The interactome of KRAB zinc finger proteins reveals the evolutionary history of their functional diversification
2019; Springer Nature; Volume: 38; Issue: 18 Linguagem: Inglês
10.15252/embj.2018101220
ISSN1460-2075
AutoresPierre‐Yves Helleboid, Moritz Heusel, Julien Duc, Cécile Piot, Christian W. Thorball, Andrea Coluccio, Julien Pontis, Michaël Imbeault, Priscilla Turelli, Ruedi Aebersold, Didier Trono,
Tópico(s)Chromosomal and Genetic Variations
ResumoResource12 August 2019Open Access Source DataTransparent process The interactome of KRAB zinc finger proteins reveals the evolutionary history of their functional diversification Pierre-Yves Helleboid Pierre-Yves Helleboid School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Moritz Heusel Moritz Heusel Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Julien Duc Julien Duc School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Cécile Piot Cécile Piot School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Christian W Thorball Christian W Thorball School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Andrea Coluccio Andrea Coluccio School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Julien Pontis Julien Pontis School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Michaël Imbeault Michaël Imbeault School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Priscilla Turelli Priscilla Turelli School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Ruedi Aebersold Corresponding Author Ruedi Aebersold [email protected] orcid.org/0000-0002-9576-3267 Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland Faculty of Science, University of Zurich, Zurich, Switzerland Search for more papers by this author Didier Trono Corresponding Author Didier Trono [email protected] orcid.org/0000-0002-3383-0401 School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Pierre-Yves Helleboid Pierre-Yves Helleboid School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Moritz Heusel Moritz Heusel Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Julien Duc Julien Duc School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Cécile Piot Cécile Piot School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Christian W Thorball Christian W Thorball School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Andrea Coluccio Andrea Coluccio School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Julien Pontis Julien Pontis School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Michaël Imbeault Michaël Imbeault School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Priscilla Turelli Priscilla Turelli School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Ruedi Aebersold Corresponding Author Ruedi Aebersold [email protected] orcid.org/0000-0002-9576-3267 Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland Faculty of Science, University of Zurich, Zurich, Switzerland Search for more papers by this author Didier Trono Corresponding Author Didier Trono [email protected] orcid.org/0000-0002-3383-0401 School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Search for more papers by this author Author Information Pierre-Yves Helleboid1,‡, Moritz Heusel2,‡, Julien Duc1, Cécile Piot1, Christian W Thorball1, Andrea Coluccio1, Julien Pontis1, Michaël Imbeault1, Priscilla Turelli1, Ruedi Aebersold *,2,3 and Didier Trono *,1 1School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 2Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland 3Faculty of Science, University of Zurich, Zurich, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41 446 333170; E-mail: [email protected] *Corresponding author. Tel: +41 216 931761; E-mail: [email protected] The EMBO Journal (2019)38:e101220https://doi.org/10.15252/embj.2018101220 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) are encoded in the hundreds by the genomes of higher vertebrates, and many act with the heterochromatin-inducing KAP1 as repressors of transposable elements (TEs) during early embryogenesis. Yet, their widespread expression in adult tissues and enrichment at other genetic loci indicate additional roles. Here, we characterized the protein interactome of 101 of the ~350 human KZFPs. Consistent with their targeting of TEs, most KZFPs conserved up to placental mammals essentially recruit KAP1 and associated effectors. In contrast, a subset of more ancient KZFPs rather interacts with factors related to functions such as genome architecture or RNA processing. Nevertheless, KZFPs from coelacanth, our most distant KZFP-encoding relative, bind the cognate KAP1. These results support a hypothetical model whereby KZFPs first emerged as TE-controlling repressors, were continuously