CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator

Abstract

Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking-Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF, with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. Here, we characterize an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, we demonstrate that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by dCTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans.

Figure 1

Drosophila CTCF encodes a protein similar to human CTCF. (A) Sequence alignment of the Zn-finger domain of dCTCF and hCTCF. Similar and identical residues (shading), zinc-coordinating amino acids (red) and amino acids with identical binding site recognition (yellow) are indicated. (B) In situ hybridization of Drosophila egg chamber with DIG-labelled antisense dCTCF RNA. Polyploid nurse cell nuclei are surrounded by dark staining of cytoplasmic dCTCF RNA (top), nuclei are identified by Hoechst staining (middle) and in the overlay (bottom). The egg chamber to the right is surrounded by follicle cells (Hoechst-stained nuclei). (C,D) dCTCF detection in syncytial blastoderm stage 3 with the rabbit anti-dCTCF-C antibody and visualization by avidin-peroxidase at two different magnifications (scale bars (B–D), 40 μm). (E) CV-1 and COS-1 cells were transfected with DNA constructs expressing the GAL DNA-binding domain (GAL), a fusion of the Zn-finger domain plus the C-terminus of chicken CTCF, a similar fusion of dCTCF or of v-erbA. Relative CAT activity is calculated with the GAL transfection defined as 100%.

Figure 2

Similar binding site specificity on vertebrate target sites. Zn-finger domains of chCTCF and dCTCF were in vitro translated and incubated with DNA. (A–D) Indicated DNA fragments were tested in electrophoretic mobility shift assay (EMSA), the β-globin FII insulator sequence (Bell et al, 1999), the CTCF-binding site in the APP gene (Vostrov & Quitschke, 1997), the sites N, FPV and A of the myc genes (Filippova et al, 1996; Lutz et al, 2003) and in the mouse ARF promoter (Filippova et al, 2002). (E) DMS methylation interference analysis was carried out with the β-globin FII insulator fragment labelled on the top or the bottom strand. After separation of bound (b) and unbound fragments (f), the DNA was analysed on a sequencing gel. The G nucleotides interfering with binding after methylation are indicated by dots, and in the sequence (F) by bold lettering.

Figure 3

In vitro and in vivo binding of dCTCF to the Fab-8 element. (A) DMS methylation interference was determined with the in vitro-translated dCTCF Zn-finger domain. Bound (b) and unbound fragments (f) labelled on the top or the bottom strand were analysed on a sequencing gel. Two groups of interfering nucleotides (marked with dots) are indicated (sites 1 and 2). (B) EMSA analysis of dCTCF binding. In vitro-translated, full-length dCTCF was incubated with the Fab-8 wild-type fragment and with the mutants mut1, mut2 and mut1+2. Interpretation of the single and double occupancy of dCTCF on the respective fragments is shown in (C), double occupancy can only be visualized with excess of protein (long gel, lane 3, (B)). The migration difference of b₁ and b₂ complexes is probably caused by site-specific DNA bending (see text). (D) Contact G nucleotides in the Fab-8 sequence (bold) are converted to AT in mutants mut1 and mut2 (see also supplementary information online). (E) Diagram (not to scale) of the Fab-8 location downstream of the Abdominal-B gene, flanked by the iab-7 and iab-8 regulator sequences. (F) In vivo binding of dCTCF to Fab-8. Chromatin from Drosophila embryos was precipitated with the rabbit anti-dCTCF-C antibody (dCTCF) and tested by PCR for the presence of Fab-8 sequences. Increasing amounts of genomic input are included for standardization. A sequence (clone 10) without dCTCF binding, mock precipitation and no DNA are included as negative controls with no signal.

Figure 4

CTCF-dependent enhancer blocking of Fab-8 in vertebrate cells and in Drosophila. (A) K562 cells were transfected with the indicated DNA constructs (B) and colony number was determined after neomycin selection. The pNI and FII constructs (Chung et al, 1993) were used as controls. Transcription of the neomycin gene (Neo) is driven by the β-globin enhancer (E). In the FII construct, two copies of the β-globin insulator (I) separate the enhancer from Neo. The Fab-8 fragment mediates enhancer blocking, whereas mutation of both CTCF-binding sites (Fab-8 mut) does not. (C–H) Fab-8 enhancer blocking in Drosophila depends on dCTCF-binding sites. Transgenic embryos with the indicated vectors were hybridized with either digoxygenin-labelled white (w) in (C,E,G) or lacZ antisense RNA probes (D,F,H). (C,D) white and lacZ expression is visualized when a 1.6 kb λ DNA fragment was inserted between the 2 × PE and iab-5 enhancers (see text). (E,F) The Fab-8 DNA insert between 2 × PE and iab-5 enhancer was analysed in 13 independent lines. Expression of the white reporter gene is restricted to the ventral mesoderm and of the lacZ gene to the abdomen. With the CTCF sites mutagenized, 22 independent lines were generated: white gene activity in the abdomen (G) and lacZ expression in the ventral mesoderm (H) are seen. (I) Chromatin was prepared from embryos carrying transgenes with either Fab-8 wt or the mut1+2 CTCF-binding sites (mut). Primers that specifically recognize transgenic (not genomic) sequences were used. ChIP using anti-CTCF-C antibody shows an increased occupancy at wild-type versus mutant sequences, in contrast to pre-immune serum and to lacZ sequences. For standardization, input dilutions were subjected to semiquantitative PCR as well.