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1.
Figure 4.

Figure 4. From: Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers.

The D4Z4-adjacent p13E-11 marker sequence is more accessible to DNaseI than untranscribed gene standards and much more accessible than D4Z4 in myoblast nuclei. The size-normalized (Supplementary Table 2) percentage of DNA remaining in the parent StyI restriction fragment is shown for each examined DNA sequence in DNaseI-treated nuclei from control fetal myoblasts (A) and FSHD myoblasts (B).

Koji Tsumagari, et al. Nucleic Acids Res. 2008 April;36(7):2196-2207.
2.
Figure 1.

Figure 1. From: Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers.

Maps of the studied D4Z4 and proximal regions. (A) Cartoon (not to scale) showing the size-polymorphic D4Z4 array and p13E-11, a marker sequence. The sequence between the D4Z4 array and the telomeric hexamer repeats is about 15–25 kb. Black or gray trapezoids, essentially identical, tandem D4Z4 repeat units (3.3-kb KpnI fragments). White trapezoid, the 0.9-kb region from the start of the array to the first KpnI site in D4Z4. (B) Maps of StyI sites for chromatin DNaseI sensitivity assays. Left, StyI restriction map of the proximal border of D4Z4 including the StyI fragment spanning the 0.8-kb p13E-11 sequence through 0.3 kb at the beginning of D4Z4. Right, StyI restriction map comparing 4q35 and 10q26 D4Z4 repeat units.

Koji Tsumagari, et al. Nucleic Acids Res. 2008 April;36(7):2196-2207.
3.
Figure 6.

Figure 6. From: Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers.

Hypothesized intrachromosomal communication by chromatin looping. (A) A disease-associated long-distance loop is hypothesized to form when the D4Z4 repeat array is contracted to less than 11 repeat units, leading to inappropriate expression in cis of an unelucidated 4q35 FSHD master gene (thick brown line). These abnormal looping interactions might upregulate transcription of the FSHD master gene in cis by direct chromatin-to-chromatin contact and/or alteration of the chromatin protein composition of the promoter of the FSHD master gene. The hypothesized long-distance looping may include interactions with the sequences distal to D4Z4, which are shown as a thick light blue line, ∼15–25 kb ending at the telomeric TTAGGG repeats (5,52). (B) Intra-array looping for arrays with more than 10 repeat units would sequester the proximal end of D4Z4 so that it would not be available for pathogenic long-distance looping. Size-dependent, topological constraints on intra-array looping could explain why the disease severity is often lower when the 4q35 array is in the 8–10 unit range relative to the 1–4 unit range. Looping interactions of D4Z4 sequences might involve G-quadruplexes.

Koji Tsumagari, et al. Nucleic Acids Res. 2008 April;36(7):2196-2207.
4.
Figure 2.

Figure 2. From: Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers.

Cancer-linked hyper- or hypomethylation in the bulk of the D4Z4 array, at the very beginning of the array, and immediately proximal to the array in representative samples. The immediately proximal region includes the 0.8-kb p13E-11 sequence, which is 0.1 kb from the beginning of the D4Z4 array. The squares denote analyzed CpG methylation-sensitive restriction sites. The most proximal HpyCH4IV and BsaAI sites in the first repeat unit of the array are shown followed by a long rectangle representing the foreshortened remainder of the array. The height of the black fill-in is approximately proportional to the average extent of methylation. Methylation in the bulk of the D4Z4 array is diagrammed for HpyCH4IV sites because the small number (six) of HpyCH4IV sites per 3.3-kb repeat unit facilitated quantitation. Other CpG-containing restriction sites were more heavily methylated in somatic controls but gave hypo- or hypermethylation in cancers similar to that seen at HpyCH4IV sites. The gray vertical bars in the D4Z4 array in cancer samples represent the subregion of the D4Z4 repeats units that is partially resistant to hypermethylation and located about 1.4 kb distal to the KpnI site (Figure 3A). See Supplementary Figures 1–3 for most of the data used in this figure.

Koji Tsumagari, et al. Nucleic Acids Res. 2008 April;36(7):2196-2207.
5.
Figure 3.

Figure 3. From: Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers.

Potential G-quadruplex sequences (PQS) in D4Z4. (A) Maps of the 3.3-kb D4Z4 repeat unit showing positions of PQS, BstUI and HpaII sites, and the alignment of the unexpectedly strong 1.4-kb band that was observed in BstUI/KpnI and HpaII/KpnI digests (Supplementary Figure 2). Diamonds, PQS; large for G5 and small for G3 runs; upward for those on the forward strand and downward for those on the complementary strand. hhspm3, a sequence that can function as a promoter; DUX4, an open reading frame without a polyadenylation signal (15,16). (B) The D4Z4 subsequence (AF117653) that contains PQS5 and PQS6. Potential unimolecular G-quadruplex structures (blue or red) are shown but there are variations possible in the exact placement of the four runs of G in this subregion that could be involved in unimolecular quadruplexes. In addition, non-adjacent compatible runs of G might participate in quadruplex formation like that seen in bi- and tetramolecular G-quadruplexes. Purple, the closest BstUI and HpaII sites to PQS5; green, mutated residues in PQS6-mut. (C) CD analysis of PQS5, PQS6 and PQS6-mut as single-stranded 23- to 32-nt oligonucleotides. (D) Illustration of one possible orientation for G residues to stack and form a hairpin-type intramolecular G-quadruplex. The numbering corresponds to PQS6 in Panel B, but various other configurations are possible that preserve the planar stacking of four G residues connected by Hoogsteen pairing (dotted lines).

Koji Tsumagari, et al. Nucleic Acids Res. 2008 April;36(7):2196-2207.
6.
Figure 5.

Figure 5. From: Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers.

D4Z4 chromatin is quite resistant and the adjacent p13E-11 chromatin is very sensitive to DNaseI in all studied FSHD and control cell populations. Nuclei (FSHD myoblasts, control fetal myoblasts, one control fibroblast cell strain) or permeabilized cells (LCLs) were treated with four or five different concentrations of DNaseI. The results are given for only one concentration of DNaseI (20, 50 or 100 U/ml) but these are representative of the results at each concentration (e.g. Figure 4) and all differences in sensitivity between different types of DNA sequences were highly significant (P < 10−4, see text). The analyzed DNA sequences for each cell type are as follows, from left to right in each bar graph: two untranscribed gene standards (Untr, CST5 and GHRHR), p13E-11 (11; bar denoted by a vertical arrow), D4Z4 (D4, 4q-type and 10q-type arrays), constitutively transcribed gene standards (Tr, B2M and HMBS), and the constitutive heterochromatin standard (Sat2). After size normalization, the percentage of DNA remaining in the parent band for each sequence was divided by that for the average of the untranscribed standards. In parentheses is the number of copies of the D4Z4 repeat unit for the allelic 4q-type arrays (no BlnI site and a single StyI site per 3.3 kb) and the allelic 10q-type arrays (one BlnI site and two StyI sites per 3.3 kb) (51). The control myoblasts and the fibroblasts had one of the 4q D4Z4 arrays replaced by a canonical 10q-type array, as is seen in some individuals, but this has no effect on phenotype (51).

Koji Tsumagari, et al. Nucleic Acids Res. 2008 April;36(7):2196-2207.

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