SUMOylation of DEC1 Protein Regulates Its Transcriptional Activity and Enhances Its Stability

Differentiated embryo-chondrocyte expressed gene 1 (DEC1, also known as sharp2, stra13, or BHLHB2) is a mammalian basic helix-loop-helix protein that is involved in many aspects of gene regulation through acting as a transcription factor. Changes in DEC1 expression levels have been implicated in the development of cancers. Using COS-7 cell, we showed that DEC1 can be modified by the small ubiquitin-like modifiers, SUMO1, 2 and 3. Two major SUMOylation sites (K159 and K279) were identified in the C-terminal domain of DEC1. Substitution of either K159 or K279 with arginine reduced DEC1 SUMOylation, but substitution of both K159 and K279 abolished SUMOylation, and more protein appeared to be retained in the cytoplasm compared to wild-type DEC1. The expression of DEC1 was up-regulated after serum starvation as previously reported, but at the same time, serum starvation also led to more SUMOylation of DEC1. In MCF-7 cells SUMOylation also stabilized DEC1 through inhibiting its ubiquitination. Moreover, SUMOylation of DEC1 promoted its repression of CLOCK/BMAL1-mediated transcriptional activity through recruitment of histone deacetylase1. These findings suggested that posttranslational modification of DEC1 in the form of SUMOylation may serve as a key factor that regulates the function of DEC1 in vivo

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells

Western blot analyses of CLOCK and ERα expression in cells treated with E2 or ICI.

<p>A, ERα and ERβ expression in MCF-7, T47D, MDA-MB-231, and MCF10A cells. B, CLOCK expression in MCF-7, T47D, MDA-MB-231, and MCF10A cells that had been treated with vehicle (control) or 1 µM E2 for 24 h. Cells were cultured in 5% charcoal striped FCS and phenol red free medium for 2 days before stimulated with E2. C, CLOCK and ERα expression in MCF-7, T47D and MDA-MB-231 cells that had been treated with vehicle, 1 µM E2 or 0.1 µM ICI alone or in combination for 24 h. Cells were cultured for 2 days in 5% charcoal striped FCS and phenol red free medium for two days before they were treated with ER ligands. D, CLOCK and ERα expression in T47D cells transfected with empty vector pcDNA3 or pcDNA3-Flag-ERβ. E, CLOCK and ERα expression in MCF-7 cells transfected with control shCon or two different shERα (shERα#1 and shERα#2). F, CLOCK expression in MCF-7 cells transfected with pcDNA3 or pcDNA3-Flag-ERα. B-F, 24 h after transfection, the cells were harvested and subjected to western blot analysis. In all experiments (A-F), β-actin expression was used as a reference. The blot shown is the representative result from three independent experiments. Image of the blot is shown in the top panel of each figure, with the quantitative analysis of the bands in the blot shown in the plot below. The levels of CLOCK or ERα signal obtained from control cells were set to 1. All experiments were repeated at least three times. Data shown in the graphs are the means ± SDs of three experiments. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05. ns, not significant).</p

ERα ligands regulate the expression of CLOCK at the transcription level.

<p>Analysis of CLOCK mRNA levels in MCF-7 cells by real-time PCR. MCF-7 cells were cultured in phenol red free medium and charcoal striped FCS medium for 2 days before being treated with E2 or ICI and the expression of <i>CLOCK</i> was then analyzed by real-time PCR. Expression of <i>CLOCK</i> was normalized against <i>GAPDH</i> mRNA level (internal control). A, Cells treated with different concentrations of E2 (10⁻¹⁰ to 10⁻⁶ M) for 8 h. B, Cells treated with 1 µM E2 for different periods of time. C, Cells treated with 1 µM E2 or 0.1 µM ICI for 12 h. D, MCF-7 cells transfected with empty vector for ERα (pcDNA3), ERα, shCon (control for shERα) or shERα#1 construct. E, MCF-7 cells were cultured in phenol red-free medium and charcoal-striped FCS medium for 2 days before being treated with E2, Act D or CHX and the expression of <i>CLOCK</i> was then analyzed by real-time PCR. Cells treated with 0.5 µg/ml Act D, 10 µg/ml CHX alone or in combination with 1 µM E2 for 12 h. A-E, Relative levels were calculated by giving an arbitrary value of 1 to the control. <i>CLOCK</i> transcript levels were normalized to <i>GAPDH</i> transcript level and expressed as arbitrary units relative to the vehicle control (set as 1). Each experiment was performed in triplicate and repeated at least three times. Data shown are the means ± SDs. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05. ns, not significant).</p

Proposed model showing the crosstalk between E2-ERα signaling and circadian rhythm.

