D4Z4 as a prototype of CTCF and lamins-dependent insulator in human cells

Abstract

Using cellular models that mimic the organizations of the subtelomeric 4q35 locus found in patients affected with Facio-Scapulo-Humeral Dystrophy (FSHD) and in healthy individuals, we recently investigated the biological function of the D4Z4 macrosatellite in this subtelomeric context.We demonstrated that D4Z4 acts as a CTCF and A-type lamins dependent insulator element exhibiting both enhancer- blocking and barrier activities, and displaces a telomere towards the nuclear periphery. This peripheral positioning activity lies within a short sequence that interacts with CTCF and A-type lamins. Depletion in either of these two proteins suppresses these perinuclear activities, revealing the existence of a subtelomeric sequence that is sufficient to position an adjacent telomere to the nuclear periphery. We discuss here the biological implications of these results in the light of our current knowledge in related fields and the potential implication of other CTCF and A-type lamins insulators in the light of human pathologies.

Keywords: A-type lamins; CTCF; D4Z4; FSHD; insulator; nuclear organization; position effect variegation; subtelomere; telomere.

Figure 1

(A) Schematic representation of the 4q35 locus involved in FSHD. The D4Z4 elements are indicated (black boxes) together with different genes present in the region (grey arrows). The 10q26 telomeric locus is 98% homolog to 4q35 in its distal part but at this locus the number of D4Z4 is variable and not associated with any pathology. (B) Schematic representation of the experimental system used to investigate the positioning of D4Z4-tagged telomeres. The constructs are derived from the pCMV vector, which carries a Hygromycin resistance gene (HyTK) and an eGFP reporter. The presence of a telomere seed (arrows) allows telomeric fragmentation, a mechanism based on the non-targeted introduction of cloned telomeres into mammalian cells. The construct can induces a double strand break and the loss of the terminal chromosomal fragment followed by the elongation of a de novo telomere from the telomere seed in telomerase positive cells. Successful de novo formation of eGFP-tagged telomeres (in 90% of the hygromycin-resistant cells) or internal integration of the CMV construct were confirmed by fluorescence in situ hybridization (FISH) on metaphase spreads. The expression of eGFP is followed by flow cytometry and cells are processed according to the 3D-FISH procedure.

Figure 2

(A) In silico comparison of the D4Z4 sequence with known CTCF sites revealed the presence of two putative sites for CTCF. The first one at the 5′ end of the repeat was described in. The sequence of the second site, located in the 3′ end of D4Z4 (position 2828–2839, CCGCCTCCGCGCGG) is shown in (A). To determine whether this candidate CTCF binding sequence is capable of binding to CTCF, Electro Moblity Shift assays (EMSA) were carried out as previously described., Incubation of decreasing amounts of C33A nuclear extracts with labeled D4Z4 oligonucleotides (lanes 1, 2) led to the formation of a DNA-protein complex. In order to compare this site to other known CTCF sites, we used unlabeled oligonucleotides corresponding to the chicken β globin FII 5′HS4 site or the TAD1 site at the mouse TCRα-Dad1 locus for competition assays (lanes 3 and 4). Molar excess of unlabeled FII or TAD1 displaces the binding of CTCF from the labeled D4Z4 sequence suggesting that the site at the 3′ end of D4Z4 binds CTCF in vitro. (B) Schematic representation of the D4Z4 element from position 1 to 3303 relative to the two flanking KpnI sites (K) (to scale). Fragments obtained after digestion of D4Z4 were cloned between the eGFP reporter and the telomeric seed. After transfection, cells were processed for 3D-FISH analysis in order to evaluate the positioning of the corresponding de novo formed telomere within the nuclear space. The histogram displays the mean positioning ± S.D shown by error bars of natural and fragmented telomeres within the nuclear volume, calculated from the positioning of the FISH signal from the center (0%) to the outer edge of the sphere after reduction of the outer signal (V_L = nuclear volume = 100%) until it overlaps with the FISH signal (V_l = x% of V_L). The 3D FISH analysis revealed that the 3′ end of D4Z4 might also be involved in the repositioning of a telomere toward the periphery of the nucleus since the T1XDFse construct that does not contain the proximal CTCF site also mediates peripheral positioning (B, BamHI; Bl, BlpI; E, EheI; F, FseI; K, KpnI). (C) Summary of the different regions of D4Z4 harboring anti-silencing or positioning activity.

Figure 3

Model for the organization of the 4q35 locus in healthy individuals and FSHD patients. Details are given in the text.