6074 Nucleic Acids Research, 2008, Vol. 36, No. 19 Figure 6. HDAC4, but not HDAC1, plays a negative role in regulating COX-2 transcription. (A) The COX-2 reporter was cotransfected with HDAC1 or HDAC4 expression vectors for 15 h in complete medium. Cell lysates were prepared for the luciferase assay. Three independent experiments are represented by the statistical analysis. (B) Two COX-2 reporter vectors, pGC207/wt (WT) and pGC207/mCEBP/CRE (mCE/ mC), were cotransfected with HDAC4 expression vectors or empty vectors. Cell lysates were harvested for the luciferase assay. The repression ratio was individually normalized with itself. (C and D) A431 cells were transfected with oligonucleotides-based siRNA of HDAC4 (HD4-1 or HD4-2), CEBPB (CE-B) or the scrambled control (C). After 30 h, these transfectants with or without EGF treatment were harvested to determine the COX-2 expression by western blot analysis (left panel of C) and RT–PCR (right panel of C) or luciferase assay (D) (RD: relative density). The product of HA/LAP1-immunoprecipitated complex showed an obvious HDAC4-interactive activity, but the HA/LAP1K174A-immunoprecipitated complex did not (Figure 7B, right panel). However, the LAP2 and LAP2K151A showed consistent and low HDAC4-interac- tive activity (Supplementary Figure S3). HDAC4 is not detectable in the HA/LIP precipitated immunocomplex by anti-HA antibodies (data not shown). This implies that the HDAC4-mediated repressive ability on the COX-2 promoter is suLAP1-dependent but not LIP- and suLAP2-dependent. suCEBPD and suLAP1 corporately function in the repression of the COX-2 promoter activity The transient COX-2 expression responds to external sti- muli. Our previous study suggests that suCEBPD can act as a negative regulator and may act through the recruit- ment of HDACs to repress COX-2 transcription (14). Herein, we further demonstrated that the suLAP1 negatively regulates COX-2 transcription. However, the structure/type of in vivo binding of these molecules, including CEBPD, CEBPB, SUMO1 and HDAC4, on the COX-2 promoter under EGF treatment was unclear. The ChIP assay, using extracts of indicated time courses from the EGF-treated A431 cells, was performed to further elucidate the scenario among SUMO1, CEBPD, CEBPB and HDAC4. Consistent with our previous study (14), the increasing binding of CEBPD was observed within a short-term 1 h of EGF treatment, and the binding returned to the basal level upon EGF treatment for 12 h. However, increased CEBPB-binding activity was observed from 3 h and extended to 12 h of EGF treatment. More importantly, the HDAC4 binding pattern is consistent with SUMO1-conjugated proteins; whereas, HDAC1 maintains consistent binding activity throughout the time frame of EGF treatment (Figure 8A). To conﬁrm whether CEBPD and CEBPB conjugated with SUMO1 and form a complex with HDAC4, the re-ChIP assay was performed. In correlation with EGF-regulated