Fig 2: Vpr activation of the ATR kinase pathway regulates ZBTB2 localization.U2OS cells were transfected with GFP-ZBTB2 and mCherry-ZASC1 and imaged 24 hours post transfection. Co-expression of WT Vpr (A) resulted in a primarily cytoplasmic ZBTB2 localization which was reversed (B) by treating with 10 μM of ATR inhibitor (CAS 905973-89-9). (C) Co-expression of the cell-cycle arrest-deficient Vpr R80A mutant failed to relocalize ZBTB2 to the nucleus. (D and E) To quantify the effects observed in (A-C), independent transfections were performed with the indicated conditions and the relative ZBTB2 fluorescence signals in the nucleus and a nucleus-adjacent cytoplasmic ring were measured in ≥100 cells and the fold change in nuclear to cytoplasmic signal reported. See Fig 9A and 9B for representative results of cells expressing ZBTB2 alone or in combination with ZASC1. (D) presents the effects of WT Vpr expression and the effect of 10 μM ATR inhibitor, and (E) presents the effects expressing the Vpr R80A cell cycle arrest deficient mutant. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported. Western blots showing the effects of (F) Vpr and ATR inhibitor or (G) WT and R80A Vpr on the levels of ZASC1, ZBTB2 and a HA-tagged CD4 control protein. Numbers indicate band intensity relative to lane expressing both ZASC1 and ZBTB2. Results are representative of three independent experiments.

Fig 3: ZASC1 regulates ZBTB2 nuclear localization.Fluorescent images of U2OS cells transfected with expression vectors encoding the indicated mCherry-fusion proteins (mCherry with a nuclear localization signal, SP1 and ZASC1 variants) and GFP- fusion proteins (firefly luciferase and ZBTB2 variants). Nuclei were stained with DAPI. Scale bar applies to all panels.

Fig 4: ZASC1 and the ATR kinase pathway regulate ZBTB2 localization.U2OS cells were transfected with GFP-ZBTB2 and either mCherry with a nuclear localization signal (A) or mCherry-ZASC1 (B-F) and imaged 24 hours post transfection. ATR kinase was activated by 320 nm UV light (C & D), 8 mM of the DNA damaging agent methyl methanesulfonate (MMS) ATR activation was inhibited by treating with 10 μM of ATR inhibitor (CAS 905973-89-9) (D, F). (G and H) To quantify the effects observed in (A-F), independent transfections were performed with the indicated conditions and the relative ZBTB2 fluorescence signals in the nucleus and a nucleus-adjacent cytoplasmic ring were measured in ≥100 cells, and the fold change in nuclear to cytoplasmic signal reported. (G) presents the effects of DNA damaging UV treatment and ATR inhibitor, and (H) presents the effects of 8 mM of the DNA damaging agent MMS and 10 μM ATR inhibitor. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported. Western blots showing the effects of (I) UV or (J) MMS treatment on the levels of ZASC1, ZBTB2 and a HA-tagged CD4 control protein. Numbers indicate band intensity relative to lane expressing both ZASC1 and ZBTB2. Results are representative of three independent experiments.

Fig 5: ZBTB2 regulates histone acetylation at the HIV-1 promoter.ChIP analysis of (A) histone H3 acetylation for the HIV-1 promoter in WT Jurkat cells infected with an HIV-1 vector with either a WT LTR or an LTR variant with all four ZASC1 binding sites mutated. ChIP analysis of (B) histone H3 acetylation and (C) total histone H3 on the HIV-1 promoter in HIV-1 vector infected WT Jurkat, Jurkat ZASC1 knockout cells and Jurkat ZBTB2 knockout cells. Immunoprecipitation and Real-time PCR analysis were performed and normalized to input controls as a percentage of starting material for immunoprecipitation and fold enrichment of experimental IPs relative to IgG controls reported. For each panel, the data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three or more independent experiments. Error bars indicate the standard deviation of the data in all panels. For (A), P-values were calculated using a standard Student’s t-test and significant changes are indicated, for (B & C) ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported.