Introduction The proper control of gene expression is paramount to all cellular events, and is orchestrated through a sophisticated balance of activating and repressing influences. Krüppel-associated box domain zinc finger proteins (KRAB-ZFP) constitute the single largest group of transcriptional repressors encoded by the genomes of higher organisms. After appearing in early tetrapods, the KRAB-ZFP family has rapidly expanded and diverged through multiple rounds of gene and segmental duplications, to give rise to more than three hundred and fifty members annotated in both mice and humans [1]–[6]. In spite of their numerical abundance, wide range of tissue-specific expression and dynamic evolutionary history, the physiological functions of KRAB-ZFPs collectively remain ill-defined, and few of their targets have been identified [3],[7]. However, emerging evidence links KRAB/KAP1-mediated regulation to processes as essential and diverse as stem cell pluripotency, early embryonic development and differentiation, genomic imprinting, response to DNA damage and control of behavioral stress [8]–[14]. Furthermore, KAP1 controls endogenous retroviruses in embryonic stem cells, a process crucial for the maintenance of genomic stability [15]. KRAB-ZFPs all harbor a so-called KRAB domain situated upstream of an array of two to forty C2H2 zinc fingers, which provide sequence-specific DNA binding ability [2]. KRAB recruits KAP1 (KRAB-associated protein 1, also known as TRIM28, Tif1β or KRIP-1) [16]–[19], which acts as a scaffold for various heterochromatin-inducing factors, such as heterochromatin protein 1 (HP1), the histone methyltransferase SETDB1, the nucleosome-remodeling and histone deacetylation (NuRD) complex, the nuclear receptor corepressor complex 1 (N-CoR1) and, at least during early embryonic development, de novo DNA methyltransferases [20]–[27]. The phylogenetically conserved family of HP1 proteins is implicated in a variety of nuclear events, such as transcriptional repression and maintenance of chromosome structure [28]. HP1 harbors two major regions: the chromo domain, which binds to repressive di- and trimethylated histone 3 lysine 9 (H3K9me2 and H3K9me3, respectively) residues, and the chromo shadow domain, involved in HP1 homodimerization and recruitment of other partners [29]–[31]. Therefore, HP1 bridges histones with other chromatin-associated proteins, that promote heterochromatin spreading [32]. An advancing front of heterochromatization can thus be propagated by the creation of HP1 binding sites, through histone methyltransferase-mediated H3K9 methylation, followed by the HP1-mediated recruitment of more histone methyltransferase for another round of H3K9 methylation/HP1 binding [33]–[36]. Little is known about the kinetics, efficiency, self-perpetuating ability and action-range of this process. Tethering of a regulated KRAB repressor domain suggested that KRAB/KAP1 induced heterochromatin formation has a rather limited spreading potential in euchromatin, as silencing could be exerted no farther than 2–3 kilobases (kb) away from the repressor binding site [37]. Yet a more recent Chromatin IP (ChIP)-on-chip analysis performed in the human testicular carcinoma cell line Ntera2 revealed close to 7,000 KAP1 binding sites, a number of which were located at the 3′end of KRAB-ZFP genes [38]. While this suggests auto-regulatory negative feedback loops for these genes, such a process would imply that KRAB/KAP1 binding can affect promoters situated at very significant distances. In agreement with such a model, large heterochromatin domains associated with both HP1β and SUV39h1 were found on chromosome 19, where most of the KRAB-ZFP gene clusters reside [39]. To examine the genomic features of KRAB/KAP1-mediated transcriptional regulation, we developed an ectopic repressor assay. In our system, promoterless lentiviral vectors serve as gene traps to drive reporter expression from cellular promoters. Drug-controllable docking of an ectopic KRAB-based repressor then allows an assessment of the effects of KRAB/KAP1 recruitment at these loci. Using this system, we found that KRAB-induced silencing can repress promoters situated several tens of kilobases away from the repressor primary docking site through reduced RNA Pol II binding. Furthermore, we observed that this phenomenon is independent of promoter strength, facilitated by HP1 and associated with spreading of heterochromatin marks between repressor binding site and targeted promoters. Finally, we could document KAP1-mediated transcriptional repression at an endogenous KRAB-ZFP gene cluster by propagation of HP1β and H3K9me3 from the 3′ end of these genes to their transcriptional start site. Our results indicate that KRAB/KAP1 induce long-range repression through the spread of heterochromatin. Results KRAB–mediated silencing can act over several tens of kilobases In order to study the features of KRAB/KAP1-induced silencing, we exposed lentivirally trapped cellular promoters to a drug-regulated KRAB-containing repressor. The tTRKRAB protein contains the KRAB domain of the human KOX1 ZFP fused to the E. coli tetracycline repressor (tTR), and binds to Tet operator sequences (TetO) in a doxycycline (dox)-controllable fashion [40],[41]. We engineered human immunodeficiency virus (HIV)-derived lentiviral (LV) gene trap vectors carrying tandem TetO repeats and a promoter-less puromycin resistance-GFP fusion reporter (puroR-GFP) downstream of a potent adenoviral splice acceptor. This design predicted that i) reporter expression would occur from the promoters of active genes targeted by the integrants, and ii) dox withdrawal would result in tTRKRAB binding to the TetO sites present in the proviruses, thus exposing the trapped promoters to potential KRAB/KAP1-mediated repression (Figure 1A). 