Multiple Positive andNegative Regulatory Elements inthe Promoter oftheMouseHomeoboxGeneHoxb-4

MouseHoxb-4 (Hox-2.6) isahomeobox genethat belongs toafamily which also includes Hoxa-4, Hoxc-4, and Hoxd-4 andthatisrelated totheDeformed geneinDrosophila melanogaster. Wehavedetermined thesequence of1.2kbof5'flanking DNAofmouse Hoxb-4 andbynuclease S1andprimer extension experiments identified twotranscription startsites, P1andP2,285and207nucleotides upstream oftheATG initiator codon, respectively. Wehaveshownthatthis region harbors twoindependent promoters whichdrive CATexpression in several different cell lines withvarious efficiencies, suggesting thatthey aresubject tocell-type-specific regulation. Through detailed mutational analysis, we haveidentified several cis-regulatory elements, located upstreamanddownstream ofthetranscription startsites. Theyinclude twocell-type-specific negative regulatory elements, whichare more active inF9embryonal carcinoma cells thaninneuroblastoma cells (regions aanddat-226to-186and+169to+205,respectively). Anadditional negative regulatory element hasbeendelimited (region bbetween +22and+113). Positive regulation isachieved bybinding ofHoxTF, a previously unknownfactor, tothesequenceGCCATTGG(+148to+155)thatisessential forefficient Hoxb-4 expression. We havealsodefined theminimalpromoter sequencesandfoundthattheyinclude two12-bp initiator elements centered aroundeachtranscription startsite. Thecomplex architecture oftheHoxb-4 promoter provides theframework forfine-tuned transcriptional regulation during embryonic development. Homeoboxgenesencodea large family oftranscription factors andare foundina widerangeofspecies, both invertebrate andvertebrate (reviewed inreferences 12,17,and 23). InDrosophila melanogaster, thegenesoftheAntennapedia/ Ultrabithorax complexes, which areclustered together, specify thephenotypes ofindividual segments along theanteroposterior axisofthebody. Inmammals, there arefourclusters of Antennapedia-class homeoboxgenes,called Hoxgenes,each