Article Figures & Data
Figures
- FIG. 1.
CTCF binding sites map within KvDMR1. (a) Imprinted domain in mouse distal chromosome 7. Imprinted genes are shown as white or gray (imprinted expression only in placenta) boxes, while black boxes represent nonimprinted genes. The arrows indicate the direction of transcription. (b) Restriction map of the mouse KvDMR1 locus with the nucleotide positions indicated as in AF119385; the thick bars above the map are the two CpG islands within KvDMR1. The thick bars below the map indicate the genomic region deleted in mice described by Fitzpatrick et al. (11) (black) and the fragment tested for enhancer blocking activity by Kanduri et al. (17) (gray). The lines with double arrowheads above the restriction map show the location of the regions analyzed in the CTCF ChIP assay (see Fig. 2a). The positions of 11 overlapping DNA fragments used as probes in an EMSA are shown as black lines below the map of the locus; the numbers refer to the names (e.g., mKD1) of forward and reverse PCR primers used to generate each EMSA probe. The asterisks indicate the positions of two CTCF binding sites (CTS) detected by EMSA and fine-mapped by methylation interference (see below). (c) EMSA analysis of CTCF binding to mouse KvDMR1 locus. DNA fragments shown in panel b were screened for binding to in vitro translated full-length CTCF (ivtFLCTCF) and 11-ZF domain of this protein. A DNA fragment (H19 DMD4) from the H19 ICR which is known to bind CTCF was used as a positive control for CTCF-binding. Only probes 1-2 and 7-8 were able to form DNA-protein complexes with full-length CTCF in these experiments. (d) Probes 1-2 (CTS1) and 7-8 (CTS2) form a complex with CTCF from HeLa cell nuclear extract. In the last lane, the complex between the extract and probes for CTS1 and CTS2 is supershifted with mouse monoclonal-CTCF antibody (36). The panels labeled “mutant” show gel shift experiments with the in vitro translated protein and nuclear extract demonstrating the effect of mutations introduced in CTS1 and CTS2 (see panel g) on the ability of these sites to bind CTCF. (e) SssI methyltransferase was used to in vitro methylate cytosine residues in CpG dinucleotides in the CTCF-positive DNA fragments (CTS1 [1-2] and CTS2 [7-8]). Prior methylation completely abrogated binding of CTCF at CTS2 and almost completely at CTS1. Digestion of unmethylated or SssI-methylated probe with methylation-sensitive enzyme BstUI demonstrates that probes were completely methylated following SssI treatment (+BstUI). (f) Methylation interference analysis of CTS1 and CTS2 sequences. Lanes F, free DNA probes separated from the CTCF-bound probes (lanes B). The nucleotides in contact with CTCF are shown as black dots. (g) CTS1 and CTS2 were mutagenized in the context of the 1-10 fragment. Most guanine nucleotides that are in direct contact with CTCF were replaced by adenosines or thymidines (shown in italics above and below the original sequence). (h) Sequence alignment of mouse CTS1 and CTS2 with sequences within fragments (KvDMR-HBi and KvDMR-FBi) previously shown to bind in vitro transcribed/translated CTCF (7). Identical nucleotides are shaded. Larger, bold nucleotides represent G (C when on opposite strand) residues in contact with CTCF in vitro as determined by methylation interference (above).
- FIG. 2.
