Promoter and11-Kilobase Upstream Enhancer Elements Responsible forHepatomaCell-Specific Expression oftheRat Ornithine Transcarbamylase Gene

geneinHepG2cells muchmore efficiently thaninCHO cells. Analysis of deletion mutantsofthe5'-flanking region inHepG2cells revealed thatthere areatleast one negative andtwo positive regulatory elements within theabout220-base-pair immediate 5'-flanking region. DNaseIfootprint analysis showed thepresenceoffactors binding tothese regulatory elements innuclear extracts ofratliver and brain, andfootprint profiles atthetwopositive elements exhibited liver-specific features. Transient expression analysis alsorevealed theexistence ofan enhancer region located 11kilobases upstreamofthetranscription start site. TheOTC enhancer was abletoactivate bothits own andheterologous promoters inHepG2butnot inCHO cells. Theenhancer was delimited toan about230-base-pair region, andfootprint analysis ofthis region revealed fourprotected areas.Footprint profiles attwoofthefour areasexhibited liver-specific features, andgelshift competition analysis showedthat a factor(s) binding tothetwoliver-specific sites isrelated to C/EBP.Theseresults suggest thatbothliver-specific promoter andenhancer elements regulate expression of theOTC genethrough interaction withliver-specific factors binding tothese elements. Theliver exhibits various tissue-specific functions suchas gluconeogenesis, ureabiosynthesis, drugdetoxication, and secretion ofplasma proteins. Thesefunctions areperformed bycooperation ofmultiple liver-specific geneproducts. Examination oftheregulation ofliver-specific genesshould provide insight intothecoordinated expression oftissue- specific genesandalso into themechanism ofunderlying cell differentiation. cis-Acting elements andtrans-acting factors regulating liver-specific transcription havebeencharacter- izedforgenesofplasma proteins, including serumalbumin (8, 9,25,31,33,42,50), a-fetoprotein (21, 22,26,48,55,64), ax-antitrypsin (11,40,46),fibrinogens (3,16),andtrans- thyretin (14). A liver-specific transcription factor, HNF1 (also designated LF-B1), that activates these genes hasbeen purified (15, 41)andcloned (18). Another liver-enriched putative transcription factor, C/EBP, thatbinds tothecis elements ofthese geneshasalsobeencharacterized (4,19, 38,67)following purification (35) andcloning (37). Mammalian ornithine transcarbamylase (OTC;carbam- oylphosphate:L-ornithine carbamoyltransferase;