renewed by turnover of their hosts' TE loads, and occasionally produced derivatives that escaped this evolutionary flushing by development and exaptation of novel functions. Synopsis In this resource, elucidation of the interactome of 101 Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) offers insights into the evolution of this largest subfamily of human transcription factors and uncovers unexpected functions of its most ancient members. Evolutionarily young KZFPs interact with the co-repressor KAP1 in order to repress transposable elements. Evolutionarily old KZFPs are weak KAP1 binders. More ancient KZFPs bind specific interactors, which include factors involved in chromatin organization and RNA metabolism. An examination of coelacanth KZFPs indicates that these proteins likely first emerged as bona fide repressors of transposable elements. Introduction KZFP genes emerged in the last common ancestor of coelacanth (Latimeria chalumnae), lungfishes, and tetrapods some 413 million years ago (MYA) (Imbeault et al, 2017). Their products harbor an N-terminal KRAB (Krüppel-associated box) domain related to that of Meisetz (a.k.a. PRDM9), a protein that originated prior to the divergence of chordates and echinoderms, and a C-terminal array of zinc fingers (ZNF) with sequence-specific DNA-binding potential (Urrutia, 2003; Birtle & Ponting, 2006; Imbeault et al, 2017). KZFP genes multiplied by gene and segment duplication to count today more than 350 and 700 representatives in the human and mouse genomes, respectively (Urrutia, 2003; Kauzlaric et al, 2017). A majority of human KZFPs including all primate-restricted family members target sequences derived from TEs, that is, DNA transposons, ERVs (endogenous retroviruses), LINEs, SINEs (long and short interspersed nuclear elements, respectively), or SVAs (SINE-variable region-Alu) (Schmitges et al, 2016; Imbeault et al, 2017). However, more ancient family members do not bind recognizable TEs but are rather found at promoters or over gene bodies (Frietze et al, 2010a,b; Imbeault et al, 2017). The KRAB domain was initially characterized as capable of recruiting KAP1 (KRAB-associated protein 1), a tripartite-motif (TRIM) protein that serves as a scaffold for a heterochromatin-inducing complex comprising notably the histone 3 lysine 9 methyltransferase SETDB1, HP1 (heterochromatin protein 1), and the histone deacetylase-containing NuRD (nucleosome remodeling deacetylase) complex (Friedman et al, 1996; Ryan et al, 1999; Schultz et al, 2001, 2002). Accordingly, many KZFPs act in association with KAP1 and associated effectors to repress TEs during the genomic reprogramming that takes place during the earliest stages of embryogenesis (Wolf & Goff, 2009; Matsui et al, 2010; Rowe et al, 2010; Castro-Diaz et al, 2014). However, KAP1 is bound neither by human PRDM9 nor by several other highly conserved KZFPs harboring additional N-terminal domains such as SCAN, which can promote oligomerization, or DUF3669, a region of still elusive function, suggesting that KAP1 binding and repressor activity are recently evolved properties of KZFPs (Okumura et al, 1997; Williams et al, 1999; Schumacher et al, 2000; Birtle & Ponting, 2006; Itokawa et al, 2009; Liu et al, 2014; Patel et al, 2016; Imai et al, 2017). Moreover, KZFP genes display broad and diverse patterns of expression and have been linked to biological events such as genomic imprinting, RNA metabolism, cell differentiation, metabolic control, and meiotic recombination (Wagner et al, 2000; Hayashi & Matsui, 2006; Quenneville et al, 2011; Zeng et al, 2012; Lupo et al, 2013; Ecco et al, 2017; Yang et al, 2017). How these other effects are accomplished is partly unknown, but they suggest that KZFPs associate with a range of cofactors extending well beyond the sole inducers of transcriptional repression. Undertaken to explore this complexity, the present study reveals the breadth and evolutionary history of the functional diversification of KZFPs. Results We selected 101 human KZFPs over a range of evolutionary ages, domain compositions, and genomic targets so as to constitute a sample representative of the whole family. Using 293T cell lines overexpressing HA-tagged versions of these proteins (Imbeault et al, 2017), we first determined their subcellular localization by indirect immunofluorescence (IF) microscopy (Table EV1). A majority of these KZFPs were almost exclusively nuclear, as illustrated for ZNF93, but some displayed unusual sub-nuclear or predominantly cytoplasmic localizations, such as the nucleolus-enriched ZNF79 or the endoplasmic reticulum (ER)-associated ZNF546 (Fig 1A). We then set out to define the protein