6070 Nucleic Acids Research, 2008, Vol. 36, No. 19 Figure 2. CEBPBs participate in silencing COX-2 gene expression. (A) A431 cells were transiently transfected with siRNA of CEBPB or a scrambled control, C, for 24 h then these transfectants were starved in serum-free medium for 6 h, followed by EGF treatment for 5 h. Western blot (left panel) and RT–PCR (right panel) were performed to detect endogenous CEBPBs and COX-2 expressions. (B) Functional domains of the transactivation domain (AD), regulatory (RD), basic (B) and leu- cine-zipper domains (ZIP) are shown in the diagram. K174 and K151 represent the sumoylation sites of LAP1 and LAP2. (C) A431 cells were transiently cotransfected with the COX-2 reporter and individual expres- sion vectors of CEBPB isoforms with or without EGF treatment for 15 h. Data shown in left panel are representative results from three indepen- dent experiments performed in triplicate. Luciferase activities are plotted in arbitrary units. The results shown in right panel are averages from three independent transfection assays. The average fold induction (mean SD, n = 3) of EGF-treated cells compared to control cells was indicated. Statistical analysis was performed by Student’s t-test. promoter activity was inhibited in the HA/LAP1 transfec- tants showing a repressive eﬀect on the COX-2 promoter activity but not in the HA/LAP2 transfectants without the EGF treatment (Figure 2C, left panel). These results sug- gest that LAP1 and LIP, but not LAP2, have consistent repressive roles regardless of EGF existence (Figure 2C). Sumoylation of LAP1 is enhanced upon long-term EGF treatment, and the sumoylation mutant of LAP1 attenuates its repressive ability on the COX-2 promoter A previous study showed that the CEBP regulatory domain motif (RDM) sequences are attachment sites for SUMO1; furthermore, these SUMO1-conjugated sites, LKAEP of LAP1- or LAP2-RDM domain, are necessary and suﬃcient for the intrinsic inhibitory function of RDMs (26). This report suggests the idea that the sumoy- lated LAP1 and sumoylated LAP2 (suLAP2) may func- tion as negative regulators in re-inactivation of the COX-2 expression upon long-term EGF treatment, which diﬀers from the LIP-mediated repressive mechanism due to the lack of transactivation domain. To verify whether SUMO1-conjugated lysine residues are involved in the regulation of COX-2 transcription, the sumoylation mutants of LAP1 and LAP2 expression vectors, HA/ LAP1K174A and HA/LAP2K151A, were generated by site-directed mutagenesis. First, an in vivo sumoylation assay of LAP1 and LAP2 showed that increases of suHA/LAP1 and suHA/LAP2 were observed when the SUMO1 expression vector was cotransfected with expres- sion vectors of HA/LAP1 or HA/LAP2 in A431 cells (Figure 3A, compare lane 2 with lane 3, and lane 6 with lane 7). This proved that SUMO1-conjugated sumoylation of LAP1 and LAP2 does occur in A431 cells. Moreover, to conﬁrm the SUMO1-conjugated speciﬁcity on the lysine 174 of LAP1 and the lysine 151 of LAP2, the HA/sumoy- lation mutants of LAP1 and HA/LAP2, LAP1K174A and HA/LAP2K151A, were recruited to perform the same in vivo sumoylation assay. The SUMO1-conjugated pat- terns were not visible in the cotransfectants of SUMO1 and HA/LAP1K174A or SUMO1 and HA/LAP2K151A (Figure 3A, compare lane 4 with lane 5, and lane 8 with lane 9). In addition, reciprocal IP assays were performed to re-conﬁrm the in vivo SUMO1-cojugated LAP1. The suHA/LAP1 is visible in the SUMO1 antibody-immuno- precipition product of HA/LAP1 transfectant, but not in the transfectants of HA/LAP1K174A or HA/LAP1 with SUMO1 siRNA (Figure 3B and Supplementary Figure S4). These results indicate that the residues of lysine 174 of LAP1 and lysine 151 of LAP2 are the SUMO1-conjugated sites in A431 cells. Meanwhile, no predictable super-shift band was observed in the HA/ LIP transfectants, indicating that it is not a SUMO1 sub- strate (data not shown). Unlike transcriptional activation of the COX-2 gene, transcriptional repression after long- term EGF treatment has, up till now, been unexplored. Therefore, the issue of whether the amounts of suLAP1 and suLAP2 are coincident with the re-inactivation of COX-2 transcription needs to be examined. In considera- tion of whether the long-term EGF treatment changes the amounts of endogenous LAP1 and LAP2 in A431 cells, the in vivo sumoylation assay was performed by exogen- ously expressing the HA/LAP1 and HA/LAP2 to reveal their sumoylated forms. The time frame for EGF-induced suLAP1- and suLAP2-increases (Figure 3C) is consistent with COX-2 transcription being re-inactivated (Figure 1). This suggests that the suLAP1 and suLAP2 may partici- pate in the re-inactivation of COX-2 transcription. We further veriﬁed whether sumoylation of both LAP1 and LAP2 function in the EGF-regulated COX-2 promoter activity. Overexpression of HA/LAP1 or HA/LAP2 reduces the COX-2 promoter activity (Figure 2C), how- ever, the overexpressed HA/LAP1K174A attenuates the repressive activity of the COX-2 promoter as compared with overexpression of HA/LAP1 (Figure 3D and Supplementary Figure S1). In addition, there was no dif- ference between the cotransfection of the COX-2 reporter with the HA/LAP2 or the HA/LAP2K151A expression vectors (data not shown). To further conﬁrm whether the sumoylation of LAP1 indeed functions on the COX-2 promoter, the CEBPB siRNA was cotransfected