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Originally published In Press as doi:10.1074/jbc.M706213200 on September 7, 2007

J. Biol. Chem., Vol. 282, Issue 46, 33336-33345, November 16, 2007
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Critical DNA Binding Interactions of the Insulator Protein CTCF

A SMALL NUMBER OF ZINC FINGERS MEDIATE STRONG BINDING, AND A SINGLE FINGER-DNA INTERACTION CONTROLS BINDING AT IMPRINTED LOCI*

Mario Renda{ddagger}, Ilaria Baglivo{ddagger}, Bonnie Burgess-Beusse§, Sabrina Esposito{ddagger}, Roberto Fattorusso{ddagger}, Gary Felsenfeld§1, and Paolo V. Pedone{ddagger}2

From the {ddagger}Dipartimento di Scienze Ambientali, Seconda Università degli Studi di Napoli via Vivaldi 43, 81100 Caserta, Italy and the §Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0540

Received for publication, July 27, 2007 , and in revised form, September 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The DNA-binding protein CTCF (CCCTC binding factor) mediates enhancer blocking insulation at sites throughout the genome and plays an important role in regulating allele-specific expression at the Igf2/H19 locus and at other imprinted loci. Evidence is also accumulating that CTCF is involved in large scale organization of genomic chromatin. Although CTCF has 11 zinc fingers, we show here that only 4 of these are essential to strong binding and that they recognize a core 12-bp DNA sequence common to most CTCF sites. By deleting individual fingers and mutating individual sites, we determined the orientation of binding. Furthermore, we were able to identify the specific finger and its point of DNA interaction that are responsible for the loss of CTCF binding when CpG residues are methylated in the imprinted Igf2/H19 locus. This single interaction appears to be critical for allele-specific binding and insulation by CTCF.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The CCCTC binding factor (CTCF)3 is an evolutionarily conserved transcription factor that is involved in various aspects of gene regulation. It was originally identified for its ability to specifically bind regulatory sequences in the promoter-proximal region of the MYC oncogene (1, 2) and in the silencer element of the chicken lysozyme gene (3, 4). It is composed of 727 amino acids and contains 11 zinc finger (ZF) domains, the first 10 of the classical Cys2-His2 types (5, 6), whereas the last has an unusual Cys2-His-Cys sequence. Through combinatorial use of its 11 ZF motifs, CTCF has been reported to bind to a variety of DNA target sites that perform distinct functions, including promoter activation (7, 8) or repression (1, 2), hormone-responsive gene silencing (3, 4), methylation-dependent chromatin insulation, and genomic imprinting (9–11).

Of these activities, the ability of CTCF to mediate insulator function has attracted the greatest interest (11). CTCF-dependent insulators have been particularly characterized in the chicken beta-globin locus and in the imprinted Igf2/H19 locus in mouse and human. The region that includes the beta-globin genes and the erythrocyte-specific enhancers is flanked on both sides by CTCF insulators (12–14); in the 5' boundary, Bell et al. (12) identified a 42-bp CTCF-binding site (FII) both necessary and sufficient for enhancer blocking activity. In the Igf2/H19 locus, the insulator in the differentially methylated domain (also called the imprinted control region) located at the 5' of the H19 gene prevents the downstream enhancers from acting on the upstream Igf2 gene on the maternal allele (15, 16). The insulator activity in mouse is mediated through four repeats (R1 to R4) that are binding sites for CTCF (15–19). The methylation of the H19 differentially methylated domain insulator on the paternal allele prevents CTCF binding, thus allowing the enhancers to activate the Igf2 gene promoter (16, 17).

Earlier studies of the interactions between CTCF and its binding sites attempted to determine critical interactions between protein and DNA by mutations in the full 11 zinc finger binding domains that either deleted individual fingers from within the cluster or sequentially deleted fingers from one end or the other (20, 21). Recent genome-wide surveys of CTCF-binding sites in vivo (22–24) have led to a much more precise definition of the consensus DNA-binding sequence. Here we have been able to demonstrate that a much smaller ZF construct as well as a much smaller DNA-binding site are sufficient to provide binding interactions similar in strength to those observed with the full 11 ZFs on the previously identified FII DNA site.

By using various mutants, we have defined in this paper the minimal CTCF domain that binds the chicken beta-globin insulator FII site, identifying the zinc fingers that are responsible for this interaction. We show that other sites such as those at the imprinted Igf2/H19 locus behave quite similarly, and we have identified a 12-bp core DNA fragment both necessary and sufficient for CTCF binding to both the FII and Igf2 R3 insulator sites. These results allow us to establish the orientation of CTCF binding at its site, and to identify the interaction, both the individual finger and the particular methylated CpG, that is disrupted when the DNA of the differentially methylated domain/imprinted control region at the Igf2/H19 locus is methylated on the paternal allele, an event that leads in vivo to monoallelic expression of Igf2.