<p>Proposed model showing the crosstalk between E2-ERα signaling and circadian rhythm.</p

Correlation between ERα and CLOCK expression in human breast tumor tissue samples.

<p>A, Representative results showing the immunohistochemical staining of ERα and CLOCK in serial sections of the breast tumor tissues. Each sample was incubated with antibody against ERα or CLOCK. Positive staining and negative staining are indicated by brown and blue staining, respectively (×200 Magnification). B, Correlation between ERα and CLOCK expression suggested by the 32 breast tumor samples. <i>χ</i>² test was used for statistical analysis. <i>P</i> values less than 0.05 were considered to indicate statistical significance.</p

Modification of DEC1 by SUMO1, 2 and 3.

<p>(A) COS-7 cells were co-transfected with Myc-tagged SUMO1, 2 or 3 and Flag-tagged DEC1, and then subjected to western blot with anti-Flag antibody. (B) MCF-7 cells were co-transfected with Flag-tagged DEC1 and Myc-tagged SUMO1 and then subjected to immunoprecipitation with anti-Flag antibody followed by western blot with anti-Myc antibody. (C) Untransfected MCF-7 cells were subjected to immunoprecipitation with anti-DEC1 antibody or anti-IgG antibody followed by western blot using anti-SUMO1 antibody. (D) MCF-7cells were cultured in the presence of 10% serum for 24 h. The medium was then replaced with fresh medium containing either 10% or 0.8% serum and further incubated for 12 h, and the cells were finally subjected to western blot with anti-DEC1 antibody.</p

Sensitivity of DEC1 SUMOylation to SENP1.

<p>(A) COS-7 cells were transfected with HA-tagged DEC1 together with 3xE-box-luc reporter and either Flag-tagged SENP1, SENP1 mutant (mu), SENP2 or SENP3. Cells were then collected and subjected to luciferase activity assay. (B) COS-7 cells were transfected as in (A) but with increasing amounts of Flag-SENP1 or SENP1 mutant. (C) COS-7 cells transfected as in (B) were subjected to western blot with anti-Flag or anti-HA antibody. **, <i>p</i><0.01</p

Effect of SUMOylation on nucleolar distribution of DEC1 and sensitivity of DEC1 SUMOylation to serum starvation.

<p>(A) MCF-7 cells were transfected with HA-tagged wild-type or mutant (mu) DEC1. Cytosolic and nuclear fractions were subjected to western blot using anti-HA antibody. Fibrillarin and β-actin were measured to monitor the efficiency of nuclear and cytosolic preparations, respectively. The graph shows the intensities of DEC1 bands in the cytosol and nucleus. (B) MCF-7 cells were stained with mouse anti-HA antibody (green) and then counterstained with DAPI (blue) for nucleus detection. MCF-7 cells were transfected with Flag-tagged wild-type DEC1 (C) or HA-tagged DEC1mutant 2K/2R (D) and grown in the presence of 10% FBS before were transferred to a medium containing 0.1% FBS. Cells were collected at the indicated times and subjected to western blot with the corresponding antibody. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

Inhibition of DEC1 ubiquitination by SUMOylation.

<p>(A) MCF-7 cells were transfected with HA-tagged wild-type DEC1 or its mutant 2K/2R and treated with CHX at the indicated time periods. Cell lysate was subjected to western blot with anti-HA antibody. (B) MCF-7 cells were transfected with HA-tagged wild-type DEC1 or 2K/2R together with Myc-tagged ubiquitin. Cell lysate was subjected to IP with anti-HA antibody followed by western blot with anti-Myc antibody. *, <i>p</i><0.05; **, <i>p</i><0.01.</p