Figure 4.
Effect of CIZ1 anchor domain on histone posttranslational modifications. (A) Graphs show the frequency of endogenous CIZ1–Xi assemblies in a cycling population of female D3T3 cells, comparing transfected and untransfected cells in the same population. Endogenous CIZ1 assemblies are detected via CIZ1-RD and classified into three categories; present, absent or intermediate. Middle and lower graphs show the frequency of repressive histone marks in cells that are, or are not transfected with GFP-C181. N is replicate analyses with nuclei scored in parentheses. Comparisons are by t test. For endogenous CIZ1 in UT and C181 cells P = 0.00023, for H3K27me3 P = 0.60, for H2AK119ub1 P = 0.0073. Error bars show SEM. (B) As in A, except that all data is derived from analysis of female primary embryonic fibroblasts (PEFs) at passages 2–3. For endogenous CIZ1 in UT and C181 cells P = 0.00033 for H3K27me3 P = 0.79, for H2AK119ub1 P = 0.016, performed on present (type 1) categories. Also shown is the effect of 5 μM PR619 on H2AK119ub1 loss, where P = 0.0099 for the no CIZ1 category (type 3). Error bars show SEM. (C) Example images of endogenous CIZ1 and histone marks (red) in untransfected (UT) and C181 transfected (green) WT PEF populations. The bar is 10 μm. (D) Lentivirus encoding C181 and/or ZSGreen was used to infect three independent populations of WT murine primary embryonic fibroblasts (PEFs) at passage 1–2. Below, expression was verified by western blot of ectopic CIZ1 (exon 17) and beta-actin in whole cell lysates over 3 days, compared to untreated control populations (UT) at days 1 and 3. Below right, live cell images of ZsGreen and brightfield images of PEFs at day 2 after transduction. Bar is 50 μm. (E) Comparison of vector-only populations to those transduced with C181 showing the frequency of cells with CIZ1–Xi assemblies (gray), H3K27me3 (red) or H2AK119ub1 (blue). n denotes replicate analyses with total nuclei inspected in parentheses. PEF cell populations are in gray. Comparisons are by unpaired t test where P < 0.001 in all cases. Error bars show SEM. Below are box and whisker plots showing mean nuclear intensity measures for cells transduced with C181 or vector control, normalized to the mean of vector-only control cells. Source data are available for this figure: SourceData F4.

Effect of CIZ1 anchor domain on histone posttranslational modifications. (A) Graphs show the frequency of endogenous CIZ1–Xi assemblies in a cycling population of female D3T3 cells, comparing transfected and untransfected cells in the same population. Endogenous CIZ1 assemblies are detected via CIZ1-RD and classified into three categories; present, absent or intermediate. Middle and lower graphs show the frequency of repressive histone marks in cells that are, or are not transfected with GFP-C181. N is replicate analyses with nuclei scored in parentheses. Comparisons are by t test. For endogenous CIZ1 in UT and C181 cells P = 0.00023, for H3K27me3 P = 0.60, for H2AK119ub1 P = 0.0073. Error bars show SEM. (B) As in A, except that all data is derived from analysis of female primary embryonic fibroblasts (PEFs) at passages 2–3. For endogenous CIZ1 in UT and C181 cells P = 0.00033 for H3K27me3 P = 0.79, for H2AK119ub1 P = 0.016, performed on present (type 1) categories. Also shown is the effect of 5 μM PR619 on H2AK119ub1 loss, where P = 0.0099 for the no CIZ1 category (type 3). Error bars show SEM. (C) Example images of endogenous CIZ1 and histone marks (red) in untransfected (UT) and C181 transfected (green) WT PEF populations. The bar is 10 μm. (D) Lentivirus encoding C181 and/or ZSGreen was used to infect three independent populations of WT murine primary embryonic fibroblasts (PEFs) at passage 1–2. Below, expression was verified by western blot of ectopic CIZ1 (exon 17) and beta-actin in whole cell lysates over 3 days, compared to untreated control populations (UT) at days 1 and 3. Below right, live cell images of ZsGreen and brightfield images of PEFs at day 2 after transduction. Bar is 50 μm. (E) Comparison of vector-only populations to those transduced with C181 showing the frequency of cells with CIZ1–Xi assemblies (gray), H3K27me3 (red) or H2AK119ub1 (blue). n denotes replicate analyses with total nuclei inspected in parentheses. PEF cell populations are in gray. Comparisons are by unpaired t test where P < 0.001 in all cases. Error bars show SEM. Below are box and whisker plots showing mean nuclear intensity measures for cells transduced with C181 or vector control, normalized to the mean of vector-only control cells. Source data are available for this figure: SourceData F4.

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