interactome of these KZFPs by affinity purification and mass spectrometry (AP-MS) essentially as previously described (Varjosalo et al, 2013) (Fig EV1A) with modifications aimed at optimizing the workflow for the analysis of DNA-associated proteins with high sensitivity. To narrow down identified proteins into a high-quality list of interactors, we used the significance analysis of interactome (Choi et al, 2011), (SAINT)-calculated false discovery rate (FDR), and spectral counts fold enrichment as well as a subcellular localization filter using our IF data for the baits and the Human Protein Atlas annotations for their preys (Thul et al, 2017). The resulting human KZFP interactome formed a high-density connectivity map of 887 high-confidence associations between the 101 baits and 219 preys displaying similar subcellular localization patterns. Confirming that our list of KZFP-binding proteins was not contaminated by loci-specific DNA-binding contaminants, KZFPs with partly overlapping genomic targets (Imbeault et al, 2017) did not share more partners than random pairs of these proteins (Fig EV1B). In order to confirm the validity of our dataset, we compared our results with the BioGRID protein–protein interaction dataset (Stark, 2005) (Table EV2). We found that 88 (about 8%) of the interactions documented in our system had been previously detected through either AP-MS or other approaches such as two-hybrid screens (Fig EV1C). Figure 1. KZFP IFs and interactome A. IF by confocal microscopy performed on KRAB-HA-overexpressing 293T cells. Staining was performed with anti-HA (Alexa-488, green) and anti-HSPA5 (Alexa-647, red) antibodies, and DNA was stained with DAPI (blue). The scale bar represents 20 μm. B. Force-directed network representing high-confidence interactions. Each bait KZFP is represented by a blue circle ("node") and linked to green nodes that represent its preys. KAP1 is represented in red and its associated proteins SIRT1, SMARCAD1, and HP1α and HP1γ in orange. All interactions represented are below the false discovery rate of 1%. The topology of the network is established by a force-directed process that follows certain rules: All nodes repel each other and are attracted to the center by artificial "gravity", and nodes with links attract each other. Weighted links between any nodes are based on the average fold change over controls. C. Zoom-in on the KAP1-centered core of the interactome. D, E. Zoom-in on KZFPs enriched with unique interactors. F. Zoom-in on the subset of KZFPs not connected to the main interactome. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. General AP-MS protocol and quality controls Graphical representation of AP-MS protocol major steps and filters used for the computational analysis. Boxplot presenting the number of shared interactors for KZFP couples sharing more than 10% of their binding sites in a reciprocal fashion compared to the number of shared interactors for the same amount of random KZFP couples. Boxplots are shown as median, and 25th (Q1) and 75th (Q3) percentiles. The upper whisker extends to the last data point less than Q3 + 1.5*IQR, where IQR = Q3–Q1. Similarly, the lower whisker extends to the first data point greater than Q1 – 1.5*IQR. Number of interactions observed in previous studies (Table EV2) associated with their corresponding KZFP. The method used for the interaction detection is indicated by a specific color. Download figure Download PowerPoint We used our AP-MS results to build a KZFP interactome global network (Fig 1B and Appendix Fig S1). It was centered on KAP1 (Fig 1C) and displayed KZFPs associating with specific preys at its periphery (Fig 1D and E), as well as three KZFPs with no detected interactors (Fig 1F). Its core encompassed the majority of the KZFP baits and their most frequent interactor, KAP1, which was frequently detected together with HP1α and HP1γ, the deacetylase SIRT1, and the ATP-dependent helicase SMARCAD1, all previously identified as KAP1 interactors (Ryan et al, 1999; Rowbotham et al, 2011; Lin et al, 2015) (Fig 1C). SIRT1 associated with the KAP1-interacting ZNF138 and ZNF793 but not with ZKSCAN3, a weak recruiter of the corepressor (Fig EV2A), consistent with such KAP1-mediated interaction. Additional proteins involved in post-translational modifications, such as phosphorylation and ubiquitylation, were repeatedly part of KAP1-comprising KZFP interactomes (Fig EV2B). Our analyses also identified mediators of nuclear import in association with more than half (56) of nuclear KZFPs, whether KAP1 was (karyopherin β, the karyopherins α KPNB1 and KPNA2) or not (karyopherin β-like importins IPO7 and IPO8) systematically