Figure 7. HDAC4 is involved in the suLAP1-mediated repression of COX-2 promoter activity. (A) The COX-2 reporter was cotransfected with the indicated expression vectors. Cell lysates were harvested for the luciferase activity assay. Reporter activities were normalized to the control transfectant of the reporter and pCDNA3. (B) The wild-type (WT) and sumoylation mutant (K174A) of HA/LAP1 were exogen- ously expressed in A431 cells. HA antibodies were used for the Co-IP assay. HDAC4, SUMO1 and HA antibodies were used for detection in the western blot analysis. COX-2 transcription, increases in the complex of HDAC4 or SUMO1-conjugated proteins including CEBPD and CEBPB were observed in the long-term 12 h of EGF treat- ment, which was similar to the results with EGF-untreated cells (Figure 8B). To further conﬁrm if CEBPDK120A and LAP1K174A can attenuate the interaction of HDAC4, the transient transfectants of HA/CEBPD, HA/CEBPDK120A, HA/LAP1 or HA/LAP1K174A were analyzed by the re-ChIP assays. The re-ChIP result demonstrated that the sumoylation mutants of CEBPD and LAP1 lost their HDAC4-binding capabilities (Figure 8C). This in vivo DNA binding assay suggests that the suCEBPD and suLAP1 have the potential to interact with HDAC4 and inactivate COX-2 transcription. Moreover, to clarify whether the suCEBPD and suLAP1 jointly regulated the inactivation of the COX-2 gene, the reporter assay was carried out by cotransfection with the COX-2 reporter and various CEBPs expression vectors as indicated. Overexpressed CEBPD reversed HA/LAP1- mediated repression of the COX-2 promoter activity (Figure 8D, compare lane 4 with lane 6), and further Nucleic Acids Research, 2008, Vol. 36, No. 19 6075 enhanced the HA/LAP1K174A-reversed COX-2 promoter activity (Figure 8D, compare lane 6 with lane 7). More importantly, an increase of COX-2 promoter activity was observed when the cells were cotransfected with LAP1K174A and CEBPDK120A expression vectors (Figure 8D, compare lane 8 with lane 7). These results suggest that suLAP1 and suCEBPD cooperate with each other in the regulation of COX-2 promoter activity. DISCUSSION CEBPB is an important transcription factor involved in cellular proliferation (45,46) and diﬀerentiation (47,48). It is also involved in COX-2 gene regulation (12,19,39). Herein, we were interested in the opposite biological phe- nomenon of EGF-induced lagging CEBPBs expression and the accompanying decrease of COX-2 transcription (Figure 1). Our goal was to clarify whether CEBPBs potentially play negative roles in COX-2 transcription. All three individual CEBPBs negatively repressed the COX-2 promoter via the exogenous expressions of LAP1, LAP2 or LIP (Figure 2C). LIP is a well-studied dominant negative regulator of CEBPs due to an absence of the transactivation domain; however, the mechanism of LAP1- and LAP2-mediated repression in transcriptional regulation is unclear. LAP1 can be sumoylated on the lysine 174 residue which was proven by an in vivo sumoy- lation assay in A431 cells (Figures 3A, B and 7B and Supplementary Figure S4). This is consistent with a pre- vious report stating that LAP1 is a SUMO1 substrate (26). The sumoylated form of LAP1 is coincident with the steady increase of LAP1 upon long-term EGF treatment (Figures 1B and 3C). Combining the LAP1K174A- mediated de-repressive eﬀect (Figure 3D and E), we spec- ulate that the increase of suLAP1 potentially plays a repressive role in COX-2 transcription upon long-term EGF treatment. As corepressors, HDACs require speciﬁc transcription factors to guide them and target DNA elements for their precise regulatory functions. Several studies have pointed out that HDAC1, HDAC3 and HDAC8 play functional roles in COX-2 transcription (49–51). suCEBPD shows a high aﬃnity in interaction with HDAC1, 3 and 4 but not HDAC2 (52). We provided evidence to show that the wild- type LAP1, and not the LAP1K174A, has a higher inter- action aﬃnity with HDAC4 (Figure 7B). In addition, LIP did not interact with HDAC4 in the Co-IP assay (data not shown). HA/LAP2K151A does not reverse LAP2- repressed COX-2 promoter activity and shows a consistent HDAC4-binding activity with wild-type LAP2 (Figure 3E and Supplementary Figure S3). The experimental results suggest diﬀerent roles for each of the three isoforms of CEBPB through interaction with HDAC4, although all of them show a repressive eﬀect on COX-2 transcription. LAP1 is not only a SUMO1 substrate (26) but also a SUMO2/3 substrate (23). Most importantly, the lysine 174 of LAP1 is the major SUMO-conjugated site. Our results demonstrate that the overexpression of SUMO1 increases the SUMO1-conjugated HA/LAP1 in A431 cells (Figure 3A). We also demonstrate that