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. 2018 Jan 2;22(1):44–58. doi: 10.1016/j.celrep.2017.12.037

Figure 2.

Figure 2

ZC3H18 Binding to Histones Is Specific and Phosphorylation Dependent

(A) SDS-PAGE gel of proteins co-precipitating with color-coded ZC3H18, PABPN1, NUP88, and NUP98 proteins expressed with a 3xF tag from HEK293 Flip-In T-Rex cell lines. As a control the HEK293 parental cell line was also used. AC was carried out in three different NaCl buffers (20 mM HEPES [pH 7.4], 0.5% Triton X-100 and 100, 300, or 500 mM NaCl, respectively) as indicated. 3xF-tagged proteins are indicated, based on their expected molecular weights, with color-coded dots and corresponding arrows. Only the ZC3H18-3xF construct co-purified histones in the 500 mM NaCl buffer (indicated by black vertical line in lane 3).

(B) SDS-PAGE gel showing the ZC3H18 isoforms and co-precipitating proteins after a two-step ZC3H18-3xF AC in 100 and 600 mM-NaCl conditions (see main text for details). Migration of ZC3H18-3xF fast- and slow-migrating forms as well as CBCN complex components are indicated as in (A). Note that histones are bound only by the slow-migrating form of ZC3H18-3xF.

(C) SDS-PAGE gel of proteins co-precipitating with ZC3H18-3xF in 100, 300, and 600 mM-NaCl containing buffers supplemented (+) or not (−) with PPI. ZC3H18-3xF isoforms denoted as in (A).

(D) ChIP-PCR analysis of ZC3H18 occupancy along the MYC gene and MYC PROMPT using amplicons from top schematics. IgG ChIP was used to assess background binding. Data are displayed as mean ± SD of two biological replicates. Significance levels between the ZC3H18 and IgG samples were assessed by a two-way ANOVA test with obtained p values presented above the bars.

(E) ChIP-PCR analysis of ZC3H18 occupancy at promoters of transcriptionally active (GAPDH, U12, BRCA1, PSMD3, MRPS15, PALB2, ZNHIT3, KRAS, and SNRPB2) and repressed (IFRG28, MYT1, and KCNA1) genes. IgG ChIP was used to assess background binding. Data are displayed as mean ± SD of two technical replicates of the representative experiment. Significance levels between the ZC3H18 and IgG samples were calculated using a two-way ANOVA test and are denoted with asterisks corresponding to the following p value ranges: ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001.