposed KZFPs displayed signs of positive selection. This was exemplified by the newly annotated pseudogene Gm8935b, where a sharp increase in mutations density was found 3’ of a premature STOP codon gained in the processed pseudogene, compared to its apparent donor gene. Moreover, we detected a distinctive enrichment in RNA-polymerase II over Gm8935b (Fig 2A, right panel). These observations led us to conclude that KZFP/rGUs resulting from retrotransposition events can be transcriptionally active and that, when positively selected, these elements could contribute to seeding new KZFP/rGUs clusters.

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Fig 2. Evolutionary relationships within the KZFP-rGUs superfamily.

(A) (Left) Schematic representation of how retrotransposition of the KZFP/rGU Gm13051 (located on chromosome 4) may have yielded the Gm8935b retrogene (on chromosome 3, to which we gave a different annotation than the one deposited in Ensembl as Gm8935, based on similarities with its putative donor gene). ChIP-seq density signal of RNA polymerase II (PolII) at the corresponding loci and genomic structures of Gm13051 and Gm8935b are shown, with thick lines depicting exons and thin lines intronic or non-transcribed sequences. KZFP/rGUs (black) and poly-A sequences (red) are annotated separately. (Right) Amino acid sequence comparison of putative GM13051 and GM8935b proteins. KRAB and ZNF domains are shaded in dark and light purple, respectively. Orange rectangle, location of stop codon in GM8935b; red rectangle, mutations located in 3’ UTR. (*) indicates full conservation, (:) amino acids with highly similar properties and (.) unrelated residues. (B) (Top) UCSC Genome Browser view of a genomic region containing KZFP/rGU-related genes GmX (annotated in present study), Gm15446, and Zfp932, located in cluster 11 on chromosome 5. In order from the top, tracks for: genes annotated in RefSeq, our de-novo transcripts annotation, KRAB-encoding sequences, KZFP/rGUs, and repeats as reported in RepeatMasker. BLAT results for segments 1–5 of Zfp932 over the studied region are shown, with red lines indicating single differences in homologous sequences. (Center) Segments 6 and 7 of Gm15446, together with their BLAT results over GmX, are highlighted by red vertical bars. Segments 8 and 9, belonging to Gm15446 and GmX are highlighted by blue and green vertical bars, respectively. Homology interruptions of segments 1, 3 and 4 are depicted by hollow bars. Lengths of DNA stretches between genes are not to scale. (Bottom) Putative evolutionary tree of Zfp932, Gm15446 and GmX, with their zinc finger signature, that is, for each ZNF the three major DNA-contacting residues. (C) Strand, name of the corresponding annotated gene and ZNF specificity residues of KZFP/rGUs for which a related transcript was detected in cluster 10. Elements are reported in linear order of appearance in the cluster. Each ZNF, represented by the triplet of its DNA-contacting amino acids, is separated from the others by a dash. Red characters indicate tandem duplications of a given triplet of amino acids. Colored underlining highlights triplets conserved in multiple putative and bona-fide KZFP genes, to mark potential paralogs and closely related genes or pseudogenes. Pink shading marks triplets repeated multiple times, in the same order, within one KZFP gene or pseudogene. Yellow shading marks the first triplets of highly homologous genes Gm13235, Gm13051, Gm13151, Zfp534, exemplifying the variability of divergence patterns observed within a cluster.

https://doi.org/10.1371/journal.pone.0173746.g002

Sequence evolution of KZFP/rGU paralogs

The analysis of KZFP/rGUs revealed that individual units accumulated various degrees of mutations, some of which compromised their coding potential. However, of 1306 C2H2-encoding transcripts that could be assigned to KZFP/rGUs in murine embryonic stem cells (mESC), 734 mapped to entries of the Ensembl listings of genes, accounting for multiple isoforms of a totality of 252 genes. Of those, more than three quarters (196/252) corresponded to bona fide KZFP genes, 13% (34/252) to non-KRAB ZFP genes and 9% (22/252) to other genes. Of 572 novel transcripts, 490 coded for a stretch of C2H2 zinc fingers preceded by a KRAB domain in the same reading frame and without intervening STOP codon (S1 File, S3 Table). This suggests that positive selection is acting to preserve the functional core of most of these elements.

