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CTCF induces histone variant incorporation, erases the H3K27me3 histone mark and opens chromatin.

Weth O, Paprotka C, Günther K, Schulte A, Baierl M, Leers J, Galjart N, Renkawitz R - Nucleic Acids Res. (2014)

Bottom Line: The insulator factor, CTCF, has been found to bind to boundaries and to mediate insulator function.This demonstrates the causal role for CTCF in generating the chromatin features found at insulators.Thereby, spreading of a histone modification from one domain through the insulator into the neighbouring domain is inhibited.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Justus-Liebig-University, 35392 Giessen, Germany.

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The LacO array of U2OS-F42B8 is positive for H3K27me3. F42B8 cells were transfected with GFP-LacI and incubated for 48 h. Fixed cells were treated with antibodies against (A) H3K9ac, (B) H4ac and (C) H3K27me3. The left panels show the GFP-tagged LacI, the middle show staining of indicated histone modification by indirect immunofluorescence, the third show the merge. Arrows point at positive signals, whereas circles represent lack of a signal.
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Figure 2: The LacO array of U2OS-F42B8 is positive for H3K27me3. F42B8 cells were transfected with GFP-LacI and incubated for 48 h. Fixed cells were treated with antibodies against (A) H3K9ac, (B) H4ac and (C) H3K27me3. The left panels show the GFP-tagged LacI, the middle show staining of indicated histone modification by indirect immunofluorescence, the third show the merge. Arrows point at positive signals, whereas circles represent lack of a signal.

Mentions: Using a LacI fusion with the green fluorescent protein (GFP), the location of the repeat array could easily be determined at the cytological level. Using antibodies directed against active (euchromatic) histone modifications, such as H3K9ac, H4ac, no signal of the GFP marked array was detectable after immunostaining (Figure 2A and B). In contrast, antibodies against the repressive (heterochromatic) mark H3K27me3 easily identified the GFP marked spot (Figure 2C). To analyse the effect of CTCF on this chromatin domain, we generated a GFP-LacI-CTCF expression vector. Also, to increase the combinatorial use of expression vectors, we generated similar fusions with the mCherry-coding region replacing GFP. Expression of Cherry-LacI clearly targets the fusion protein to a localized structure within the nucleus (Figure 3A). Targeting was sensitive to treatment with IPTG (Supplementary Figure S4), a substance known to interfere with DNA binding of the LacI repressor. Expression of Cherry-LacI-CTCF caused an increase in the labelled nuclear structure (Figure 3). Cherry-LacI co-localizes with GFP-LacI-CTCF (Supplementary Figure S5), allowing the conclusion that the structure seen in Figure 3 is the LacO array, which is strongly enlarged when compared to the Cherry-LacI or GFP-LacI bound array. We quantified the LacI-CTCF induced expansion of the array and found that in more than 80% of the transfected cells such an array enlargement could be observed (Supplementary Figure S6). In order to verify whether this expansion is caused by chromatin opening of the heterochromatic array we carried out a FAIRE assay (32). Expression of GFP-LacI-CTCF causes a 3-fold increase in solubilized array DNA as compared to GFP-LacI expression. A CTS negative control site in the genome did not respond to CTCF expression (Figure 3D). In order to quantify the chromatin opening activity we measured the size of the array within the micrographs using the Volocity software. We measured a 10- to 20-fold increase of the 2D area, when expressing LacI-CTCF (Figure 3E and F). Thus, we conclude that in contrast to LacI, the LacI-CTCF factor is actively opening a heterochromatic region inserted in the genome.


CTCF induces histone variant incorporation, erases the H3K27me3 histone mark and opens chromatin.

Weth O, Paprotka C, Günther K, Schulte A, Baierl M, Leers J, Galjart N, Renkawitz R - Nucleic Acids Res. (2014)

The LacO array of U2OS-F42B8 is positive for H3K27me3. F42B8 cells were transfected with GFP-LacI and incubated for 48 h. Fixed cells were treated with antibodies against (A) H3K9ac, (B) H4ac and (C) H3K27me3. The left panels show the GFP-tagged LacI, the middle show staining of indicated histone modification by indirect immunofluorescence, the third show the merge. Arrows point at positive signals, whereas circles represent lack of a signal.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231773&req=5

Figure 2: The LacO array of U2OS-F42B8 is positive for H3K27me3. F42B8 cells were transfected with GFP-LacI and incubated for 48 h. Fixed cells were treated with antibodies against (A) H3K9ac, (B) H4ac and (C) H3K27me3. The left panels show the GFP-tagged LacI, the middle show staining of indicated histone modification by indirect immunofluorescence, the third show the merge. Arrows point at positive signals, whereas circles represent lack of a signal.
Mentions: Using a LacI fusion with the green fluorescent protein (GFP), the location of the repeat array could easily be determined at the cytological level. Using antibodies directed against active (euchromatic) histone modifications, such as H3K9ac, H4ac, no signal of the GFP marked array was detectable after immunostaining (Figure 2A and B). In contrast, antibodies against the repressive (heterochromatic) mark H3K27me3 easily identified the GFP marked spot (Figure 2C). To analyse the effect of CTCF on this chromatin domain, we generated a GFP-LacI-CTCF expression vector. Also, to increase the combinatorial use of expression vectors, we generated similar fusions with the mCherry-coding region replacing GFP. Expression of Cherry-LacI clearly targets the fusion protein to a localized structure within the nucleus (Figure 3A). Targeting was sensitive to treatment with IPTG (Supplementary Figure S4), a substance known to interfere with DNA binding of the LacI repressor. Expression of Cherry-LacI-CTCF caused an increase in the labelled nuclear structure (Figure 3). Cherry-LacI co-localizes with GFP-LacI-CTCF (Supplementary Figure S5), allowing the conclusion that the structure seen in Figure 3 is the LacO array, which is strongly enlarged when compared to the Cherry-LacI or GFP-LacI bound array. We quantified the LacI-CTCF induced expansion of the array and found that in more than 80% of the transfected cells such an array enlargement could be observed (Supplementary Figure S6). In order to verify whether this expansion is caused by chromatin opening of the heterochromatic array we carried out a FAIRE assay (32). Expression of GFP-LacI-CTCF causes a 3-fold increase in solubilized array DNA as compared to GFP-LacI expression. A CTS negative control site in the genome did not respond to CTCF expression (Figure 3D). In order to quantify the chromatin opening activity we measured the size of the array within the micrographs using the Volocity software. We measured a 10- to 20-fold increase of the 2D area, when expressing LacI-CTCF (Figure 3E and F). Thus, we conclude that in contrast to LacI, the LacI-CTCF factor is actively opening a heterochromatic region inserted in the genome.

Bottom Line: The insulator factor, CTCF, has been found to bind to boundaries and to mediate insulator function.This demonstrates the causal role for CTCF in generating the chromatin features found at insulators.Thereby, spreading of a histone modification from one domain through the insulator into the neighbouring domain is inhibited.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Justus-Liebig-University, 35392 Giessen, Germany.

Show MeSH