Functional Properties of the P-Globin Locus Control Region in

In this report, we compare the function of the human p-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for ”classical “ enhancer activity, (2) a colony assay that detects ”productive integration events, “ and (3) an assay that de-tects the ability of LCRfragments to confer hemin inducibility on linked, stably integrated y-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an ‘y-globin promoter driving the neomycin phosphotrans-ferase gene (y-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named p locus activation region [pLAR]) had activity in all three assays; of the individual LCR sites, only site I I was highly active in all three assays. One region within site I I, consisting of tandem AP-1INF-E2 consensus elements, had- 10 % as much colony assay activity as the entire pLAR. UAN ET AL ’ AND Forrester et aI2 defined a series of T DNAse I hypersensitive sites upstream and down-stream from the P-like globin gene cluster that were shown to be erythroid-specific and developmentally stable. The hypersensitive site (HS) at-6.1 kb (with respect of the €-globin gene transcription initiation site) was designated site I, the site at-10.9 kb site 11, the site at-14.9 kb site 111, and at-18.0 kb, site IV. Dhar et a13 recently demon-strated that many of these sites are DNAse I hypersensitive in nonerythroid and even nonhematopoietic cell lines; the-14.9 kb site is the only one that is uniquely hypersensitive in erythroid cell lines. Forrester et a14 demonstrated that chromosome 11 contained all of the information required to reconstitute these hypersensitive sites in mouse erythro-leukemia cells, and coined the term locus activation region (LAR) to designate this function. Grosveld et a15 called the same sequences the “dominant control region ” (DCR) and named the sites 1 (-18 kb), 2 (- 14.9 kb), 3 (- 10.9 kb), and 4 (-6.1 kb). (Recently, consensus names were given to the sites at the 7th Switching Conference as follows:-6.1 kb, 5