Retracing the full evolutionary history of KZFP/rGU clusters remains a complicated task, due to both persistent retrotransposition activity of many mouse TEs and confounding effects of partially overlapping genomic rearrangements (S2B Fig). We thus designed an additional approach to date the emergence of mouse-specific and closely related KZFP/rGUs. For this, we focused on the regions directly bordering KZFP/rGUs and studied their sequence conservation after splitting them in segments, which we considered as separate units. Coupling our analysis to newly annotated KZFP/rGUs-containing transcripts helped us circumscribe the region of interest to the one enclosed by gene borders, although segments numbers and limits were arbitrarily fixed. As an example, cluster 11 contains three KZFP/rGUs: Zfp932, Gm15446 and a putative gene not annotated in the RefSeq nor in other genes repositories, which we named GmX. Zfp932 and Gm15466 are known paralogs, recently characterized through genomic and functional studies as responsible for recognizing partly overlapping sets of TEs [35]. By defining discrete segments of DNA within the regions spanning Zfp932 and Gm15466, we could determine by BLAT [36] that the next most conserved sequence was spanning GmX. Taking Zfp932 (Fig 2B, segments 1–5) as main viewpoint allowed us to single out a series of new segments (Fig 2B, segments 6–9) that disrupted homology between the three genes. Segments 6–7 were present in Gm15446 and GmX, but not in Zfp932, while segment 8 was restricted to Gm15446 and segment 9 to GmX. Furthermore, several nucleotide differences were found within the studied segments of paralogs Zfp932 and Gm15446, and even more when comparing them to GmX. Taking into account the sum of these observations, we could draw a tree representing the likely sequential emergence of these genes, with Zfp932 followed by Gm15446 and then GmX. Interestingly, the ZNF signature of GmX strongly differs from those of Zfp932 and Gm15446, which are highly similar. Accordingly, segment 5, which spans the KZFP/rGU of each of these three genes, was the least conserved within the whole cluster. A broader examination of the specificity residues, that is, of the amino acids at positions -1, 3 and 6 of the DNA-contacting alpha helix, encoded by KZFP/rGUs of cluster 10 similarly revealed complex mechanisms of diversification of neighboring poly-zinc fingers, with evidence for duplications, deletions, inversions and mutations of ZNF-coding sequences. Triplets of the DNA-contacting residues belonging to one ZNF could be duplicated in tandem up to 4 times, and combinations of 2 to 7 sequential triplets could be repeated within the same gene or pseudogene. These repetitions were different between highly homologous signatures, which were not residing in adjacent KZFP/rGUs and could even be encoded on the opposite strand (Fig 2C). Together, these data support the model of a high level of plasticity within poly-zinc fingers coding sequences, with fast emergence of new zinc fingerprints, which either get fixed by positive selection or lose their coding potential and degenerate.

Transcriptional regulation of KZFP/rGUs

We next assessed by RNA-Seq the transcription of KZFP/rGUs in three distinct murine cell types: mESC, hepatocytes and MEFs. Each displayed a specific pattern of KZFP/rGU expression, with mESC transcribing the highest number of elements (Fig 3A, S4 Table). The ZNF-coding region of KZFP genes was previously demonstrated to recruit KAP1, and to be secondarily subjected to trimethylation of histone 3 on lysine 9 (H3K9me3) [37, 38]. ChIP-seq analyses confirmed the extensive binding of KAP1 at KZFP/rGUs in mESC, MEF and Hepa-1.6, a murine hepatoma cell line that we used as a surrogate for hepatocytes (Fig 3B). The density of KAP1 peaks correlated with that of KZFP/rGUs over many clusters in all three tissues, including clusters completely devoid of annotated protein-coding genes (S3A–S3C Fig). However, KZFP/rGUs themselves only accounted for a fifth to at most a third of all of KAP1 peaks within these regions, where they were largely outnumbered by TEs (Fig 3C).