Figure 1
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  		FIGURE 1.
A, sequence alignment of four well characterized CTCF DNA-binding sites (7, 12, 19). The FII oligonucleotide includes the CTCF-binding site within the insulator located at the 5' boundary of the chicken beta-globin locus (12); the R3 and R4 sequences are derived from two of the four repeats present in the mouse imprinted control region at the 5' of the H19 gene that are binding sites for CTCF and through their insulator activity regulate imprinting at the Igf2/H19 locus (19); the APP oligonucleotide includes the CTCF recognized sequence present in the promoter of the human amyloid beta-protein precursor gene (7). The 12-bp core site is underlined; an asterisk indicates the bases that are identical in all the four DNA sequences. B, gel mobility shift analysis of CTCF ZF 1–11 DNA binding to the FII 45-bp oligonucleotide; the DNA binding specificity has been investigated by competition experiments. Purified CTCF ZF 1–11 protein (6 pmol) was incubated with 55 fmol of the labeled FII 45-bp oligonucleotide in the absence (lane 1) or presence of a 100-fold excess of unlabeled specific oligonucleotide FII 45 bp (lane 2) and a 100-fold excess of an unlabeled oligonucleotide with a nonspecific sequence (NS, lane 3) and then subjected to the gel shift analysis. C, gel mobility shift titration of CTCF ZF 1–11 with the FII 45-bp oligonucleotide (see "Experimental Procedures") (lower panel) and Scatchard analysis of the gel shift binding data (upper panel). The ratio of bound to free DNA is plotted versus the molar concentration of bound DNA in the reaction mixture.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Purification of the Proteins—DNA fragments encoding the different segments of the human CTCF protein to be expressed as maltose-binding protein (MBP) fusion were generated by PCR using the plasmid p4B 7.1 (12) as a DNA template. Oligonucleotides were synthesized on the basis of the published sequence (GenBankTM accession number NM_006565). The following oligonucleotides were used as primers: primer 1, 5'-CGGAATTCGGTGTAAAGAAGACATTCCAGTG-3', and primer 2, 5'-AACATGGTCGACTCAGCCATCTGGGCCAGCACAATTATC-3' for CTCF ZF 1–11; primer 3, 5'-CGGAATTCGAGAAGCCATTCAAGTGTTC-3', and primer 4, 5'-AACATGGTCGACTCAGCGCTTCTCATTCTTGTGTGAC-3', for CTCF ZF 4–8; primer 3 and primer 5, 5'-AACATGGTCGACTCATTGCTCAATATAGGAATGCTGC-3', for CTCF ZF 4–7; primer 3 and primer 6, 5'-AACATGGTCGACTCAGGCCACATTTTCTGTGTGCTTC-3', for CTCF ZF 4–6; primer 3 and primer 7, 5'-AACATGGTCGACTCAAGGCTTTTCCCCTGAATGGG-3', for CTCF ZF 4–5; primer 8, 5'-CGGAATTCGAGCGTCCGTTTCAGTGCAG-3', and primer 4 for CTCF ZF 5–8; primer 8 and primer 5 for CTCF ZF 5–7; primer 9, 5'-CGGAATTCGAAAAGCCTTATGAATGTTATATTTG-3', and primer 4 for CTCF ZF 6–8. All the PCR products were digested with the restriction enzymes EcoRI and SalI and cloned into EcoRI- and SalI-digested pMal C2G (New England Biolabs) bacterial expression vector. All the plasmids obtained were sequenced to confirm that there were no mutations in the coding sequences. The fusion proteins were expressed in the Escherichia coli BL21 host strain. The transformed cells were grown in rich medium plus 0.2% glucose (according to protocol from New England Biolabs) at 37 °C until the absorbance at 600 nm was 0.6–0.7, at which time the medium was supplemented with 200 µM ZnSO4 (CTCF ZF 1–11) or 100 µM ZnSO4 (all the others), and protein expression was induced with 0.3 mM isopropyl 1-thio-beta-D-galactopyranoside and was allowed to proceed for 2 h. The cells were then harvested, resuspended in 1x PBS (pH 7.4) (25), 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM aprotinin, and 10 µg/ml lysozyme, sonicated, and centrifuged for 30 min at 27,500 relative centrifugal force. The supernatant was then loaded on amylose resin (New England Biolabs) according to the manufacturer's protocol. Following washes with 1x PBS, purified fractions were eluted in maltose elution buffer (10 mM maltose, 100 mM Tris (pH 8.0), and 100 mM NaCl). The CTCF ZF 4–7 and CTCF ZF 5–8 proteins were expressed also as GST fusions. The coding sequences were generated by PCR as described for the MBP fusion proteins. The DNA fragments were then digested with the restriction enzymes EcoRI and SalI and cloned into EcoRI- and SalI-digested pGex 6P1 (Amersham Biosciences) bacterial expression vector. The fusion proteins were expressed in the E. coli BL21 host strains. The cells were grown in LB medium at 37 °C until the absorbance at 600 nm was 0.6–0.7 at which time the medium was supplemented with 100 µM ZnSO4, and protein expression was induced for 2 h with 1 mM isopropyl 1-thio-beta-D-galactopyranoside at 28 °C. The cells were then harvested, resuspended in 1x PBS (pH 7.4) (25), 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 µM aprotinin, and 10 µg/ml lysozyme, sonicated, and centrifuged for 30 min at 27,500 relative centrifugal force. The supernatant was then loaded on a glutathione-Sepharose resin (Amersham Biosciences) according to the manufacturer's protocol. Following washes with 1x PBS, purified fractions were eluted in glutathione elution buffer (10 mM glutathione, 100 mM Tris (pH 8.0), and 100 mM NaCl). To remove the GST from the CTCF ZF 4–7 and CTCF ZF 5–8 fusion proteins, according to the manufacturer's advice, a cleavage reaction mixture containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 unit/100 µg of GST fusion protein of PreScission Protease (Amersham Biosciences) was loaded on the resin where the fusion proteins were bound, incubated for 12 h at 4 °C, and centrifuged for 5 min at 500 relative centrifugal force, and the supernatant containing the protein without the GST tag was then recovered.


