Fig. 4: The effects of heat treatment and hybridization on denaturing cross-linked chromatin and decross-linking.

a Analysis of the decross-linking of chromatin after exposure to heat treatment. Two methods for crosslinking chromatin were used, including 3% (w/v) formaldehyde and 20 mg L⁻¹ NiSO₄ (Con), and 3% (w/v) formaldehyde and 20 mg L⁻¹ NiSO₄ combined with ultraviolet radiation treatment (UV). The cross-linked chromatin was incubated in LS buffer [with 10% (v/v) formamide, 0.5% (w/v) SDS] at 90 °C for different times, and the decross-linked DNA was quantified using qPCR. Increased Ct value variation indicates increased decross-linking of DNA and protein. b Determination of the time required for denaturing the cross-linked chromatin. After sonication, the sonicated cross-linked chromatin was added with SYBR Green and incubated at 90 °C. The denatured chromatin was monitored at 5 s intervals by visualization with SYBR Green fluorescence using a real-time PCR instrument. Five replicates were performed (n = 5 independent experiments). The reduction of fluorescence reflected the denaturation of chromatin. c Analysis of the decross-linking of chromatin during hybridization. The chromatin cross-linked with 3% (w/v) formaldehyde combined with NiSO₄ (Con), and the same cross-linked chromatin further cross-linked with ultraviolet radiation (UV) before sonication were used for analysis. These two kinds of cross-linked chromatin were incubated in LS buffer [with 10% (v/v) formamide, 0.5% (w/v) SDS] at 37 °C for 12 h, and the decross-linked DNA in different time points were harvested by Tris-phenol and chloroform extraction, and quantified using qPCR. The y-axis indicates the CT value variation (i.e., ΔCt), ΔCT = Ct decross-linked DNA in the control at 0 h [cross-linked with 3% (w/v) formaldehyde together with 20 mg L⁻¹ NiSO₄]− Ct decross-linked DNA of samples [including chromatins crosslinked with 3% (w/v) formaldehyde combined with NiSO₄ and 3% (w/v) formaldehyde combined with NiSO₄ and ultraviolet radiation] at different time points. Therefore, ΔCT > 0 indicates increased decross-linking of chromatin. ΔCT < 0 means decreased decross-linking of chromatin. The amount decross-linked chromatin in the sample cross-linked with formaldehyde together with ultraviolet radiation was less than that in the control; therefore, its ΔCt value was negative. Three replicates (sample size of 50 seedlings) were performed (n = 3 independent experiments). Error bar indicates standard deviations of the mean measurements. Asterisks indicate significant difference between Con and UV treatment (*P < 0.05, ***P < 0.001, t-test). The promoter region of AtCAT3 used for the qPCR evaluation was site 1 and is shown in Fig. 7l and Supplementary Data 4.