Ectopically tethered CP190 induces large-scale chromatin decondensation

Abstract

Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. In this study, we analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. We propose that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF.

Figure 1. Ectopically tethered CP190 induces large-scale chromatin decondensation at the lacO array in mammalian cells.

(a). GFP-LacI-insulator fusion constructs. (b). The F42B8 cell clone of U2OS cells with a LacO repeat cluster integrated close to the centromere, which constitutes a heterochromatic domain, were transiently transfected with GFP-LacI, or GFP-LacI fused to chCTCF, dCTCF or CP190. Cells were fixed and stained with Hoechst stain and observed under fluorescence microscopy. (c). F42B8 cells were transfected with GFP-LacI, GFP-LacI-chCTCF, or GFP-LacI-dCTCF alone or with Gal4-CP190 and analyzed as above. Note that GFP-LacI-dCTCF is able induce decondensation in presence of Gal4-CP190 (indicated by the white arrow). (d). Quantitative analysis of the decondensation of the lacO array (size of the array in μm²) upon targeting of insulator fusion proteins measured by the Volocity software.

Figure 2. GFP-LacI-dCTCF induces decondensation of the lacO array by recruiting Gal4-CP190.

F42B8 cells were transfected as above, with Gal4-CP190 and GFP-LacI, GFP-LacI-chCTCF, or GFP-LacI-dCTCF. Cells were fixed post 48 hours of transfection and analysed by immuno-staining using anti-CP190 antibody (Bx63). As seen in panel 3, only LacI-dCTCF is able to recruit CP190 to the lacO array (shown by green arrows). LacI-CP190 (bottom panel) was used as a positive control for staining.

Figure 3. dCTCF and CP190 result in decondensation of an integrated LacO array in S2 cells.

S2 lacO cells were transfected with GFP-LacI, GFP-LacI-dCTCF and GFP-LacI-CP190. Induction of the metallothionein promoter (pMT vectors) was performed by addition of 500 μM CuSO₄ for 24 hrs. (a). The cells were fixed and stained with Hoechst to visualize the nucleus. The left panels show the GFP tagged proteins and the right show the merge with Hoechst. (b). The size (in μm²) of representative arrays (~50) was measured by the Volocity software and presented as a box plot, whereas whiskers are defined according to Tukey. Significance was controlled with a two tailed Mann-Whitney-test (*** p ≤ 0.001).

Figure 4. Tethering of CP190 does not lead to increased luciferase activity.

HEK 293T cells were transiently transfected with pGL3-luc-7lacO reporter (shown on top) and the indicated LacI fusion constructs. After 24 hours of transfection, the cells were harvested and lysed to measure luciferase activity relative to beta- galactosidase activity. Bars represent fold change luciferase activity of insulator-LacI fusion constructs upon targeting to 7×lacO repeat. Values are the mean standard error of two independent experiments.