Figure 2
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  		FIGURE 2.
Identification of the CTCF minimal DNA binding domain. A, gel mobility shift titration of CTCF ZF 4–8 with the FII 45-bp oligonucleotide (see "Experimental Procedures") (lower panel) and Scatchard analysis of the gel shift binding data (upper panel). The ratio of bound to free DNA is plotted versus the molar concentration of bound DNA in the reaction mixture. B, gel mobility shift DNA binding analysis of different CTCF ZF 4–8 C-terminal deletion mutants: CTCF ZF 4–8 (lane 1), CTCF ZF 4–7 (lane 2), CTCF ZF 4–6 (lane 3), and CTCF ZF 4–5 (lane 4). C, gel mobility shift DNA binding analysis of different CTCF ZF 4–8 N-terminal deletion mutants: CTCF ZF 4–8 (lane 1), CTCF ZF 5–8 (lane 2), and CTCF ZF 6–8 (lane 3). D, gel mobility shift analysis of CTCF ZF 5–7 DNA binding. E and F, gel mobility shift DNA binding analysis of CTCF ZF 4–7 and CTCF ZF 5–8 after the proteolytic cleavage of the GST tag. The DNA binding specificity and metal requirement for binding were investigated. The purified CTCF ZF 4–7 cut (E) and CTCF ZF 5–8 cut (F) fragments (1.5 pmol) without the GST tag were incubated with 55 fmol of the labeled FII 45-bp oligonucleotide in the absence (lane 1) or presence of a 100-fold excess of unlabeled specific oligonucleotide FII 45 bp (lane 2), a 100-fold excess of an unlabeled oligonucleotide with a nonspecific sequence (NS, lane 3), or 50 mM EDTA (lane 4) and then subjected to the gel shift analysis.

 
Gel Mobility Shift Analysis—Unless otherwise specified, 0.9 pmol of each of the purified proteins were incubated for 10 min on ice with 55 fmol of the specified labeled duplex oligonucleotide in the presence of 25 mM Hepes (pH 7.9), 50 mM KCl, 6.25 mM MgCl2, 1% Nonidet P-40, 5% glycerol, and 200 ng of double-stranded poly(dI/dC-dI/dC) (Roche Applied Science). After incubation, the mixture was loaded on a 5% polyacrylamide gel (29:1 acrylamide/bisacrylamide ratio) and run in 0.5x TBE (25) at 4 °C (200 V for 2 h, 15 min). As a nonspecific competitor for competition experiments, the oligonucleotide NS, 5'-TGGCCAGGGCCGCGCCGTGGCGGGGCCAGGGCGCGGGGCT-3', was used. Protein concentration was determined by a modified version of the Bradford procedure (26) (Bio-Rad protein assay). In the case of the CTCF ZF 1–11 protein, an apparent higher protein concentration (6 pmol) was required likely because not all the protein sample was correctly folded. The affinity of CTCF ZF 1–11 and CTCF ZF 4–8 for FII 45 bp and of CTCF ZF 4–8 for FII core oligonucleotides was measured by a gel mobility shift assay by performing a titration of the proteins with the oligonucleotides. In the case of CTCF ZF 1–11, in a volume of 20 µl, 3 pmol of the protein was incubated with 0.08, 0.12, 0.14, 0.16, and 0.18 pmol of the duplex FII 45 bp; in the case of CTCF ZF 4–8, in a volume of 20 µl, 0.9 pmol of the protein was incubated with 0.08, 0.12, 0.14, 0.16, 0.17, and 0.18 pmol of the duplex FII 45 bp and 0.08, 0.12, 0.14, 0.16, 0.18 and 0.20 pmol of the duplex FII core, respectively. Scatchard analysis of the gel shift binding data was performed to obtain the Kd values (27). All numerical values were obtained by computer quantification of the image using a Amersham Biosciences Typhoon Trio+ apparatus.


Figure 3
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  		FIGURE 3.
DNA binding analysis of the CTCF deletion mutants on different DNA-binding sites. A, R3 31-bp oligonucleotide; B, R4 31-bp oligonucleotide; and C, APP 45-bp oligonucleotide were used as probes in gel mobility shift experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Minimal Set of Fingers for Strong Binding—In proteins containing multiple fingers, it is clear that adequate specificity and affinity can be provided by a few critical fingers and that different fingers can have very different roles in a polydactyl complex (6, 28). To define the minimal region of the CTCF protein able to bind the CTCF-binding site FII within the chicken beta-globin insulator, a site that is an essential component of the enhancer-blocking activity of that element (12), fragments encoding all 11 ZFs of CTCF (CTCF ZF 1–11), and various deletion mutants were cloned into the pMal expression vector. The proteins were expressed in E. coli as a MBP fusion and purified. A gel mobility shift analysis was performed on the purified proteins to determine their ability to bind DNA.

As expected, the CTCF ZF 1–11 protein binds a 45-bp-long oligonucleotide duplex, which includes the FII site (FII 45-bp, for the sequence see Fig. 1A), producing a single complex (Fig. 1B, lane 1). This result confirms that the N-terminal and C-terminal regions of the protein that flank the ZF domains are not required for DNA binding. The binding specificity of the purified CTCF ZF 1–11 protein was demonstrated by competition experiments with unlabeled oligonucleotides; the complex is competed by addition of a 100-fold excess of unlabeled FII 45-bp oligonucleotide (Fig. 1B, lane 2) but not by the same amount of an unrelated oligonucleotide sequence (NS, Fig. 1B, lane 3). The affinity of the CTCF ZF 1–11 protein for the FII 45-bp oligonucleotides was measured using a gel mobility shift assay. Titration of the protein with the oligonucleotide is shown in Fig. 1C; Scatchard analysis (27) of these data leads to an apparent dissociation constant of 3.3 ± 1.0 x 10–10 M.