present (Fig EV2B and C). Click here to expand this figure. Figure EV2. KZFP common interactors SIRT1 interaction validation. HA-tagged KZFPs transduced cell lines were used for immunoprecipitations. HA immunoprecipitation of weak KAP1 interactor ZKSCAN3 and candidates ZNF138 and ZNF793, followed by the detection of endogenous SIRT1 in the immunoprecipitates through Western blot using an anti-SIRT1 antibody. Input = cellular lysate, IP = immunoprecipitate. Western blot using an anti-HA antibody on the IPs at the bottom. Venn diagram representing all the common preys that were detected in 5 and more KZFP interactomes. In pink are shown the common preys that only appear in interactomes alongside KAP1, in blue the prey that only appears in interactomes devoid of KAP1, and in purple the preys that are in both type of interactomes. IPO7 interaction validation. HA-tagged KZFPs transduced cell lines were used for immunoprecipitations. HA immunoprecipitation of predominantly cytoplasmic ZNF283 and candidates ZNF577 and ZNF765, followed by the detection of endogenous IPO7 through Western blot using an anti-IPO7 antibody. Input = cellular lysate, IP = immunoprecipitate. Western blot using an anti-HA antibody on the IPs at the bottom. Source data are available online for this figure. Download figure Download PowerPoint In line with the known dimerization potential of SCAN (Williams et al, 1999), another sub-network emerged that was based on associations between proteins harboring this domain (Fig 2A). Nine out of 17 SCAN-KZFPs were found in such complexes, which for six of them also included the non-KRAB SCAN-containing ZFP ZNF24, thought to play a role in transcription and DNA replication (Jia et al, 2013; Lopez-Contreras et al, 2013). These results concur with some of the putative SCAN-mediated interactions noted in previous studies (Huttlin et al, 2015; Schmitges et al, 2016). Figure 2. SCAN and DUF3669 domains are involved in oligomerization Force-directed network representing the SCAN interactome displaying SCAN-containing baits and their SCAN-containing preys. HA immunoprecipitation of stably expressed ZNF282-HA in cells previously transfected with ΔDUF3669-ZNF398-GFP and WT-ZNF398-GFP followed by the detection of ZNF398 constructs in the IPs through Western blot using an anti-GFP antibody. Bottom: Western blot using an anti-HA antibody on the IPs. Input = cellular lysate, IP = immunoprecipitate. Left: DUF3669-only and KRAB domain constructs used. Right: HA immunoprecipitation in cells previously co-transfected with ZNF398-DUF3669-HA and either ZNF282-KRAB-Flag or ZNF282-DUF3669-Flag followed by the detection of either of these protein constructs in the IPs through Western blot using an anti-Flag antibody. Bottom: Western blot using an anti-HA antibody on the IPs. Source data are available online for this figure. Source Data for Figure 2 [embj2018101220-sup-0007-SDataFig2.pdf] Download figure Download PowerPoint The capture of ZNF282 by ZNF398 attracted our attention, as both of these KZFPs contain a DUF3669 domain, and interactions between DUF3669-containing KZFPs were previously recorded (Gao et al, 2008; Kang & Shin, 2015; Huttlin et al, 2017) (Appendix Fig S2). Confirming the ability of DUF3669 to mediate protein–protein interactions, we could co-immunoprecipitate HA-tagged ZNF282 with full length but not DUF3669-deleted version of GFP-tagged ZNF398 (Fig 2B), and we could pull-down the ZNF398 DUF3669 domain with the corresponding fragment of ZNF282, but not with its KRAB counterpart (Fig 2C). We thus conclude that the DUF3669 domain can trigger associations between KZFPs, although in vitro experiments with purified proteins will be needed to confirm that it is through a direct interaction. Outside of the KAP1-centered network core, we delineated other interactomes displaying uncommon associations (Fig 1D and E). First, we focused on preys captured by only two to four KZFPs (Fig 3A). Those found together with KAP1 related to nucleosome formation and modification or involved other TRIM family proteins or cytoplasmic chaperones sharing subcellular localizations with their baits as for ZNF546, ZNF304, and ZNF283 (Fig 1A, Appendix Fig S3A). Several subunits of translation-promoting eukaryotic initiation factor 3 (eIF3) were also part of the interactomes of the weak KAP1 recruiters ZNF79, ZKSCAN4, and ZNF746. Figure 3. A sub-population of KZFPs displays rare and unique interactors Venn diagram representing all preys detected in less than 5 and more than 1 KZFP interactomes. In blue, are shown the preys that only appear in interactomes alongside KAP1, in red the preys that only appear in interactomes devoid of KAP1, and in