CTCF binds to the unmethylated paternal allele of KvDMR1 in vivo. (a) Chromatin immunoprecipitated from C57BL/6J × SD7 F1 MEFs with monoclonal CTCF antibody was analyzed by real-time PCR using PCR primers flanking CTS1 or primers amplifying two control regions (1.2 kb upstream or 1.6 kb downstream of CTS1, as shown in Fig. 1b). (b) The same CTCF ChIP material and that prepared from SD7 × C57BL/6J F1 MEFs was amplified using primers that flank a single-nucleotide polymorphism (SNP) between C57BL/6J and SD7 mice and directly sequenced. While both A and G alleles are present in input chromatin, only the paternal allele (G in the C57 × SD7 cross and A in the SD7 × C57 cross) was detected in the immunoprecipitated chromatin. (c) Real-time PCR analysis of chromatin immunoprecipitated from PLFs derived from mice heterozygous for a deletion of KvDMR1 (11). ChIP was carried out using a monoclonal CTCF antibody or polyclonal antibody against dimethylated lysine 9 of histone 3. A region from Myc promoter (12) served as a positive control for CTCF binding. Enrichment of CTCF-bound chromatin at KvDMR1 was much less in PLFs from mice with a maternal deletion than in PLFs from wild-type mice (lower and upper histograms, respectively). Since the enrichment of CTCF was also drastically reduced at the internal control Myc locus in the same cells, it is likely that this reduction is due to loss of chromatin during the washing of protein A/G agarose beads or the recovery of the immunoprecipitated DNA. Ab, antibody.
- FIG. 3.
Mapping of KvDMR1 repressive activity in an enhancer-blocking assay. (a) The extent of DNA fragments tested for different functional activities are shown as black lines with the name of each fragment indicated. For comparison, the genomic region deleted in mice described in Fitzpatrick et al. (11) is shown as a black bar. The light gray bars are regions of the locus tested for insulator/silencer activity by others (27, 43). (b) Schematic representation of the E-p-neo basic vector used for generation of the experimental constructs (see text for details; SCS is a Drosophila insulator element [SO]). (c) The test-fragments indicated in panel a were inserted in the SalI/ClaI (“in” position) site in the indicated orientations with respect to the endogenous locus, stably transfected into Jurkat cells, and plated on soft agar; neo-resistant colonies were counted after 3 to 4 weeks. Enhancer-blocking activity was assessed as the number of the neo-resistant colonies for a given construct relative to the number of colonies formed with the E-p-neo construct (taken as 100%). Each transfection was done in triplicate. Individual constructs were transfected in two to three independent experiments.
- FIG. 4.
A transcriptional promoter within KvDMR1 can be uncoupled from enhancer-blocking activity. (a) Restriction map of KvDMR1 locus. The arrow above the map points toward direction of Kcnq1ot1 transcription. The sequence of the locus harboring promoter activity is shown below the restriction map with arrows facing down indicating the 5′ ends of individual fragments tested for promoter activity; all these fragment have the same 3′ end specified by an arrow pointing up. The broken arrows show transcription start sites as determined by RACE analysis. The heavy broken arrow shows the position of the major transcription start site (see text). The bolded and italicized sequence represents the overlap between the fragments tested in the promoter assay and those representing the minimal repressive element. (b) Promoter activity of different fragments from KvDMR1 locus (positions of the fragments are shown in Fig. 3a) was evaluated by a luciferase reporter assay. All test fragments were cloned into pGL3-Basic vector upstream of a luciferase gene. The luciferase activity is shown as the increase in activation relative to the activity from the vector alone. All constructs were transiently transfected into Jurkat cells in triplicate. (c) Enhancer-blocking activity of the fragment containing the full promoter sequence (pGL/400) was measured in enhancer-blocking assay as described in the legend of Fig. 3c.
- FIG. 5.
An enhancer-like element located 5′ of the KvDMR1 CpG island. (a) Restriction map of KvDMR1 region as shown in Fig. 1b. The gray bars below the sequence represent the fragments tested for enhancer activity; for comparison, fragments with repressive activity (1-10 and 1-16) are shown in black. A summary of the different functional elements defined in this study is presented in the lower panel. (b) Fragments shown in panel were inserted in pGL3-promoter vector upstream (SmaI site) or downstream (SalI site) of the luciferase reporter. Luciferase activity of each test construct is shown as the increase in activation relative to the activity of the vector alone. Because of the high background level obtained with the vector alone in Jurkat cells, the experiment was carried out in HeLa cells. Each transfection was done in triplicate.