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Fig 3. KAP1 binds to KZFP/rGUs and neighboring TEs.

(A) Heatmap of RNA-Seq depicting expression levels of KZFP-rGUs-derived transcripts in WT mES cells, liver cells and MEFs. Exact values were reported in S4 Table. (B) Positional correlation between KZFP/rGUs and KAP1 peaks in mES cells, Hepa 1.6 cells and MEFs, over a window of 10 kb. In the legend, indication of the p-values obtained by Fisher’s exact test. (C) Distribution analysis of KAP1 peaks located within KZFP/rGUs clusters per tissue. Selected TEs (“Sel. TEs”) comprise repeats annotated in RepeatMasker excluding Satellite, Simple, Low Complexity, Unknown repeats and subgroups counting less than a 100 hits among all KZFP/rGUs clusters. Peaks overlapping yet other entities, for instance genes, are listed as “other”. (D) Normalized RNA-Seq average coverage over significantly dysregulated KZFP-rGUs, considering a flanking region of 1.5 kb upstream and 3.5 kb downstream of each element, in mES (top), liver (center) and MEF (bottom) WT and Kap1 KO cells. Shaded curves represent the 95% confidence interval around the mean. (E) Mean log2 of the expression fold change upon Kap1 removal in mES cells of elements present in KZFP/rGUs clusters: KZFP/rGUs, ERVKs, ERV1s and the rest of TEs (“others”) (dots), as well as their total average (dashed line). The analysis was performed per cluster, with the number of elements in each category reported on the right (categories with fewer than 5 elements were not considered).

https://doi.org/10.1371/journal.pone.0173746.g003

Upon Kap1 knockout (KO), many KZFP/rGUs were upregulated in all three tissues, albeit with some differences: in mESC, 145 out of 150 dysregulated elements increased in expression, while for liver and MEF the ratios were 66/67 and 77/124, respectively (Fig 3D). Noteworthy, KZFP/rGUs upregulation upon KAP1 depletion was significant in mESC and liver only for elements situated within clusters, but not for their more isolated counterparts, while in MEF it was significant in neither setting (S3D Fig). We explored more in depth the apparently collective behavior of KZFP/rGUs located within a same cluster upon KAP1-depletion. For this, we scrutinized cluster 10, which contained the highest fraction of upregulated elements in these systems (23/25 in mESC, 15/25 in liver and MEF). We first found that, in spite of a high degree of homology between these KZFP/rGUs, their promoter regions were markedly divergent (S3E Fig). Second, only a third of these units (8/25) were deregulated in all three tissues, all the others behaving in a cell-specific manner. KZFP/rGUs transcription, as its perturbation, was not restricted to elements that were part of previously annotated protein-coding genes, and it always occurred in the sense orientation, indicating that transcriptional read-through from other units, which would have contributed both sense and antisense transcripts, was not significantly affecting our analysis (S3F Fig).

Noteworthy, along with the higher density of KAP1 peaks measured within KZFP/rGUs clusters compared to OLFR and VMNR genes clusters, we found an important frequency of upregulated TEs in Kap1 KO tissues (S3G Fig). TEs are subjected to KAP1-mediated repression via sequence-specific tethering by KZFPs [15, 35, 39–41]. Elements of the ERVK and ERV1 subgroups were found to be preferential sites of KAP1 recruitment within KZFP/rGUs clusters, but other TEs and KZFP/rGUs were equally upregulated upon KAP1 removal (Fig 3E and S3H and S3I Fig), indicating that de-repression then extended to the whole cluster when KAP1-targeted TEs lost the regulator.