10.1371/journal.pgen.1000869.g001 Figure 1 KRAB–mediated silencing can act over several tens of kilobases. (A) Mechanism of how endogenous genes are targeted by tTRKRAB using the lentiviral vector-based “Trapping/Silencing” (TrapSil) system: TetO-containing gene traps carrying the promoterless puroR-GFP gene only express this reporter if after proviral integration they “trap” an actively transcribing gene. The TetO sites further allow binding of the ectopic repressor tTRKRAB to the gene traps after dox removal, while the trap reporter serves as a direct read-out for the effects of tTRKRAB–mediated “silencing”. (B) tTRKRAB–expressing HeLa cells were transduced with LV TrapSil vectors and 23 clones expressing the trap reporter were isolated. Mean fluorescence intensity (MFI) GFP values of these individual clones, cultured with and without dox, were determined and the ratios of these values were used to calculate the silencing efficiency (% silencing = 1- ((MFI GFP –dox)/(MFI GFP +dox))) depicted below the x-axis for each clone. (C) GFP-mediated cell sorting was used to isolate populations of HeLa cells exhibiting either a “repressible” (>90% silencing) or an “irrepressible” ( 90% silencing) are depicted as black triangles above the baseline, whereas the irrepressible counterparts (<10% silencing) are depicted as light grey triangles below the baseline. (0.27 MB TIF) Click here for additional data file. Figure S4 HP1β and H3K9me3 spread from the KRAB-binding site to the promoter. ChIP analyses quantifying the relative enrichment (% of input) of both HP1β and H3K9me3 were performed for the repressible clone IX in the presence and absence of tTRKRAB binding. The interrogated sequence at the ephrin receptor B4 (EPHB4) locus spanned from the proviral tTRKRAB binding sites (light grey circles) to the trapped promoter. qPCR amplicons are depicted as letters and are not drawn to scale. All values are expressed as means +SEM of triplicate experiments. Fold changes were calculated as ratios of -dox/+dox enrichments, with the ratios of the respective positive controls set as 1. The controls consisted of p53BP2 for H3K9me3 and of ZNF556 for HP1β (Table S4). (0.12 MB TIF) Click here for additional data file. Figure S5 HP1β and H3K9me3 spread along the 50 kb-spanning ZNF77/57 locus upon tTRKRAB binding. ChIP analyses quantifying the relative enrichment (% of input) of both HP1β and H3K9me3 were performed for the repressible clone XI in the presence and absence of dox. The ZNF77-1 promoter drives the expression of the integrated TrapSil provirus (depicted as light grey circles). The interrogated sequence spanned the whole ZNF77/57 locus and the respective qPCR amplicons are depicted as letters and are not drawn to scale. All values are expressed as means +SEM of triplicate experiments. Fold changes were calculated as ratios of -dox/+dox enrichments, with the ratios of the respective positive controls set as 1. The controls consisted of p53BP2 for H3K9me3 and of ZNF556 for HP1β (Table S4). (0.30 MB TIF) Click here for additional data file. Figure S6 ChIP analyses of different TrapSil clones at control loci. (A–D) We ensured that there were similar amounts of ChIP material in the +dox compared to the -dox samples for each TrapSil clone by analyzing HP1β or H3K9me3 relative enrichment levels at control loci. These control loci are: p53BP2, ZNF554, ZNF555, ZNF556, ZNF77-1, and ZNF77-2 and were probed in the TrapSil clones (A) XVI, (B) I, (C) XI, and (D) IX. (0.37 MB TIF) Click here for additional data file. Figure S7 Quantification of the KAP1 knockdown efficiency in a stable HeLa cell line. HeLa cells were stably transduced with lentiviruses expressing shRNA targeting either KAP1 or GFP. The levels of knockdown were quantified by using (A) qPCR measurements normalized to EEF1α and (B) western blot analyses for KAP1 levels with PCNA as a loading control. The qPCR values are expressed as means +SEM of triplicate experiments. (0.12 MB TIF) Click here for additional data file. Figure S8 ChIP analyses of knockdown HeLa cell lines at control loci. We ensured that there were similar amounts of ChIP material in the shKAP1, compared to the shGFP cell lines, by analyzing HP1β or H3K9me3 relative enrichment levels at the control gene p53BP2 and at the human satellite 2 repeats (Sat2). (0.08 MB TIF) Click here for additional data file. Table S1 Characterization and integration site mapping of LV-based TrapSil HeLa and MEF clones. (0.08 MB DOC) Click here for additional data file. Table S2 Integration site mapping of the repressible and the irrepressible LV TrapSil populations. After proviral integration site mapping, the position of TrapSil integrants was determined relative to the trapped promoter for both the repressible and the irrepressible populations. More specifically, we computed two gene lists (LV repressible and LV irrepressible), which included all the trapped promoters with a single annotated transcriptional start site (TSS), which were further used to build Figure 1C. Each of these gene lists are represented by a table containing gene name (column 1), gene length (column 2), and integrant coordinate relative to the TSS of the gene (column 3). The gene name column can contain more than one identifier due to UCSC known gene annotation, which merges different gene annotations. (0.06 MB XLS) Click here for additional data file. Table S3 List of primers used in this study. (0.05 MB XLS) Click here for additional data file. Table S4 Fold change values calculated for different TrapSil clones by comparing ChIP enrichments in the presence or absence of tTRKRAB binding. (0.04 MB XLS) Click here for additional data file.
