TheFunctional DomainsoftheMurineThy-iGenePromoter

TheThy-i genepromoter resembles a "housekeeping" promoter inthatitislocated within a methylationfree island, lacks acanonical TATAbox,anddisplays heterogeneity inthe5'-end termini ofthemRNA.Using transgenic mice, we showthatthis promoterdoesnotconfer any tissue specificity andisactive onlyina position-dependent manner. Itcan onlybeactivated ina tissue-specific manner byelements thatlie downstream oftheinitiation site. Wehaveanalyzed thefunctional domains oftheminimal Thy-i promoterand showthat thedominant promoter elements consist ofmultiple binding sites forthetranscription factor Spl, an inverted CCAAT box,andsequencesproximal tothetranscription start site. DNaseIandgelmobility shift assaysshowthebinding ofanumberofnuclear factors tothese elements, including SplandCP1.Ourresults showthatthestructure ofthis promoter onlypermits productive interactions ofthetwotranscription factors SplandCP1withthebasal transcription machinery inthepresenceofenhancer sequences. Themurine Thy-1antigen, amemberoftheimmunoglobulin superfamily, isacell surface glycoprotein anchored on themembrane through aphospholipid tail (47, 66,70). Inthe mouse, Thy-1isexpressed athigh levels intheneuronal and lymphoid systems (55, 57,70), being found onthesurface of allneurons, inwhichitrepresents aterminal differentiation marker (54). Inthelymphoid system, Thy-1ispresent athigh levels onmostcortical thymocytes andmature T lymphocytes. Thisexpression pattern istheresult ofbothtissuespecific anddevelopmental regulation exerted atthetranscriptional level (44, 62). Thepromoter islocated within amethylation-free, CpGrich island (HTFisland) (4)andlacks acanonical TATAbox (23), features which arefound often in"housekeeping" gene promoters (18) butless frequently intissue-specific genes (19). Initiation oftranscription occurs frommultiple sites the distribution ofwhich differs inmurine brain andthymus (62). Inapparent contrast totheabsence oftissue specificity and its housekeeping characteristics, theThy-i genepromoter is nevertheless characterized bythepresence ofa tissuespecific DNaseI-hypersensitive site, located 80bpupstream ofthestart site (62). Byusing transgenic mice, anumberofThy-i transcriptional elements havebeenidentified. Expression ofthegene inearly thymocytes iscontrolled byenhancer sequences located within themurine third intron (67). However, this thymocyte-specific enhancer isnotsufficient tomaintain expression ofthegeneinmature Tcells (44), whichrequires thepresence ofdistinct regulatory sequences (67). Transcriptional activation oftheThy-i geneinthemajority of neuronal cells isdictated bycis-acting sequences present at the3'endofthefirst intron (7,67). Thus, tissue-specific and developmental transcriptional activation oftheThy-i promoterisachieved through separate elements whichfunction independently ofeachother andarelocated downstream of theprincipal site ofinitiation. Experiments intransgenic micehavealso established that a300-bp region oftheThy-i promoter (-270 to+36)contains all oftheelements required