Mechanisms of KZFP/rGUs clusters control

The histone lysine methyltransferase (KMT) SETDB1, a mediator of KAP1-induced repression, was also enriched at KZFP/rGUs, where its recruitment markedly dropped upon Kap1 knockdown, in agreement with previous findings for canonical KZFP genes [38] (S4A Fig). Furthermore, the same range of KZFP/rGUs was upregulated in Kap1 and Setdb1 KO mESC (Fig 4A), indicating that the transcriptional dysregulation recorded at these loci was likely mediated by the canonical KAP1-SETDB1 complex, known to lead to H3K9me3 deposition and silencing of underlying elements [21, 37, 42–44].

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Fig 4. KAP1 and SETDB1-mediated transcriptional regulation of KZFP/rGU clusters.

(A) Normalized RNA-Seq average coverage over KZFP-rGUs, considering a flanking region of 1.5 kb upstream and 3.5 kb downstream of each element. Shaded curves represent the 95% confidence interval around the mean. RNA-Seq data is plotted for KZFP/rGUs upregulated (main panels) or unaffected (smaller top right inserts) in either Kap1 (left) or Setdb1 (right) KO ES cells. (B) UCSC Genome Browser view of H3K9me3 ChIP-Seq profiles over KZFP/rGUs in cluster 10 in WT and Kap1 KO cells (light and dark color shades, respectively) for MEFs (top track), and liver cells (bottom track). Underneath, tracks for single KZFP/rGUs and for clusters are shown. (C) Heatmap illustration of RNA-Seq signal over KZFP/rGUs in cluster 10, reported linearly in their genomic order, for MEFs, liver and ES cells. (D) (Left) UCSC Genome Browser view of two KZFP genes with RNA-Seq profiles of WT MEFs (blue) and H3K9me3 of WT and Kap1 KO MEFs (red and pink, respectively). Below the RNA-Seq profile, genes annotated in RefSeq and the KZFP/rGUs tracks are displayed. The 3’ end of KZFP genes is highlighted by red-shaded vertical bars. (Right) Normalized H3K9me3 ChIP-Seq enrichment over (upper panel) KZFP/rGUs in WT and Kap1 KO MEFs, and (lower panel) KZFP/rGUs highly and lowly expressed in WT MEFs, including 5.5 kb on each side of KZFP/rGUs.

https://doi.org/10.1371/journal.pone.0173746.g004

Although KAP1-dependent deposition of H3K9me3 could be so extensive as to appear as covering entire KZFP/rGUs clusters both in MEF and liver, the underlying elements were actively transcribed (Fig 4B). Remarkably, neighboring elements within a cluster could display from rather homogeneous to very dissimilar expression levels in a given tissue, and the same element could be highly expressed in one cell type and barely detectable or completely silenced in another (Fig 4C). A closer examination revealed that H3K9me3 was not homogeneously distributed over clusters, being very high over the 3’ end of KZFP genes and KZFP/rGUs but practically absent from their promoters (Fig 4D, left). Loss of H3K9me3 upon Kap1 removal was observed over the entire cluster, including every 3’ end of KZFP/rGUs (Fig 4D, top right). Nevertheless, there was no correlation between its loss and the upregulation of the underlying element in Kap1 KO cells, and this mark was particularly abundant over highly transcribed KZFP/rGUs (Fig 4D, bottom right, S4C Fig).