purple the preys that are in both types of interactomes. This table displays all the KZFPs associating with 3 or more unique interactors, the identity of these interactors as well as the features associated with them. Venn diagram representing the binding sites overlaps between ZNF764, TFIIIC subunit GTF3C2, and CTCF. The GTF3C2 bedfile was obtained from Encode (ENCFF002CYL), and CTCF bedfile was obtained from Encode version 3. The overlap and resulting P-values were obtained using the Bedtools Fisher exact test. Histograms representing the percentage of KZFPs (identified in Fig 3B) binding sites falling in TEs (red) or TSSs (blue). KZFPs binding sites on L1PA5 and L1PA6. Top: Boxes containing the interactors of KZFPs found enriched on L1PA5 and L1PA6. Middle: plot showing the average ChIP-exo signals (scaled between 0 and 1) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment (MSA). Bottom: schematic representation of L1PA5 and L1PA6 different domains. Download figure Download PowerPoint We then focused on the 16 KZFPs interacting with three or more unique preys, i.e., preys that were detected in association with only one KZFP, reasoning that their interactors would provide more detailed hints on their biological roles (Fig 3B). Of note, 15 interactions between a KZFP and a unique interactor were already observed in published datasets (Table EV2). Several of these unique preys pointed to RNA-related functions such as unique partners detected with the eIF3-associated ZKSCAN4 and ZNF746 or the RNA-processing paraspeckle-forming proteins PSPC1, NONO, and SFPQ for ZNF213, corroborated by their similar sub-nuclear localization patterns in these structures (Hata et al, 2008) (Appendix Fig S3B). Furthermore, factors involved in cell-cycle regulation (cyclin and associated kinases) bound ZNF20, and components of the chromosome-organizing SMC complexes associated with ZNF597 and ZNF3. Finally, TFIIIC subunits were part of the interactome of ZNF764, previously found to cooperate with CTCF in establishing genomic boundaries (Moqtaderi et al, 2010). Correspondingly, chromatin immunoprecipitation/deep sequencing (ChIP-seq) studies found the three proteins co-localized significantly on the genome (Fig 3C). We further documented the genomic recruitment of the 16 KZFPs interacting with 3 or more unique preys, completing a previously established dataset (Imbeault et al, 2017) with additional ChIP-seq studies (Table EV3). A majority was found at TEs or gene transcription start sites (TSS) (Fig 3D), but some were rather associated with other entities such as imprinting control regions (ICRs) for ZNF445 (Takahashi et al, 2019) and CTCF binding sites for ZNF764. In addition, few or no ChIP-seq peaks were detected for ZNF446, ZNF546, ZNF213, and ZNF597, indicating different modalities or absence of DNA binding. Except for the MER51A/E-binding ZNF20, TE-binding KZFPs displaying unique interactomes were enriched over LINEs, which often recruited several of them (Imbeault et al, 2017) (Appendix Fig S4). For instance, ZNF93, ZNF765, and ZNF248 bound to L1PA6 and L1PA5 LINE1 integrants, for the first two over their 5′ untranslated and for the third over their ORF2-coding regions (Fig 3E). The presence of escape mutations in the LINE1 lineage (Jacobs et al, 2014; Imbeault et al, 2017) indicates that ZNF93, ZNF765, and ZNF248 initially acted as bona fide inhibitors of transposition. Yet, their persistent association with L1PA5/PA6 integrants suggests that these KZFPs, while perhaps still repressing transcription, do so no longer to block retrotransposition, since their target retroelements have long lost all spreading potential. ZNF765 is recruited over the L1PA5/PA6 promoter region, and two of its interactors stand out as potential regulators of LINE1 transcripts: the RNA-binding proteins interactor ZKSCAN4 and ZC3H18, known to be involved in RNA export, anabolism, and catabolism (Chi et al, 2014; Winczura et al, 2018). Furthermore, L1PA6 and L1PA5 integrants also recruit over their 3′ half two KZFPs with conventional KAP1-centered interactomes, ZNF382 and ZNF84, for which there is no sign of mutational escape, strongly suggesting that these KZFPs, in spite of their repressor potential, never limited the spread of these retrotransposons. We then turned our attention toward KAP1, the most common partner of KZFPs. Interestingly, our network reflected a range of affinities between individual KZFPs and the corepressor (Fig 4A), translating in differential KAP1 recruitment strengths (KAP1FC). With an arbitrary cut-off at three, this parameter derived from spectral counts enrichment delineated two groups of KZFPs, which we qualified as