We compared this result with the DNA binding capability of proteins with a reduced number of fingers. Interestingly, similar high affinity binding to the FII site, with an apparent Kd of 1.0 ± 0.2 x 10–10 M, can also be obtained with a protein that includes only the ZF domains from 4 to 8 (CTCF ZF 4–8, Fig. 2A). This result indicates that ZFs outside the region that includes the fingers from 4 to 8 do not contribute significantly to DNA binding affinity to the FII site. Starting from the construct CTCF ZF 4–8, we made different C-terminal (Fig. 2B) and N-terminal deletion mutants (Fig. 2C) to identify the minimal DNA binding domain. The protein that includes ZFs 4–7 (CTCF ZF 4–7) still binds the FII site with high affinity (Fig. 2B, lane 2), indicating that when ZF 4–7 are present, a ZF 8 contribution is not essential for FII DNA binding. The protein that includes ZF 4–6 (CTCF ZF 4–6, Fig. 2B, lane 3) is able to bind the FII site but with lower affinity compared with the CTCF ZF 4–7 protein (Fig. 2B, compare lane 2 and lane 3), indicating that ZF 7 plays an important role in stabilizing the interaction with the FII DNA site. However, a further deletion of ZF 6 produces the protein CTCF ZF 4–5, which does not bind DNA (Fig. 2B, lane 4), indicating that ZF 6 is involved in FII DNA interaction.

We next determined the effect of deleting fingers from the N terminus of the cluster CTCF ZF 4–8 (Fig. 2C). When ZF 4 is removed, the resulting protein CTCF ZF 5–8 is also capable of FII DNA binding, but with reduced affinity compared with CTCF ZF 4–8 (Fig. 2C, lanes 1 and 2), indicating an important role for ZF 4 in FII DNA recognition. The protein CTCF ZF 6–8, obtained by further deleting ZF 5, does not bind the FII site (Fig. 2C, lane 3), indicating that ZF 5 is essential for FII high affinity recognition.

The above results show that the proteins CTCF ZF 4–7 and ZF 5–8, each containing 4 ZF domains, are both able to bind the FII site, although CTCF ZF 4–7 shows higher DNA binding affinity (compare Fig. 2B, CTCF ZF 4–7, lane 2, with Fig. 2C, CTCF ZF 5–8, lane 2). Inasmuch as these two proteins share the ZFs 5–7, we tested the CTCF ZF 5–7 construct for its DNA binding affinity to the FII site. Interestingly, the CTCF ZF 5–7 protein does not bind the FII 45-bp oligonucleotide (Fig. 2D, lane 2); this result indicates that ZF 8, which we have shown to be dispensable for CTCF high affinity binding to the FII site when ZF 4 is present (compare Fig. 2B, CTCF ZF 4–8 and CTCF ZF 4–7, lanes 1 and 2), becomes essential when ZF 4 is missing (compare Fig. 2D, CTCF ZF 5–8 with CTCF ZF 5–7, lanes 1 and 2). On the other hand ZF 4, which we have shown to be important in stabilizing the CTCF ZF 4–8 DNA binding on the FII site (compare Fig. 2C, CTCF ZF 4–8 with CTCF ZF 5–8, lanes 1 and 2), becomes essential when ZF 8 is missing (Fig. 2B, compare CTCF ZF 4–7 lane 2 with Fig. 2D, CTCF ZF 5–7, lane 2). The results indicate that to obtain high affinity FII DNA binding, four zinc fingers are necessary, with the protein CTCF ZF 4–7 showing the highest DNA affinity. The only three zinc finger-containing protein, which shows appreciable DNA binding to the FII site in the binding condition we have tested, is the construct CTCF ZF 4–6. Considering that CTCF ZF 4–6 and CTCF ZF 5–7 share ZF 5 and 6, we can argue that ZF 4 makes a stronger contribution to DNA binding affinity at this site compared with ZF 7. Similarly, the observation that CTCF ZF 4–7 binds more strongly than CTCF ZF 5–8 to the FII DNA site suggests that in this DNA interaction ZF 4 makes a more significant contribution than ZF 8.


Figure 4
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  		FIGURE 4.
Identification of the minimal core sequence recognized by CTCF in the FII site. Gel mobility shift analysis of CTCF deletion mutants DNA binding to the FII 23-bp oligonucleotide (A) and to the FII core oligonucleotide (B); the sequences of the FII 23-bp and FII core oligonucleotides are indicated; the core sequence is underlined, and the bases that have been mutated are indicated in italics. C, gel mobility shift titration of CTCF ZF 4–8 with the FII core oligonucleotide (see "Experimental Procedures") (lower panel) and Scatchard analysis of the gel shift binding data (upper panel). The ratio of bound to free DNA is plotted versus the molar concentration of bound DNA in the reaction mixture.

 
To exclude the possibility that the MBP moiety might influence the DNA capability of the fusion proteins, we expressed both the CTCF ZF 4–7 and CTCF ZF 5–8 proteins as GST fusions and tested their ability to bind the FII 45-bp oligonucleotide; both proteins are able to bind the probe as GST fusions (data not shown) and after the GST moiety was removed by proteolytic cleavage (see "Experimental Procedures") (Fig. 2, E, CTCF ZF 4–7 cut, lane 1, and F, CTCF ZF 5–8 cut, lane 1). The binding specificity of the purified proteins without the tag was demonstrated by competition experiments with unlabeled oligonucleotides; the complexes are competed by addition of a 100-fold excess of unlabeled FII 45-bp oligonucleotide (Fig. 2, E, lane 2, and F, lane 2) but not by the same amount of an unrelated oligonucleotide sequence (NS, Fig. 2, E, lane 3, and F, lane 3). Addition of 50 mM EDTA to the binding reactions (Fig. 2, E, lane 4, and F, lane 4) abolishes the DNA binding activity, confirming that binding of the proteins to DNA is metal-dependent.


