TRANSCRIPTIONAL REGULATION OF THE HUMAN ERYTHROID 5-AMINOLEVULINATE SYNTHASE GENE - IDENTIFICATION OF PROMOTER ELEMENTS AND ROLE OF REGULATORY PROTEINS

We have characterized the 5'-flanking region of the human erythroid-sp ecific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and sh own that the first 300 base pairs of promoter sequence gives maximal e xpression in erythroid cells, Transcription factor binding sites clust ered within this promoter sequence include GATA motifs and CACCC boxes , critical regulatory sequences of many erythroid cell-expressed genes , GATA sites at -126/-121 (on the noncoding strand) and -102/-97 were each recognized by GATA-1 protein in vitro using erythroid cell nuclea r extracts, Promoter mutagenesis and transient expression assays in er ythroid cells established that both GATA-1 binding sites were function al and exogenously expressed GATA-1 increased promoter activity throug h these sites in transactivation experiments, A noncanonical TATA sequ ence at the expected TATA box location (-30/-23) bound GATA-1- or TATA -binding protein (TBP) in vitro, Conversion of this sequence to a cano nical TATA box reduced expression in erythroid cells, suggesting a spe cific role for GATA-1 at this site. However, expression was also marke dly reduced when the -30/-23 sequence was converted to a consensus GAT A-1 sequence (that did not bind TBP in vitro), suggesting that a funct ional interaction of both factors with this sequence is important. A s equence comprising two overlapping CACCC boxes at -59/-48 (on the nonc oding strand) was demon strated by mutagenesis to be functionally impo rtant. This CACCC sequence bound Spl, erythroid Kruppel-like factor, a nd basic Kruppel-like factor in vitro, while in transactivation experi ments erythroid Kruppel-like factor activated ALAS2 promoter expressio n through this sequence, A sequence at -49/-39 with a 9/11 match to th e consensus for the erythroid specific factor NF-E2 was not functional , Promoter constructs with 5'-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as i n erythroid cells, indicating that an enhancer sequence located elsewh ere or native chromatin structure may be required for the tissue-restr icted expression of the gene in vivo.