We thus examined the impact of KAP1 depletion on the prevalence of H3K27ac, a histone mark associated with active promoters and enhancers, within KZFP/rGUs clusters. In clusters where a high fraction of KZFP/rGUs were dysregulated upon Kap1 KO in MEF cells, many new sites became enriched for this mark, most of which were just upstream of previously unidentified transcriptional start site (TSS, Fig 5A, left). ChIP-qPCR for H3K4me1, another histone modification marking actively transcribed promoters, confirmed the activation of numerous KZFP/rGUs genes in these clusters upon Kap1 deletion (Fig 5A, bottom right panel). Other regions within KZFP/rGUs clusters, not corresponding to any TSS, gained H3K27ac (Fig 5B and S5A Fig). These did not overlap with KZFP/rGUs themselves, but with TEs from all subclasses (S5B Fig). Upon KAP1 depletion, H3K27Ac was notably, but not exclusively, enriched at ERV1 and ERVK integrants normally situated in the vicinity of a KAP1 binding site (Fig 5C). Removal of KAP1 thus appeared to unleash enhancers, many located within TEs, with secondary transcriptional activation of KZFP/rGUs and other TEs situated nearby (Fig 6). Of note, proximity to a KAP1 binding site was not critical for the dysregulation of KZFP/rGUs, indicating that the regulatory mechanisms acting on these units are long-range (S6A Fig). Moreover, the transcriptional dysregulation of KZFP/rGUs was fully reversible upon re-expressing Kap1 in Kap1 KO MEF, but their upregulation was comparable or more pronounced immediately after deletion than at later times, suggesting that after long-term culture some compensatory mechanisms could dampen the transcriptional phenotype of Kap1 KO cells (S6B and S6C Fig). Upregulation of KZFP/rGUs upon Kap1 removal was confirmed on cDNAs generated by priming with random hexamers, suggesting that Poly(A)- transcripts, known to generate from ZFP genes [45], are not following a different transcriptional regulation compared to their Poly(A)+ counterparts (S6C Fig).

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Fig 5. KAP1 influence at KZFP/rGU clusters.

(A) (Top) UCSC Genome Browser view of a region containing a high density of KZFP/rGUs (black bars), many of which are dysregulated in MEF Kap1 KO cells (pink bars). From the top, tracks for KZFP/rGUs, H3K27ac peaks in MEF WT and Kap1 KO cells (light and dark green bars, respectively), and genes annotated in Ensembl and RefSeq are displayed. (Bottom, left) zoom-in of H3K27ac ChIP-Seq profiles and peaks upstream of two distinct KZFP/rGUs upregulated upon Kap1 deletion. Transcripts annotated in the frame of this study are reported below the track of dysregulated KZFP/rGUs. (Bottom, right) ChIP-PCR of H3K4me1 over several newly revealed TSSs within this region in MEF WT and Kap1 KO cells. Enrichments are normalized to those over Gapdh promoter, Beta-Tubulin being an additional promoter whose transcription is not affected by Kap1 removal (control). (B) UCSC Genome browser views of H3K27ac profiles in MEF (superimposing WT in red and Kap1 KO in green) over part of KZFP/rGUs cluster 3. From top, tracks for genes annotated in Ensembl and RefSeq, de-novo KZFP/rGUs transcripts annotation, H3K27ac ChIP-Seq profiles, KZFP/rGUs and repeats as reported by RepeatMasker. Genomic stretches gaining H3K27ac signal upon Kap1 KO do not overlap with any TSS. (C) H3K27ac ChIP-Seq normalized average coverage in WT (red) and Kap1 KO (green) MEFs over: left, ERV1 and ERVK elements located within KZFP/rGUs clusters and less than 3 kb away from a KAP1 peak in the same tissue; center, H3K27ac peaks within KZFP/rGUs clusters, not matching any KZFP/rGU transcript nor annotated promoter, less than 3 kb away from a KAP1 peak; right, without any restriction for KAP1 peak proximity. Shaded curved represent the 95% confidence interval around the mean.

https://doi.org/10.1371/journal.pone.0173746.g005

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Fig 6. Model for transcriptional regulation of KZFP/rGUs clusters.