Figure 5
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  		FIGURE 5.
ZF 4 positioning and CTCF orientation on the FII core site. Comparison of CTCF ZF 4–8 (A), CTCF ZF 4–7 (B), CTCF ZF 4–6 (C), and CTCF ZF 5–8 (D) DNA binding to the FII core (lane 1) and FII core mut 1 (lane 2) oligonucleotides. E, comparison of CTCF ZF 4–6 DNA binding to the FII core (lane 1) and FII core mutT (lane 2) oligonucleotides. In gel shift experiments presented in A, B, and D, 0.2 pmol of the purified proteins were used, whereas in C and E 0.9 pmol of the purified CTCF ZF 4–6 protein were used. The sequences of the FII core, FII core mut 1, and FII core mutT oligonucleotides are indicated; the core sequence is underlined, and the bases that have been mutated are indicated in italics.

 
Other CTCF-binding Sites—To assess if the CTCF ZF 4–8 region could also be involved in the recognition of other biologically significant CTCF DNA target sites, we first tested the different protein deletion mutants for their capability to bind one of the CTCF sites responsible for the chromatin insulator function of the mouse Igf2/H19 imprinting control region (site R3 according to Engel et al. 19; see Fig. 1A). Essentially, the DNA binding results obtained with the different CTCF deletion mutants on a 31-bp-long probe, including the R3 sequence (R3 31bp, Fig. 3A), are similar to those obtained with the FII site, with the most significant difference being that the CTCF ZF 4–6 protein, which is still able to bind the FII sequence (Fig. 2B, lane 3), is not able to bind the R3 site (Fig. 3A, lane 7). Interestingly, the CTCF ZF 4–8 DNA binding domain is also able to bind an oligonucleotide, including the R4 CTCF site of the Igf2/H19 imprinting control region (19) (R4 31bp, Fig. 3B) and one derived from the CTCF recognized sequence present in the promoter of the human amyloid beta-protein precursor (APP) gene (7) (APP 45-bp, Fig. 3C; for the sequences see Fig. 1A). The alignment of all of these sites reveals that 8 bases in a 12-bp-long sequence are shared among all of them (Fig. 1A); we defined this 12-bp sequence as the core site.

Length of the Minimum DNA-binding Site—It has been proposed that CTCF-binding sites would not be detected by gel shift analyses with 20–30-bp double-stranded oligonucleotides because these probes would be too short to accommodate the CTCF protein and guarantee efficient CTCF binding in vitro (1, 29). Having demonstrated that 4 ZFs are sufficient for CTCF high affinity DNA binding, we reasoned that we could reduce the size of the oligonucleotide and still get high affinity binding. First, we derived from the FII 45-bp probe a 23-bp oligonucleotide (FII 23 bp), which included the core site, and used it as a probe in a gel shift experiment (Fig. 4A). Interestingly, the binding of the different CTCF deletion mutants to the FII 23-bp oligonucleotide is similar to that observed with the FII 45 bp (compare Fig. 4A and Fig. 2, B and C). To test if the core site, present in the FII 23-bp probe, was indeed responsible for the recognition of the target site by the CTCF proteins, we designed a new oligonucleotide (FII core, Fig. 4B) in which the core was preserved and the flanking sequences were changed with respect to the wild type FII 23-bp sequence. Interestingly, all the CTCF deletion mutants bind the FII core (Fig. 4B) just as they do the FII 23-bp site (Fig. 4A). Moreover, we determined by gel mobility shift assay the affinity of the CTCF ZF 4–8 protein for the FII core oligonucleotide (Fig. 4C) and found a Kd value of 1.7 ± 0.3 x 10–10 M very similar to the one obtained for the interaction of the same protein with the FII 45-bp oligonucleotide; these results clearly indicate that all the specific DNA contacts that the CTCF ZF 4–8 protein makes with the FII target site can be mapped within the core sequence.


Figure 6
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  		FIGURE 6.
ZF 7 positioning and CTCF orientation on the FII core site. Comparison of CTCF ZF 4–8 (A), CTCF ZF 4–7 (B), CTCF ZF 5–8 (C), and CTCF ZF 4–6 (D) DNA binding to the FII core (lane 1) and FII core mut 2 (lane 2) oligonucleotides. 0.2 pmol of the purified proteins were used in A and B experiments, 0.9 pmol in C and D experiments. The sequences of the FII core and FII core mut 2 are indicated; the core sequence is underlined and the bases which have been mutated are indicated in italics.

