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First published online 27 November 2002
doi: 10.1242/jcs.00234

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RNAi reveals anti-apoptotic and transcriptionally repressive activities of DAXX

Jennifer S. Michaelson^* and Philip Leder

Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
^* Present address: Department of Exploratory Science, Biogen Inc., 12 Cambridge Center, Cambridge, Massachusetts 02142, USA

View larger version (73K): [in a new window]   Fig. 1. Treatment with Daxx-specific RNAi species results in depletion of DAXX protein. Western blot analysis of whole-cell lysates prepared from RNAi treated cells. Samples in the upper panels are detected with anti-DAXX antibody, with the arrow indicating the DAXX band; samples in the lower panels are detected with anti-ß-actin antibody as a loading control. Mock transfections were performed with buffer alone. hDx1, hDx2 and mDx2 refer to RNAi species, as described in Materials and Methods. Unless otherwise specified, cell lysates were prepared from HeLa cells, and were collected 72 hours post-transfection. In B, cells were harvested 3 days (3d), 5 days (5d) or 9 days (9d) following RNAi transfection. In D, cells were transfected with mouse Daxx cDNA (+mDaxx) 24 hours following RNAi treatment. Cell lysates were prepared from 293 (E) and U2OS (F) cells following one round (1X) or two rounds (2X) of transfection.

View larger version (15K): [in a new window]   Fig. 2. RNAi-mediated depletion of DAXX results in increased levels of apoptosis. A percent apoptosis is calculated as the percentage of total cells comprising the sub-G1 peak as determined by FACS analysis cell-cycle profiling following propidium iodide staining. Averages and standard deviations were calculated from a minimum of two independent transfection experiments. (A) HeLa cells were fixed and stained 48, 72 or 96 hours post-RNAi treatment. (B) HeLa cells were fixed and stained 72 hours post-RNAi treatment. (C) Western blot analysis of PARP in HeLa cell extracts prepared 96 hours following RNAi treatment. Full-length PARP migrates at 112 kDa; cleaved PARP migrates at 85 kDa.

View larger version (17K): [in a new window]   Fig. 3. Bcl-2 rescues apoptosis in cells depleted of DAXX by RNAi. Mock- or hDx2-transfected HeLa cells were fixed and stained 72 hours post-RNAi treatment. DNA transfections were performed 4 hours after RNAi treatment. Percent apoptosis is calculated as the percentage of total cells comprising the sub-G1 peak as determined by FACS analysis cell-cycle profiling following propidium iodide staining. Averages and standard deviations were calculated from a minimum of two independent transfection experiments. In B, the sub-G1 peak of GFP-positive cells only is calculated.

View larger version (9K): [in a new window]   Fig. 4. Depletion of DAXX by RNAi results in transcriptional de-repression. HeLa cells were transfected with RNAi (or mock transfected), followed 24 hours later by DNA transfection with Met-Luciferase and ß-gal constructs. Luciferase activity was measured after an additional 24 hours. In A, fold-de-repression was calculated by the fold increase of luciferase activity for hDx2-transfected relative to mock-transfected samples, with the mock-treated luciferase being set arbitrarily as 1. In B, DNA transfections in some cases included mouse Daxx cDNA (mDaxx), with the indicated microgram amount per well of six-well plate. All values represent averages of three independent transfections with standard deviation. All experiments were normalized by co-transfection with ß-gal.

View larger version (17K): [in a new window]   Fig. 5. Depletion of DAXX by RNAi induces target-specific transcriptional de-repression. (A) HeLa cells were transfected with hDx2 or mDx2 followed 24 hours later by DNA transfection with reporter plasmids. Fold de-repression was calculated by the fold increase of luciferase activity for hDx2-transfected relative to mDx2-transfected samples, with the mDx2-treated luciferase value for each reporter being set arbitrarily as 1. (B) HeLa cells were transfected with reporter plasmids, with or without mouse Daxx cDNA. Fold repression is calculated by computing the fold difference between cells transfected without DAXX (mock transfected) relative to cells transfected with Daxx. All values represent averages of three independent transfections with standard deviation. All experiments were normalized by co-transfection with ß-gal.

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