6076 Nucleic Acids Research, 2008, Vol. 36, No. 19 Figure 8. suLAP1 and suCEBPD play a negative coordinating role in the transcriptional regulation of COX-2 gene, and were consistent with the HDAC4 binding in the long-term EGF treatment. Sheared formaldehyde-cross-linked chromatin with or without EGF treatment from A431 cells was immunoprecipitated with speciﬁc antibodies as indicated and the COX-2 promoter region was processed for PCR ampliﬁcation with speciﬁc primers. As a positive control, PCR ampliﬁcation was also carried out with input DNA from chromatin before the IP step. The detailed procedures of the ChIP (A) and re-ChIP (B) assay are described in ‘Materials and methods section’. (C) A431 cells were transfected with various expression vectors as indicated for 24 h. The re-ChIP assay was performed to determine the chromatin/protein complex of HDAC4 and CEBPD or LAP1 on COX-2 promoter. The detailed procedures are described in ‘Materials and methods section’. (D) A431 cells were cotransfected with the COX-2 reporter and various expression vectors as indicated. Lysates of the transfectants were harvested for the luciferase assay. SUMO1-LAP1 is detectable in the HA/LAP1-immunopre- cipated complex but not in the HA/LAP1K174A-immuno- precipated complex (Figure 3B, 7B and Supplementary Figure S4). Furthermore, the knockdown of SUMO1 attenuates LAP1-mediated repression of the COX-2 pro- moter (Supplementary Figure S5). These data suggest that SUMO1-LAP1 can function in the repression of the COX- 2 promoter activity. However, we still could not rule out the possibility that the SUMO2/3-conjugated LAP1 may also participate in the regulation of COX-2 transcription because the SUMO2/3 is also detectable in the HA/LAP1- immunoprecipated complex but not the HA/LAP1K174A- immunoprecipated complex (Supplementary Figure S6). This issue requires further investigation. In addition, Supplementary Figure S2 shows that the suCEBPD has a higher HDAC4-interactive activity than wild-type CEBPD. This provides explanation that the LAP1K174A cannot fully recover HDAC4-exerted repressive activity (Figure 3D, 3E and 7A), and is also consistent with the result that the co-overexpression of CEBPK120A and LAP1K174A can enhance COX-2 promoter activity (Figure 8D). These results suggest that the suCEBPD and suLAP1, but not suLAP2 or LIP, respond to HDAC4-mediated repression of COX-2 transcription. This result also provides evidence implying that the CEBPD and LAP1 are both candidates in responding to the HDAC4-mediated transcriptional regulation including chromatin remodeling. The CEBP and CRE motifs play critical functional roles in the transcriptional regulation of the COX-2 gene (4,14,16). CEBPD was shown to directly bind to these two motifs by an in vitro DNA binding assay. This current study demonstrates that CEBPBs can also bind to these two sites (Figure 4B), suggesting the possibilities of CEBPBs switching or coordination on the CEBP or CRE motifs of the COX-2 promoter during the time- dependent EGF treatment (Figure 9). Several papers demonstrate that exogenously overexpressed CEBPD or CEBPB are potential positive regulators on the COX-2 transcription. However, exogenously expressed CEBPD has greater potential to be an acute responsive protein than CEBPB in the induction of the COX-2 gene