 
CTCF Orientation on Its Binding Site—To define the orientation of the CTCF ZF DNA binding domain on the FII core site, and to map which of the fingers was able to interact with the individual bases, we designed oligonucleotides bearing various mutations in the core site and used them in gel mobility shift assays performed with the different CTCF deletion mutants. First, we tested the FII core mut 1 oligonucleotide, mutated in the DNA triplet located at the 3' end of the core (Fig. 5); interestingly, this mutation does not affect the DNA binding of the CTCF ZF 5–8 protein (Fig. 5D), which lacks ZF 4, whereas it strongly reduces the DNA binding affinity of the CTCF ZF 4–8, CTCF ZF 4–7, and CTCF ZF 4–6 proteins (Fig. 5, A–C), which all have the ZF 4; this result strongly suggests that ZF 4 is recognizing the 3' end site of the FII core. Having determined that ZF 4 recognizes the 3' end of the core, we reasoned that the other ZFs could read the DNA sequence in a 3' -> 5' direction. We have previously shown that the CTCF ZF 4–6 protein, which is still able to bind the FII sequence, is not able to bind the R3 site of the mouse Igf2/H19 imprinted control region (Fig. 3A, lane 7). The sequence comparison between the FII and the R3 site (Fig. 1A) shows that there are three differences in the core sequence, the most 3' of which is a G to T transversion at position 9. When the same G to T mutation is introduced in the FII core site (FII core mutT), CTCF ZF 4–6 DNA binding is strongly inhibited (Fig. 5E); this result suggests that a single base mutation in the 3'-half of the core sequence can be responsible for the different binding of the CTCF ZF 4–6 to the FII and R3 DNA sites, confirming that CTCF ZF 4–6 recognizes the 3' of the FII core and the CTCF protein recognizes the R3 core site with a similar orientation, despite the differences in the DNA sequence.


Figure 7
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  		FIGURE 7.
Identification of the bases of the triplet located at the 5' end of the FII core site that are important for CTCF DNA binding. Gel mobility shift analysis of CTCF ZF 4–8 (A), CTCF ZF 4–7 (B), and CTCF ZF 5–8 (C) DNA binding to the FII core (lane 1), FII core mut 2 (lane 2), FII core mut 3 (lane 3), FII core mut 4 (lane 4), and FII core mut 5 (lane 5) oligonucleotides. 0.2 pmol of the purified proteins were used in A and B, and 0.9 pmol in C. The sequences of the FII core, FII core mut 2, FII core mut 3, FII core mut 4, and FII core mut 5 are indicated; the core sequence is underlined, and the bases which have been mutated are indicated in italics.

 
We then tested the binding of various CTCF finger deletion mutants, using as a probe the FII core mut 2 oligonucleotide, in which the first 3 bases at the 5' end of the core were mutated; this mutation strongly reduces the DNA binding affinity of the CTCF ZF 4–8, CTCF ZF 4–7, and CTCF ZF 5–8 proteins (Fig. 6, A–C), but it does not affect the DNA binding of the CTCF ZF 4–6 protein (Fig. 6D). Considering that all the proteins whose DNA binding is inhibited by the mutation of the first 3 bases of the core have ZF 7, whereas only the DNA binding of the CTCF ZF 4–6 protein, which lacks ZF 7, is not affected by this mutation, we can conclude that ZF 7 is the one recognizing the most 5' triplet of the FII core. To understand if all 3 bases mutated in the FII core mut 2 oligonucleotide were important for CTCF DNA binding, we used as probes in similar gel shift experiments point mutants of the FII core oligonucleotide in which each of the first 3 bases of the core was singly mutated (FII core mut 3, FII core mut 4, and FII core mut 5, see Fig. 7); interestingly, the mutation of the cytosine in position 2 (FII core mut 4) has the strongest inhibitory effect on CTCF ZF 4–8, CTCF ZF 4–7, and CTCF ZF 5–8 DNA binding (Fig. 7, A–C), suggesting that the contact of ZF 7 with this base, or its complementary guanine, is essential for high affinity DNA binding. We note that mutation of position 3 of the core (FII core mut 5, T replacing G) does not prevent the binding, consistent with the recently published consensus (22) in which A, T, or G can be found at this position.

Effect of DNA Methylation, Disruption of a Critical Finger-DNA Interaction—CTCF DNA binding to different target sequences, including the FII site, has been demonstrated to be sensitive to DNA methylation (16, 17). Interestingly, Bell and Felsenfeld (16) already demonstrated that methylation on both strands of the first CpG in the FII core sequence alone significantly reduced CTCF binding. This CpG dinucleotide includes on the upper strand the cytosine in the second position of the core, which if mutated together with its complementary guanosine inhibits CTCF binding (Fig. 7, see FII core mut 4). To test if the methylation of this base, like the mutation, would affect CTCF DNA binding, we produced an oligonucleotide, FII core met, which differs from FII core only in the methylation of the cytosine in position 2 of the core on the upper strand. Interestingly, the methylation of this single base strongly inhibits DNA binding of the CTCF ZF 1–11 protein (Fig. 8A), as well as that of the CTCF ZF 4–8, CTCF ZF 4–7, and CTCF ZF 5–8 deletion mutants (Fig. 8, B–D), although it has no effect on CTCF ZF 4–6 DNA binding (Fig. 8E). These results allowed us to identify in the FII core a methylation signal that affects CTCF DNA binding and to show that this inhibition acts by preventing ZF 7 DNA contacts. The observation that the methylation of the cytosine (FII core met) has the same effect as the mutations of this base and its complementary guanine (Fig. 7, FII core mut 4) strongly suggests that ZF 7 is making contact with the cytosine on the upper strand and that the addition of a methyl group is interfering with this interaction.


Figure 8
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  		FIGURE 8.
Analysis of the sensitivity to the cytosine methylation of the FII core site of the different CTCF deletion mutants DNA binding. Gel mobility shift analysis of CTCF ZF 1–11 (A), CTCF ZF 4–8 (B), CTCF ZF 4–7 (C), CTCF ZF 5–8 (D), and CTCF ZF 4–6 (E) DNA binding to the FII core (lane 1) and FII core met (lane 2) oligonucleotides. 6 pmol of the purified protein were used in A, 0.2 pmol in B and C, and 0.9 pmol in D and E. The sequences of the FII core and FII core met are indicated; the core sequence is underlined; the cytosine that has been methylated is indicated. The FII core met oligonucleotide is methylated only on the upper strand.

