Transcriptional regulation of globin genes depends upon a variety of cis-acting regulatory DNA sequences and transacting protein or other factors. Several of the cis-acting sequences have been identified through naturally occurring and laboratory synthesized mutations or deletions. Transacting factors which bind to some of these cis-acting sequences have been cloned using DNA binding assays. As an alternate approach to the isolation of transcription regulatory proteins, we used a functional assay to screen a CDNA library from erythroid-like cells. A CDNA library was constructed from K562 erythroleukemic cells. 1.5 x 105 CDNA clones were screened for specificity to hemin induced K562 cells producing high levels of globin MRNA and hemoglobin protein. Of the 80 clones identified to be specific for induced K562 cells, 45 were found to be full length. These CDNA clones were inserted into an eukaryotic expression vector and driven by an SV40 viral promoter. To test for functional activity these clones were co-transfected with a globin promoter/reporter gene construct into HeLa cells. Six clones were found to transactivate globin promoter activity. Four were homologous to the ferritin heavy chain, one to the ferritin light-chain, and one to no known DNA or corresponding protein sequence. The 5' region of the ferritin cDNA clones extended up to 145 bases upstream from the coding region, but did not contain the iron response element usually associated with ferritin mRNA. These clones could activate transcription of other promoter/reporter gene constructs, but to a lesser extent than the effect on globin promoters. Transfection of the ferritin clones into K562 cells increased the endogenous epsilon globin level. We are now working on functional studies to determine if the transcriptional activation of these clones is dependent upon specific cis-acting globin DNA sequences.
NIH ExPorter
NIH ExPorter