Distinct Cytoplasmic and Nuclear Functions of the Stress Induced Protein DDIT3/CHOP/GADD153

DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders

DDIT3 induced cell cycle arrest.

<p>Flow cytometry analysis of DDIT3morEGFP expressing HT1080 cells cultured with and without tamoxifen for 12 hours. Cells with nuclear DDIT3 were accumulated in G1-phase and depleted from the S- and G2-phases. Numbers of diploid cells in G1-, G2- and M-phase are shown in black and S-phase cells are shown as striped. Aneuploid cells are shown in the white overlaying histogram.</p

Subcellular localization of DDIT3.

<p>(A) Immunoblot analyses of nuclear (Nu) and cytoplasmic (Cy) extracts of human liposarcoma cell line GOT3 and normal human fibroblasts F470 following 8 hours of tunicamycin (2 µg/ml,Tunica) treatment. Cytoplasmic accumulation of DDIT3 is seen in both cell lines compared to untreated cells (Control). GAPDH and Lamin A are cytoplasmic and nuclear markers, respectively. (B) Confocal microscopy (upper panel) of HT1080 cells containing DDIT3morEGFP before and 1 hour after addition of tamoxifen (100 nM). A translocation of the EGFP tagged recombinant DDIT3 protein from the cytoplasm to the nucleus can be seen after the addition of tamoxifen (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033208#pone.0033208.s012" target="_blank">Movie S1</a>). Immunoblot analyses (lower panel) of nuclear and cytoplasmic extracts. GAPDH and H1A were used as cytoplasmic and nuclear markers, respectively.</p

Genes regulated by cytoplasmic and nuclear DDIT3.

<p>(A) Genes regulated 2 and 8 hours after nuclear translocation of DDIT3 in DDIT3morGFP expressing HT1080 cells. (B) Genes regulated both by cytoplasmic DDIT3 and nuclear DDIT3. White bars represent regulation by cytoplasmic DDIT3. Grey and black bars represent the regulation of nuclear DDIT3 compared to levels in cells with cytoplasmic DDIT3 at 2 and 8 hours after tamoxifen addition, respectively.</p