A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing

Meylan et al. BMC Genomics 2011, 12:378 http://www.biomedcentral.com/1471-2164/12/378 RESEARCH ARTICLE Open Access A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1mediated silencing Sylvain Meylan1,2†, Anna C Groner1,2†, Giovanna Ambrosini1,3, Nirav Malani4, Simon Quenneville1,2, Nadine Zangger1,2, Adamandia Kapopoulou1,2, Annamaria Kauzlaric1,2, Jacques Rougemont1, Angela Ciuffi5, Frederic D Bushman4, Philipp Bucher1,3 and Didier Trono1,2* Abstract Background: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences. Method: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression. Results: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes. Conclusion: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression. Keywords: KAP1, KRAB-zinc finger proteins, transcriptional repression, chromatin, heterochromatin, histone modifications Background Gene expression is modulated through the alteration of chromatin states by epigenetic regulators. Krüppel-associated box zinc finger proteins (KRAB-ZFPs), which together constitute the single largest group of transcriptional repressors encoded by the human genome, * Correspondence: † Contributed equally 1 School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Full list of author information is available at the end of the article partake in this process [1-3]. The KRAB-ZFP family is evolutionary recent and has expanded and diverged through multiple rounds of gene and segment duplications, to give rise to more than three hundred and fifty annotated members in humans [4-7]. Despite their abundance, KRAB-ZFPs and their transcriptional targets remain largely uncharacterized except for a few [8-10]. KRAB-ZFPs carry a C-terminal array of two to forty C2H2 zinc finger motifs, each potentially capable of recognizing a triplet of nucleotides in a sequence-specific manner [1], while their N-terminal KRAB domain © 2011 Meylan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Meylan et al. BMC Genomics 2011, 12:378 http://www.biomedcentral.com/1471-2164/12/378 recruits the KAP1 (KRAB associated protein 1) corepressor [11-14]. KAP1 (also named TIF1b, KRIP-1 or TRIM28) binds KRAB and homotrimerizes through its N-terminal RBCC (Ring finger/B box/Coiled-Coil) domain, while its C-terminus acts as a scaffold for various heterochromatin-inducing factors, such as heterochromatin protein 1 (HP1), the histone methyltransferase ESET (also known as 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 [15-22]. This results in local loss of histone acetylation, enrichment in histone 3 lysine 9 trimethylation (H3K9me3) and increased chromatin compaction [23,24]. Using chromatin immunoprecipitation (ChIP) and a tiling array, KAP1 has been documented to bind more than 7000 sites in a human testicular embryonal carcinoma cell line [25]. A more recent publication additionally revealed that KAP1 chromatin targeting falls into different categories, only a subset of which is dependent on its RBCC domain and consequently on its association with KRAB-ZFPs [26]. KAP1 is dynamically associated with both heterochromatin and euchromatin. It is thought to organize constitutive heterochromatin and to stimulate its propagation, as evidenced by its co-localization with HP1 in pericentromeric heterochromatin domains [16,27]. Using a combination of gene trapping and a drug-controllable KRAB-containing repressor, we recently demonstrated that KRAB/KAP1 can induce long-range repression through HP1-dependent heterochromatin spreading [28]. However, while some promoters located tens of kilobases (kb) from KAP1 docking sites were silenced by this mechanism, others were resistant. Here, we investigated the basis for this differential behavior by comparing the genomic context and the pre-existing levels of specific chromatin marks at repressed and non-repressed genes. This analysis revealed that genes most susceptible to KRAB/KAP1induced silencing were in genomic regions of high gene activity. More specifically, repression was most efficient at sites with increased levels of pre-existing repressive histone marks at promoters and gene bodies, embedded within gene-rich regions with high levels of transcription. Results Characterization of thousands of KRAB/KAP1-targeted gene traps To study the impact of specific genomic features on KRAB/KAP1-induced silencing, we used the recently described trapping/silencing (TrapSil) system [28]. Here, retrovirally-trapped cellular promoters are exposed to a Page 2 of 14 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)-controlled manner [29,30] (Figure 1A). We engineered retroviral-based gene trap vectors carrying tandem TetO repeats and a promoterless GFP-puromycin resistance fusion reporter. This design predicts that i) reporter expression occurs from the promoters of active genes targeted by the integrants ("trapping”), and ii) Dox withdrawal results in tTRKRAB binding to the TetO sites present in the provirus, thus exposing the trapped promoters to KRAB/KAP1mediated silencing ("silencing”) (Figure 1A) (...truncated)