Figure 6.

Cellular stress and PLZF-induced L1 regulation. (A) Localization of the PLZF protein by immunofluorescence and western blotting during heat-shock stress in KG1 haematopoietic cells. KG1 cells were submitted to heat shock (20 min at 42°C, 0 h) and then allowed to recover for 1 or 3 h (1 and 3 h). PLZF immunofluorescence was performed as described in Guidez et al (2005) at these time points. PLZF, which is found in nuclear foci (0 h) under normal conditions, is detected in the cytoplasm of stressed cells (1 and 3 h) by both immunofluorescence (1) and western blotting analysis (2). Actin was used as negative control for translocation from the nucleus to the cytoplasm. (B) PLZF interaction with L1 sequences. PLZF DNA association with L1 sequences was evaluated by ChIP following heat-shock treatment. Negative (IgG, lane 5) and positive (anti-histone H3, lane 2) control antibodies were used to assess the specificity of the immunoprecipitation, while acetylation levels (anti-Ac histone H3, lane 4) and PLZF DNA binding (anti-PLZF, lane 3) indicated an increase in histone acetylation and a loss of PLZF DNA binding to L1 sequences under stress conditions. Lane 1 represents 10% of the input DNA used for PCR detection. (C) L1 expression during cellular stress. L1 expression was determined by qPCR relative to GAPDH expression. (D) (1) PLZF expression modulates the L1 retrotransposition frequency. The 293T cells transfected with the L1-EGFP were co-transfected with empty (−) or PLZF^WT, PLZF^ON and PLZF^OFF mutant expression vectors. The percentage of cells expressing EGFP was measured by flow cytometry. Percentages of EGFP-positive cells (% of EGFP+ cells) were scored on day 8 in the absence or presence of PLZF expression followed by heat shock-induced cellular stress (dotted boxes). (2) Effect of L1 retrotransposition during cellular stress. 294T cells, transfected with the L1-EGFP reporter plasmid in the presence of an empty (−) or PLZF expression (PLZF^WT) vectors, were submitted to heat shock (20 min at 42°C) and returned to culture. Percentage of EGFP-positive cells was scored by flow cytometry and retrotransposition frequencies were compared to the control cells (transfected with the empty expression vector under normal cell culture conditions).

Source data for this figure is available on the online supplementary information page.

Source data for Figure 6A1 ^{(1.8MB, doc)}

Source data for Figure 6C2 ^{(525.5KB, doc)}