strong and weak KAP1 binders (Figs 4A and EV3A and B). Upon examining which KZFP features correlated with KAP1 recruitment, we first noticed that family members with < 40% of ChIP-seq peaks on TEs interacted on average less strongly with KAP1 (Fig 4B). The KRAB domain typically comprises an obligatory A-box bearing the residues necessary for KAP1 recruitment and a facultative B-box (Urrutia, 2003). B-box displaying KZFPs predominantly yielded high KAP1FC values, whereas their B-box-less counterparts were split among strong and weak KAP1 binders (Fig EV3C), confirming the enhancing but non-essential role of this subdomain (Vissing et al, 1995). We built a phylogenetic tree based on the KRAB-A-boxes of 346 protein-coding human KZFPs (Fig 4C). A majority clustered as a homogeneous group harboring very few amino acid differences, and we termed these standard-KRAB KZFPs (sKZFPs). A heterogeneous set of variant KRAB (vK) emerged that displayed a very significant degree of divergence from the KRAB A-box consensus sequence. We identified such 35 vKZFPs in the human proteome (in addition to two KZFPs, ZNF333 and ZFP28, harboring both a standard and variant KRAB). Only one out of the 18 vKZFPs subjected here to AP-MS had a KAP1FC value above three, whereas the opposite was observed for 81 out of 83 tested sKZFPs (Fig 4D). By assessing their evolutionary ages (Imbeault et al, 2017), we determined that among tested KZFPs, those harboring a vK domain segregated in oldest age bins, conserved from placental mammals (105 MY) up to sauropsids (320 MY), whereas all but two of their sK-containing counterparts were 105 MY Old (MYO) or younger (Figs 4D and EV3D). We then confirmed that all sKZFPs, including the ones not tested in our study, presented significantly higher evolutionary ages (Fig EV3E). Older age also correlated with unusual interactomes: 13 out of 16 KZFPs interacting with more than three unique interactors were 105 MYO or older, and KZFPs displaying no interactors were all older than 105 MY (Figs 4E and 1F). Moreover, we detected less than four interactors (whereas the average number for the 101 KZFPs was 8.8) for more than half of these ancient KZFPs. For some, this might have been due to lack of expression of their functional partners in 293T cells, although these KZFPs themselves did not display abnormally low levels in these cells (Appendix Fig S5A). In contrast, more than half of the KZFPs associated with more than three unique interactors segregated in a small cluster characterized by a higher expression in testis and blood according to the GTEx database (The GTEx consortium, 2013) (Appendix Fig S5B). In sum, the most conserved KZFPs were weak KAP1 binders and displayed functionally diversified or partner-depleted interactomes indicative of non-canonical roles. Figure 4. Old KZFPs display an unusual and conserved KRAB domain devoid of KAP1 binding KAP1 force-directed network. The distance KZFP-KAP1 is inversely proportional to the measured KAP1 enrichment over controls (KAP1FC). KZFPs with KAP1FC values above three were colored in blue and the ones below in green. Boxplot representing the KAP1FC value of our baits in function of the percentage of their binding sites falling in TEs. Mann–Whitney two-sided rank test. Human KZFPs KRAB domain A-boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal Zappo color scheme (residues sharing common physicochemical properties display the same color: http://www.jal-view.org/help/html/colourSchemes/zappo.html). This figure also displays a zoom-in on the variant KRAB domain cluster (right). On this zoom-in, the tested vKZFPs, whose interactomes were defined in our study, were marked by an asterisk. Box plot representing KAP1FC values in function of KZFPs age. On a superimposed swarm plot, the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts, by blue dots. Mann–Whitney two-sided rank test. Boxplot representing the number of interactors of KZFPs in function of their evolutionary age. On a superimposed swarm plot, individual KZFPs number of preys were represented by dots. When green, the interactome of this KZFP contained 3 or more unique interactors (Fig 3B). Boxplot representing the average dn/ds ratios for all vK (red) and sK (blue) domains displaying the same evolutionary ages. Mann–Whitney two-sided rank test. Data information: Boxplots are shown as median, and 25th (Q1) and 75th (Q3) percentiles. The upper whisker extends to the last data point less than Q3 + 1.5*IQR, where IQR = Q3–Q1. Similarly, the lower whisker extends to the first data point greater than Q1 – 1.5*IQR. Download figure Download PowerPoint Click
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