 
Paternal specific methylation of CpG sites within the CTCF-binding sites present in the chromatin insulator upstream of H19 (R1 to R4 in mouse according to Engel et al., 19) eliminates CTCF binding, restricting the activity of this regulatory element to the maternal allele, and thus regulating the imprinting of the H19 and Igf2 genes (15, 16). In the R3 31-bp oligonucleotide we used (Fig. 3A) there are four CpGs (Fig. 1A), the first of which is outside the core sequence; by comparing CTCF ZF 4–7 DNA binding to the R3 31-bp oligonucleotide (Fig. 9A, lane 1) with that to R3-derived oligonucleotides in which all the four CpG were methylated on both strands (R3 fully met, Fig. 9A, lane 2), only on the upper strand (R3 for met, Fig. 9A, lane 3) or on the lower strand (R3 rev met, Fig. 9A, lane 4), we confirmed the results obtained by Hark et al. (15) that the methylation signal that affects CTCF DNA binding to this site is on the upper strand. To understand which of the methylated cytosines in the R3 site upper strand was responsible for CTCF DNA binding inhibition, we tested in gel mobility shift experiments R3 oligonucleotides in which only one of the 4 possible cytosines was methylated (R3 for met 1, R3 for met 2, R3 for met 3, and R3 for met 4). The methylation of the cytosine of the first CpG outside the core sequence does not affect CTCF DNA binding (Fig. 9B, oligo R3 for met 1, lane 3); of the three cytosines in the CpGs located in the R3 core sequence, only the methylation of the first (Fig. 9B, R3 for met 2, lane 4) and second cytosines (Fig. 9B, R3 for met 3, lane 5) inhibits the protein binding, with the first being the one with the strongest effect, whereas the methylation of the third CpG in the core had no effect (Fig. 9B, R3 for met 4, lane 6).

We then tested if also in the case of the R3 site, the CTCF DNA binding domain would be able to bind the R3 core site just as it does the entire wild type 31-bp sequence. Indeed, the protein CTCF ZF 4–7 is able to bind with high affinity the R3 core oligonucleotide, which is 23 bp long and has the R3 core flanked by scrambled DNA sequences (Fig. 9C, lane 1); as expected, methylation of the first CpG of the core site in the R3 core oligonucleotide (R3 core met) inhibits CTCF ZF 4–7 DNA binding (Fig. 9C, lane 2); this results is similar to the one obtained with the methylation of the first CpG of the FII core (see Fig. 8C). Taken together these results further confirm that the DNA binding modality on the R3 site is similar to the one described on the FII site, thus suggesting that the methylation of the first CpG of the R3 core is preventing the interaction of ZF 7.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have defined the CTCF DNA binding domain responsible for the interaction of this protein with various biologically relevant DNA-binding sites; this domain includes ZFs from 4 to 8. Interestingly, ZF 8, which we have shown to be dispensable for CTCF high affinity binding to the FII site when ZF 4 is present, becomes essential when ZF 4 is missing, suggesting that the contribution of this finger to CTCF DNA binding affinity could vary depending on the particular DNA site. Moreover, we have identified in the FII CTCF site of the chicken beta-globin insulator a 12-bp core site, conserved in various other CTCF DNA-binding sites, which is recognized by the CTCF DNA binding domain (CTCF ZF 4–8) with high affinity, comparable with that measured with an oligonucleotide bearing a wild type 45-bp sequence. We demonstrated that ZF 4 recognizes the 3' end of the identified core site, whereas ZF 7 recognizes the 5' end; it seems likely that ZF 8 interacts with the DNA sequence outside the core, providing additional but nonsequence-specific DNA contacts. These results allowed us to orient the protein on its target DNA and obtain the first results revealing how individual CTCF ZFs are contributing to the interaction with the target sites. Considering that the data in the literature indicate that most of the Cys2-His2 zinc fingers bind 3-bp units (28), the 12-bp core identified in this study could be specifically recognized by ZF 4 to ZF 7 oriented on the DNA sequence in the 3' to 5' direction.


Figure 9
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  		FIGURE 9.
Definition of the methylation signal that affects CTCF binding to the R3 site and identification of the core site recognized by the protein. A, gel mobility shift analysis of CTCF ZF 4–7 DNA binding to the R3 31 bp (lane 1), R3 fully met (lane 2), R3 for met (lane 3), and R3 rev met (lane 4) oligonucleotides. The four CpGs present in the R3 31-bp oligonucleotide are methylated on both strands in R3 fully met, only on the upper strand in R3 for met, and only on the lower strand in R3 rev met. B, gel mobility shift analysis of CTCF ZF 4–7 DNA binding to R3 for met 1 (lane 3), R3 for met 2 (lane 4), R3 for met 3 (lane 5), and R3 for met 4(lane 6) oligonucleotides. C, gel mobility shift analysis of CTCF ZF 4–7 DNA binding to the R3 core (lane 1) and R3 core met (lane 2) oligonucleotides. The sequences of the R3 31 bp, R3 for met, R3 for met 1, R3 for met 2, R3 for met 3, R3 for met 4, R3 core, and R3 core met oligonucleotides are indicated; the core sequence is underlined, the cytosines that have been methylated are indicated, and the bases that have been mutated in R3 core and R3 core met are indicated in italics; in B and C the complementary oligonucleotides used to obtain the double strands probes were not methylated.

