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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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CTCF-induced decondensation and demethylation of the array occur very rapidly. F42B8 cells were transfected with GFP-LacI-CTCF in the presence of 150 μM IPTG and incubated for 12 h. IPTG was removed from the medium for indicated time periods (A–D) followed by treatment with 4% PFA. Immunostaining was performed with an antibody against H3K27me3 and the nucleus was stained with Hoechst. In (A) an untransfected cell is shown. Arrows point at positive signals, whereas circles indicate the lack of a signal. (E) The size of ∼50 arrays was measured using the Volocity software and shown as a box plot. (F) The H3K27me3 value was detected as the ratio of signal intensity of the array to signal intensity of the whole nucleus defined by Hoechst staining. (G) The data sets of the size and the signal intensity are plotted as arbitrary units, whereas the mean values are set as 0 (lowest) and 1 (highest), respectively.
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Figure 6: CTCF-induced decondensation and demethylation of the array occur very rapidly. F42B8 cells were transfected with GFP-LacI-CTCF in the presence of 150 μM IPTG and incubated for 12 h. IPTG was removed from the medium for indicated time periods (A–D) followed by treatment with 4% PFA. Immunostaining was performed with an antibody against H3K27me3 and the nucleus was stained with Hoechst. In (A) an untransfected cell is shown. Arrows point at positive signals, whereas circles indicate the lack of a signal. (E) The size of ∼50 arrays was measured using the Volocity software and shown as a box plot. (F) The H3K27me3 value was detected as the ratio of signal intensity of the array to signal intensity of the whole nucleus defined by Hoechst staining. (G) The data sets of the size and the signal intensity are plotted as arbitrary units, whereas the mean values are set as 0 (lowest) and 1 (highest), respectively.

Mentions: To determine the sequence of the molecular events of H3K27me3 removal and of chromatin domain opening we needed to determine the time required to establish these changes. We could not simply use different time points after transfection to do so, since expression of the fusion proteins takes time and has to be distinguished from the time after DNA binding. Therefore, again, we made use of the IPTG substance to interfere with DNA binding of the GFP-LacI-CTCF fusion. Removal of IPTG allows for DNA binding and, therefore, the time after DNA-binding required to induce the molecular changes at the chromatin array can be determined. The size of the LacO array did not change significantly immediately after IPTG removal and for up to 2 h of GFP-LacI-CTCF binding (Figure 6B an E). However, at 4 h the array size started to increase and continued to do so up to 40 h (Figure 6C–E). Longer incubations did not further increase the size of the array (not shown), and as such the major changes in domain opening are observed between 4 and 40 h of LacO binding of CTCF.


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)

CTCF-induced decondensation and demethylation of the array occur very rapidly. F42B8 cells were transfected with GFP-LacI-CTCF in the presence of 150 μM IPTG and incubated for 12 h. IPTG was removed from the medium for indicated time periods (A–D) followed by treatment with 4% PFA. Immunostaining was performed with an antibody against H3K27me3 and the nucleus was stained with Hoechst. In (A) an untransfected cell is shown. Arrows point at positive signals, whereas circles indicate the lack of a signal. (E) The size of ∼50 arrays was measured using the Volocity software and shown as a box plot. (F) The H3K27me3 value was detected as the ratio of signal intensity of the array to signal intensity of the whole nucleus defined by Hoechst staining. (G) The data sets of the size and the signal intensity are plotted as arbitrary units, whereas the mean values are set as 0 (lowest) and 1 (highest), respectively.
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Figure 6: CTCF-induced decondensation and demethylation of the array occur very rapidly. F42B8 cells were transfected with GFP-LacI-CTCF in the presence of 150 μM IPTG and incubated for 12 h. IPTG was removed from the medium for indicated time periods (A–D) followed by treatment with 4% PFA. Immunostaining was performed with an antibody against H3K27me3 and the nucleus was stained with Hoechst. In (A) an untransfected cell is shown. Arrows point at positive signals, whereas circles indicate the lack of a signal. (E) The size of ∼50 arrays was measured using the Volocity software and shown as a box plot. (F) The H3K27me3 value was detected as the ratio of signal intensity of the array to signal intensity of the whole nucleus defined by Hoechst staining. (G) The data sets of the size and the signal intensity are plotted as arbitrary units, whereas the mean values are set as 0 (lowest) and 1 (highest), respectively.
Mentions: To determine the sequence of the molecular events of H3K27me3 removal and of chromatin domain opening we needed to determine the time required to establish these changes. We could not simply use different time points after transfection to do so, since expression of the fusion proteins takes time and has to be distinguished from the time after DNA binding. Therefore, again, we made use of the IPTG substance to interfere with DNA binding of the GFP-LacI-CTCF fusion. Removal of IPTG allows for DNA binding and, therefore, the time after DNA-binding required to induce the molecular changes at the chromatin array can be determined. The size of the LacO array did not change significantly immediately after IPTG removal and for up to 2 h of GFP-LacI-CTCF binding (Figure 6B an E). However, at 4 h the array size started to increase and continued to do so up to 40 h (Figure 6C–E). Longer incubations did not further increase the size of the array (not shown), and as such the major changes in domain opening are observed between 4 and 40 h of LacO binding of CTCF.

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