The Krüppel-associated Box Repressor Domain Can Trigger de Novo Promoter Methylation during Mouse Early Embryogenesis

Abstract

The Krüppel-associated box (KRAB) domain is a transcriptional repression module responsible for the DNA binding-dependent gene silencing activity of hundreds of vertebrate zinc finger proteins. We previously exploited KRAB-mediated repression within the context of a tet repressor-KRAB fusion protein and of lentiviral vectors to create a method of external gene control. We demonstrated that with this system transcriptional silencing was fully reversible in cell culture as well as in vivo. Here we reveal that, in sharp contrast, KRAB-mediated repression results in irreversible gene silencing through promoter DNA methylation if it acts during the first few days of mouse development.The human genome encodes several hundreds of KRAB 4 -containing ZFPs, a vertebrate-specific family of DNA-binding factors that likely plays major roles in gene regulatory networks important for ontogenesis, differentiation, and cell growth (1-4). When tethered to DNA via their zinc finger motifs, KRAB-ZFPs recruit the KAP1 (KRAB-associated protein 1) corepressor (also known as TIF1␤, transcription intermediary factor 1␤; KRIP1, KRAB-interacting protein 1; or TRIM28, tripartite motif protein 28) via their KRAB domain. KAP1 is characterized by the presence of a RING finger, B-boxes, a coiledcoil region, a PHD finger, and a bromodomain (5-7). The first three of these motifs define the so-called RBCC or TRIM domain, which is both necessary and sufficient for homo-oligomerization and direct binding to KRAB. Upon recruitment to DNA loci, KAP1 functions as a scaffold for the formation of a multimolecular complex comprising HP1 (heterochromatin protein 1), histone deacetylases, and histone methyltransferases, which induces transcriptional repression through the formation of heterochromatin (8, 9). The primordial importance of this process from the earliest stage of mammalian development is supported by the embryonic lethality of the mouse KAP1 knock-out, which results in failure to gastrulate and death at day E5.5 (10). Considering the obvious importance of KRAB-mediated gene control, it is quite remarkable that only very few gene targets of the KRAB-containing ZFPs have so far been identified (11).We described previously a conditional gene regulation system based on the use of the tetracycline-controllable trans-repressor tTRKRAB, a chimeric protein built by fusing KRAB with the DNA binding domain of the Escherichia coli tetracycline repressor (tetR). Upon binding tetracycline operator (tetO) sequences, tTRKRAB induces transcriptional repression, which can silence RNA polymerase I, II, and III promoters situated within a radius of a few kilobases, thus allowing for doxycycline-regulated gene expression and knockdown (12-15) (Fig. 1A). Using lentiviral vectors as vehicles for this system, we demonstrated that KRAB-induced silencing is fully reversible in vitro and in vivo even after prolonged periods of repression in diverse contexts such as in a variety of established human and murine cell lines, in primary cells, including mouse ES cells, and ...

The Krüppel-associated Box Repressor Domain Can Induce Reversible Heterochromatization of a Mouse Locus in Vivo

“…Dox Treatment and Analysis-Mouse embryonic fibroblasts were isolated as described (24) and where applicable were grown in the presence of 1 g/ml dox (Sigma). Dox was administered to mice in their drinking water, which was also supplemented with 4% glucose at a concentration of either 2 g/liter or 0.2 g/liter.…”

“…Subsequent work revealed that, in these circumstances, the tethering of KRAB to the vicinity of a promoter led to its de novo DNA methylation, and hence to its stable silencing even in the absence of repressor binding (24). Importantly, in these cases KRAB was targeted to the vicinity of a CpG-island containing promoter in the context of a lentiviral vector.…”

Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes

“…Will the disease still develop if a correction of SMN2 splicing is discontinued after the first phases of post-natal life? Mouse models for SMA 28,29 and transgenic mice expressing the tTR-KRAB regulator 30 are available. If these genotypes are combined with the regulatable U7-ESE-B cassette, many of the above questions could be addressed, simply by providing doxycycline through drinking water at different times during ontogenesis.…”

“…Whether and how precisely this will be feasible will depend on the repression and induction kinetics observed in vivo (in our cell culture system B48 h and B120 h were necessary for full induction and repression, respectively) and on whether the silencing will not be irreversible in vivo. 30 Efficient transfer of the therapeutic U7-ESE-B cassette to motoneurons will need to be developed to approach a true somatic gene therapy for SMA. This may well (a and b) HS2 cells transduced with the regulatable U7 and KRAB lentiviral vectors, equilibrated in the repressed state, were induced with 1 mg ml À1 doxycycline.…”

Erase–Maintain–Establish: Natural Reprogramming of the Mammalian Epigenome

“…Initially termed binding factor A, this 1.3-MDa multisubunit complex contains TRIM28 as a repressor subunit (Reik 2007) and the KRAB-ZFP809 that serves as its sequence-specific DNA-binding partner (Wolf and Goff 2009). TRIM28 links the DNA methylation and KRAB-ZFP repressive machineries as shown by tethering a doxycycline-controllable KRAB domain to a lentiviral construct in vivo (Wiznerowicz et al 2007). Here, a green fluorescent protein (GFP) reporter gene was found to be expressed only if recruitment of the repressive KRAB-ZFP was prevented by adding doxycycline in a narrow time window right after fertilization (between E0.5 and E3.5).…”