 
We note that earlier investigations that made use of sequential deletions from either end of the 11 ZF full binding domains have reported a variety of critical contacts, in some cases involving many more fingers (21). We believe that binding can be partially stabilized by nonsequence-specific interactions between peripheral fingers and outlying DNA sequences. As we have shown, however, the critical interactions for strong binding are confined to a very small DNA sequence and a 5-finger central region of CTCF. Our results should also be compared with those of Quitschke et al. (20), who made use of long constructs in which individual fingers were deleted from the interior to show that the central fingers 5–7, and more weakly fingers 8–10, could contribute to binding to the APP gene promoter sequence; moreover, in agreement with the CTCF DNA binding data on the APP gene promoter (20), our results provide direct confirmation that the N-terminal end of the zinc finger domain is aligned toward the transcriptional start site.

We also found that methylation of the cytosine in position 2 of the core site inhibits ZF 7 DNA binding on both the FII and R3 sites, strongly affecting CTCF DNA binding affinity. Having tested the effect on CTCF DNA binding of the methylations of all the four CpGs present in the R3 site, one of which is located outside the core, we have demonstrated that only the methylations of the cytosines on the upper strand of the first and second CpGs in the core inhibit CTCF binding, with the first having a stronger effect. Considering that the CTCF-binding sites present in the Igf2/H19 insulator are very well conserved, it is likely that we have identified the most important methylation signals that affect CTCF DNA binding in these sequences, and we can affirm that ZF 7 DNA binding is inhibited by these methyl groups.

Recent studies on genome-wide localization of CTCF-binding sites have provided a redefinition of the CTCF consensus sequence (22–24). Kim et al. (22) have identified a 20-bp motif that is present in over 75% of the experimentally identified vertebrate CTCF-binding sites; according to the authors, CTCF binding in vivo is mediated mostly by this single consensus motif, which is also highly conserved evolutionarily as demonstrated by comparing potential CTCF-binding sites in different vertebrate genomes. A similar CTCF-binding motif was demonstrated by Xie et al. (24) to be significantly enriched in conserved noncoding regions of the human genome, and these repeated regulatory motifs have been proposed to be involved in insulator function to limit the spread of gene activation; a comparable CTCF-binding site consensus sequence has also been proposed for the Drosophila CTCF protein (23). Interestingly, the 12-bp core sequence we have identified as essential for FII and R3 sites CTCF DNA recognition is entirely consistent with this CTCF consensus motif; in fact, CTCF-binding sites randomly selected from the genome (22) have sequences with strong homology to those shown in Fig. 1A. This strongly suggests that our results concerning the modality of CTCF DNA recognition and zinc finger usage at the FII and R3 sites can be extended to most of the relevant CTCF DNA sites. Inasmuch as our core site is positioned exactly in the middle of the previously identified consensus, we suggest that it serves as the point of contact by the CTCF protein in vivo. Within that consensus, variations in the core sequence recognized by ZFs 4–7 may in some cases determine whether other ZFs are also necessary for high affinity interaction, although fingers 4–7 seem sufficient for strong interaction with all the sites we examined, comparable with that observed with peptides containing all 11 fingers. Nevertheless, a small yet significant population of in vivo CTCF-binding sites lacks this motif; it is probable that CTCF might have a distinct binding mode at these sites and may use different fingers to recognize these sequences (22). Future experiments are required to identify the ZFs employed by CTCF to recognize these less represented binding sites.

Interestingly, it has been reported that different proteins, including CHD8, Sin3A, and YB-1 (30–32) bind to the zinc finger cluster of CTCF, suggesting that the zinc finger domain mediates not only DNA binding but also protein-protein interactions; on the basis of our results, the zinc finger domains outside the region from ZF 4 to ZF 8 are good candidates to be responsible for these interactions essential for CTCF activity.

Co-crystal structures of several transcription factors with multiple ZFs bound to DNA have helped in understanding the positioning and nature of amino acids responsible for DNA contacts (6, 28); in these polydactyl DNA complexes not all the ZFs that bind DNA behave alike; some are positioned in the major groove to contact base pairs, whereas others traverse the DNA minor groove either making few stabilizing contacts with the DNA phosphodiester backbone or not contacting the nucleic acid at all. Only the resolution of the co-crystal structure of the identified CTCF minimal DNA binding domain with its target site will clarify the contribution of each CTCF ZF to the interaction with DNA.


   FOOTNOTES
 
* This work was supported in part by Grants PRIN 2006 from Ministero dell'Istruzione dell'Università e della Ricerca (MIUR) (to R. F. and P. V. P.), FIRB 2003 from MIUR (to P. V. P.), L. R. N. 5 2003 from Regione Campania (to P. V. P.), and by the Intramural Research Program, NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: NIDDK, National Institutes of Health, Bldg. 5, Rm. 212, Bethesda, MD 20892-0540. Tel.: 301-496-4173; Fax: 301-496-0201; E-mail: gary.felsenfeld{at}nih.gov. 2 To whom correspondence may be addressed. Tel.: 39-0823-274598; Fax: 39-0823-274605; E-mail: paolov.pedone{at}unina2.it.

3 The abbreviations used are: CTCF, CCCTC binding factor; ZF, zinc finger; MBP, maltose-binding protein; PBS, phosphate-buffered saline; GST, glutathione S-transferase; mut, mutant; APP, amyloid beta-protein precursor. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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