(Top) Schematic representation of KZFP/rGUs clusters main features in WT and Kap1 KO cells. From top: KZFP/rGUs cluster limits, profiles of H3K9me3 and H3K27ac enrichment (full and hollow curves, respectively); KZFP/rGUs, showing the characteristic local high density of elements; KAP1 peaks, correlating with the KZFP/rGUs-dense region; track of TEs. (Bottom, left) an example illustrating the precise distribution in WT and Kap1 KO cells of H3K27ac and H3K9me3 profiles, and their correspondence with to KZFP/rGU-derived transcripts, where thicker lines represent coding sequences and thin lines non-coding sequences, with arrows indicating directionality of the transcripts. KRAB-encoding sequences and KZFP/rGUs are outlined separately, below the annotated transcripts. (Bottom, right) Molecular model of KAP1-mediated regulation of KZFP/rGUs clusters. In WT cells, the complex KAP1-SETDB1, possibly coupled to other HMTs, binds KZFP/rGUs and discrete sets of TEs. KZFP/rGUs accumulate H3K9me3 at their 3’end, but their promoter is devoid of this mark and can be bound by transcription factors and the RNA-polymerase II machinery, generating transcripts (higher panel). Upon Kap1 deletion, SETDB1 is no longer recruited, H3K9me3 levels drop, and H3K27ac becomes enriched over TEs and promoters allowing for a general increase in transcription, albeit still under the differential influence of specific transcription factors (lower panel).

https://doi.org/10.1371/journal.pone.0173746.g006

Cell culture and mouse work

mESCs and MEFs wild-type (WT) and KO for Kap1 were cultured and generated as previously described [57, 59] (strain C57BL/6J). Hepatocyte-specific Kap1 KO mice were generated and genotyped according to [60] (strain C57BL/6J). Murine hepatoma cell line Hepa 1.6 cells were cultured using standard methods.

Plasmids and lentiviral vectors

For KAP1 knockdown experiments, pLKO vector encoding shKAP1 and the empty vector as control were used. For KAP1 overexpression, pSicoR-KAP1-HA vector was used. 48 h after transduction, infected cells were selected with 1 μg mL⁻¹ puromycin in growth medium for an additional 72 h. For de novo Kap1 excision, a non-integrating pHAGE2 Cre-IRES-PuroR lentiviral vector was used. Lentiviral vectors production protocols are available at http://tronolab.epfl.ch and backbones at Addgene (http://www.addgene.org).

RT-PCR and RNA-Seq

Total RNA was extracted and DNase-I treated using a spin column-based RNA purification kit (Macherey-Nagel). cDNA was prepared with SuperScript II reverse transcriptase (Invitrogen). Primers were used for SYBR green qPCR (Applied Biosystems) and the sequences are provided in S2 File. For sequencing of mRNA (poly(A)+), 100-bp single-end RNA-seq libraries were prepared using the Illumina TruSeq Stranded or Unstranded mRNA reagents (Illumina). Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents. Sequencing was performed in 100-bp reads runs by Illumina HiSeq 2500. Further information about the mapping and analysis procedures is provided in S2 File.

ChIP-qPCR and ChIP-Seq

ChIP and library preparation were done according to (Ecco, Cassano et al. 2016), with modifications as described in S2 File. Sequencing was performed in 100-bp reads run on Illumina HiSeq 2500. Primers sequences used for ChIP-qPCR are provided in S2 File.

Bioinformatics analyses and statistics

R version 3.1.2 (http://www.R-project.org) or GraphPad Prism version 6.0 and 7.0 (http://www.graphpad.com) were used for statistical analyses and graphical representations of the data. Detailed bioinformatics analyses are provided in S2 File.

Ethics statement

Experimental protocols were performed according to European Council Guidelines and the Swiss Federal Veterinary Office. Acceptable standards of human animal care and the experimental design of this study were approved by the Ethics Committee for Animal Care of the Vaud Region in Switzerland (licenses 25350 and 22919).

Data access

All next-generation sequencing data have been submitted to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) database under the accession number GEO: GSE87734.