Oka K, Ishimura-Oka K, Chu M J, Chan L
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Gene. 1996 Nov 21;180(1-2):69-80. doi: 10.1016/s0378-1119(96)00408-8.
The expression of the hepatic lipase (HL) gene is highly tissue specific. In order to identify cis-acting elements which regulate the expression of this gene in the liver, multiple deletion mutants of the 5'-flanking region of the HL gene fused to the human growth hormone gene were transfected in HepG2 cells, which normally produce HL. Transient expression assays indicated the presence of negative (at nucleotides (nt) -1576(/)-1342 and -623(/)-407) and positive (at nt -1862(/)-1576 and -50(/)-9) regulatory elements. Transfection of HeLa cells, which do not produce HL, with the same deletion constructs resulted in a similar pattern of promoter activities. However, additional negative (nt -138/-50) and positive (nt -407(/)-138) elements were found. DNase I footprint analysis of the proximal and distal HLpromoter sequences with HepG2 and HeLa cell nuclear extracts identified seven protected regions: A, nt -1540(/)-1527; B, -1505(/)-1473; C, -1467(/)-1460; D, -592(/)-577; E, -565(/)-545; F, -234(/)-220; and G, -70(/) -48. Sites A, B, C, D and E were located within regions containing negative regulatory elements. In order to determine which nuclear factor interacts with the negative elements, sites B, D and E were mutated and the effects of mutation on competition in a gel retardation assay and on promoter activity were studied. When the binding motif for AP1 in sites B, D and E was mutated, the specific DNA-protein complexes were not competed with the mutant oligonucleotides and promoter activity increased twofold. The magnitude of the increase is less than expected from the deletion analysis, and simultaneous mutations did not cause further increase in promoter activity, which suggests that other sites are involved in this negative modulation. These results suggest that the transcription of the HLgene in HepG2 cells is negatively modulated by multiple cis-acting negative elements and AP1-like nuclear factor may play some role in this modulation.
肝脂肪酶（HL）基因的表达具有高度的组织特异性。为了鉴定调控该基因在肝脏中表达的顺式作用元件，将与人生长激素基因融合的HL基因5'侧翼区的多个缺失突变体转染到正常产生HL的HepG2细胞中。瞬时表达分析表明存在负调控元件（位于核苷酸（nt）-1576（/）-1342和-623（/）-407处）和正调控元件（位于nt -1862（/）-1576和-50（/）-9处）。用相同的缺失构建体转染不产生HL的HeLa细胞，得到了相似的启动子活性模式。然而，还发现了额外的负调控元件（nt -138 / -50）和正调控元件（nt -407（/）-138）。用HepG2和HeLa细胞核提取物对近端和远端HL启动子序列进行DNase I足迹分析，确定了七个受保护区域：A，nt -1540（/）-1527；B，-1505（/）-1473；C，-1467（/）-1460；D，-592（/）-577；E，-565（/）-545；F，-234（/）-220；G，-70（/）-48。位点A、B、C、D和E位于含有负调控元件的区域内。为了确定哪种核因子与负调控元件相互作用，对位点B、D和E进行了突变，并研究了突变对凝胶阻滞试验中的竞争和启动子活性的影响。当位点B、D和E中AP1的结合基序发生突变时，特异性DNA-蛋白质复合物不能与突变的寡核苷酸竞争，启动子活性增加了两倍。增加的幅度小于缺失分析预期的结果，同时突变并未导致启动子活性进一步增加，这表明其他位点参与了这种负调控。这些结果表明，HepG2细胞中HL基因的转录受到多个顺式作用负调控元件的负调控，AP1样核因子可能在这种调控中发挥一定作用。
