liver-enriched transcription-inhibitory protein (LIP). These variants are the result of an alternative translation initiation due to a leaky ribosomal scanning mechanism (20,21). LAP1 and LAP2 contain an N-terminal regula- tory domain that is able to regulate the transcriptional transactivation; however, their behavior is thought to be diﬀerent within cells (22,23). Moreover, LIP is considered to be a dominant-negative regulator of both LAP1/2 and the remainder of the CEBP family members, because of the lack of a transactivation domain. Recent studies have shown that LAP1 not only functions as a transcriptional activator but can also act as a transcriptional repressor to inhibit gene transcription, such as peroxisome proliferator- activated receptor beta /delta (PPAR /d) (24) and cyclin D1 (25). Modiﬁcation of LAP1 by the small ubiquitin modiﬁer (SUMO) family members, SUMO1 (26), SUMO2 and SUMO3 (23), has been recently reported, and this modiﬁcation has been proposed as important for its inhibitory function. However, the chromatin-remo- deling enzymes involved in the sumoylated LAP1 (suLAP1)-mediated transcriptional repression and target genes are unclear. Histone deacetylases (HDACs) participate in transcrip- tional repression through the recruitment/interaction with repressor/corepressor and the deacetylation of histones, resulting in local modiﬁcation of chromatin structures. In mammalian cells, there are two major categories of HDACs classiﬁed according to the structures and homol- ogy of the yeast counterparts (27). The widely expressed class I HDACs, including HDAC1, 2, 3 and 8, are similar to yeast RPD3 and are exclusively localized in the nuclei. Class II HDACs are composed of HDAC4, 5, 6, 7, 9 and 10, which are expressed in a tissue-speciﬁc manner and shuttle between the cytoplasm and nuclei in response to external signaling (28). Both classes of HDACs can inter- act with mSin3 and NuRD to form a large repressor com- plex (29), which can be recruited by speciﬁc DNA-binding factors to bind to the gene promoters (30). In addition, other studies indicate that class II HDACs participate in repression of gene expression in cell-speciﬁc development not only through their deacetylase activity but also by interacting with proteins and corepressors, such as CtBP (31), NCoR/SMRT (32,33) and HP-1 (34). HDAC4 plays a pivotal role in cell development through posttrans- lational modiﬁcation of sumoylation. For example, HDAC4 can interact with the myocyte enhancer factor-2 (MEF2) family, enhancing their sumoylation, then subse- quently repressing MEF2-dependent transcription (35,36). We previously reported that CEBPD modulates the basal and epidermal growth factor (EGF)-induced COX- 2 transcription (14). Herein, we further demonstrated that the increases of LAP1 and its sumoylation form are coin- cident with the re-inactivation of the COX-2 gene expres- sion upon long-term EGF treatment. Therefore, we suggest that not only sumoylated CEBPD (suCEBPD) but also suLAP1 may collaboratively participate in the repression of COX-2 transcription. In addition, we showed that EGF can regulate the nucleocytoplasmic shuttling of HDAC4 and SUMO1 by immunoﬂuorescence assay, and the time frame of CEBPBs induction coincides with this reimporting of HDAC4 and SUMO1 into Nucleic Acids Research, 2008, Vol. 36, No. 19 6067 the nuclei. Furthermore, the LAP1K174A shows a lower HDAC4-interaction aﬃnity yet can enhance COX-2 pro- moter activity compared with the wild-type LAP1. An in vivo DNA-binding assay shows the spatial and temporal binding activity of SUMO1, CEBPD, CEBPB and HDAC4 on the COX-2 promoter followed EGF treat- ment. Using the same in vivo DNA-binding approach, we further demonstrated that the LAP1K174A and CEBPDK120A attenuated the HDAC4-binding ability to the COX-2 promoter. These results suggest that the suLAP1 and suCEBPD both act as mediators in modulat- ing HDAC4 recruitment on the COX-2 promoter. MATERIALS AND METHODS Materials Human EGF was purchased from Peprotech (Rocky Hill, NJ, USA). The COX-2, CEBPB, CEBPD, HDAC1, HDAC4, glyceraldehydes 3-phosphate dehydrogenase (GAPDH) and hemagglutinin [HA; for immunoprecipita- tion (IP)] antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against HA (for western blot) were purchased from BM (Boehringer, Mannheim, Germany) or Babco (Richmond, CA, USA). Antibodies recognizing p300 were purchased from Transduction Laboratories (Lexington, KY, USA). Antibodies against SUMO1 were purchased from Zymed Laboratories (South San Francisco, CA, USA). Antibodies against SUMO2/3 were purchased from Abcam (Cambridge, MA, USA). Lipofectamine 2000, Dulbeco’s modiﬁed Eagle’s medium (DMEM), Opti- MEM medium, the Trizol RNA extraction kit and SuperScriptTM III were obtained from Invitrogen (Carlsbad, CA, USA). The Arrest-In reagent was pur- chased from Open Biosystems (Huntsville, AL, USA). Monoclonal anti-HDAC1 antibodies, Protein A-agarose and streptavidin–sepharose beads were purchased from Upstate (Charlottesville, VA, USA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT, USA). The Taq DNA polymerases, the in vitro tran- scription/translation kit and luciferase assay system were from Promega (Madison, WI, USA). Expression plasmid pcDNA3/HA was a gift from Dr Hsin-Fang Yang-Yen (IMB, Academia Sinica, Taiwan). All oligonucleotides for DNA aﬃnity precipitation assay (DAPA) and electro- phoretic mobility shift assay (EMSA) were synthesized by MDBio Inc. (Taipei, Taiwan). N-ethylmaleimide (NEM), an inhibitor of SUMO proteases, was obtained from Sigma (St Louis, MO, USA). Plasmid construction The numbering used to designate residues in CEBPB is based on the human CEBPB sequence (NM_005194). Expression vectors encoding HA-LAP1 (full-length C/ EBPB), HA-LAP2 (residues 24–345) and HA-LIP (resi- dues 199–345) were constructed in pcDNA3/HA which was ampliﬁed from the A431 cDNA with speciﬁc primers as follows: LAP1F/BamHI: 50-GGATCCCAACGCCTG GTGGCCTGG-30, LAP2F/BamHI: 50-GGATCCGAAG TGGCCAACTTCTAC-30, LIPF/BamHI: 50-GGATCCG