6072 Nucleic Acids Research, 2008, Vol. 36, No. 19 Figure 4. Bindings of CEBPBs parallel with HDAC4 binding upon long-term EGF treatment. (A) Schematic representation of the sequences of CEBP and CRE motifs in this study. (B) In vitro translation of HA/CEBPD, HA/LAP1, HA/LAP2 and HA/LIP was performed, and the mixtures with these products and DNA probes were subjected to a gel-shift (EMSA) assay as described in ‘Materials and methods section’. An arrowhead indicates the retarded CEBPs-C/EBP or CEBPs-CRE complex. The in vitro-translated lysate without an expression vector is shown for the negative control (lanes 1 and 6). (C) Nuclear extracts were harvested from A431 cells at the indicated times and were treated with or without EGF, then incubated with biotin-labeled CRE or C/EBP oligonucleotides. The precipitated products were pulled down by streptavidin–sepharose and analyzed by western blot with antibodies recognizing p300, HDAC4 or CEBPBs. CEBPB isoforms and HDAC4 can bind to the CEBP and CRE motifs of the COX-2 promoter Our previous study showed that the CEBP and CRE motifs (Figure 4A) both played critical roles in the EGF-regulated COX-2 gene expression in A431 cells (14). To verify whether CEBPBs can participate in COX-2 transcription through these two motifs, a gel- shift assay was carried out using individual in vitro-trans- lated HA/CEBPB isoforms and labeled CEBP or CRE probes. The three CEBPB isoforms bound directly to the CEBP (Figure 4B, lanes 3–5) and CRE (Figure 4B, lanes 8–10) motifs. In addition, HDACs have been proven to be recruited by sumoylated transcription factors, functioning as corepressors to silence gene transcription (41). Our pre- vious study implied that the CEBPD has the ability to modulate the binding of chromatin remodeling enzymes, histone acetyltransferases (HATs) or HDACs, through its posttranslational modiﬁcation of acetylation or sumoyla- tion. However, the components involved in the suCEBPD- and suLAP1-mediated inactivation of the COX-2 gene are uncertain. suCEBPD has been identiﬁed to contain higher HDAC4-interactive activity by DAPA (Supplementary Figure S2). To further determine the binding activity of chromatin remodeling enzymes, such as p300, HDAC4 and CEBPBs on the CEBP or CRE motifs followed by the EGF treatment, a DAPA was performed to show the change of dynamic transcriptional complex. The pull- down results of CEBP and CRE motifs show similar bind- ing patterns (Figure 4C). Brieﬂy, the transient-increased p300-binding activity is observed from 1 h and returned to the basal form after 4 h of EGF treatment. Similar to the lagging induction of CEBPBs, the apparent binding of CEBPB isoforms was also detected from 4 h and extended to 12 h upon EGF treatment. Interestingly, the binding activity of HDAC4 is consistent with the CEBPBs binding in the lagging stage of EGF treatment. These results sug- gest that the lagging bindings of CEBPBs and HDAC4 may play a negative role in the regulation of COX-2 tran- scription. Additionally, HDAC4 has been implicated in interaction with SUMO1-conjugated substrates (36) and the nucleocytoplasmic shuttling of HDAC4 is reportedly controlled by signaling molecules (42,43). However, which external stimulator can induce HDAC4 redistribution is less studied. In our system, an equal distribution of HDAC4 between nucleus and cytoplasm was observed to pre-exist in nucleus during the EGF-starvation stage. Importantly, the HDAC4 showed a lagging import from 4 h and this extended to 8 h upon EGF treatment (Figure 5A, upper panel). On the other hand, the SUMO1-conjugated proteins are predominant in the nucleus, but transiently exported to the cytoplasm during short-term EGF treatment up until 4 h (Figure 5A, lower panel). The Figure 5B further indicates that HA/LAP1 is coexisting with endogenous HDAC4 and SUMO1 during the long-term EGF treatment. Combining this information with that of Figure 3C and Figure 4C, we suggest that the nuclear importation of HDAC4 and SUMO1 coincides with the LAP1 induction and the amount of sumoylated LAP1 during the inacti- vated period of COX-2 gene transcription. HDAC4, but not HDAC1, plays a functional role in silencing COX-2 HDAC4 is a SUMO E3 ligase and displays a transcrip- tional repressor activity (32,36). However, its interactive transcription factors and the detailed repressive mechan- ism of transcriptional regulation are still less known. Since the sumoylation of LAP1 plays a negative regulatory role