Regulation of the beta globin gene is important for better understanding hemoglobinopathies such as sickle cell anemia and beta thalassemia, yet this regulation is poorly understood. We are currently @i studying two regions of DNA which appear to be important in this respect, one extending from the cap site upstream to -600 bp and the other the second intervening sequence (IVS2). The area upstream of the beta globin gene can be subdivided into several regions. The first 106 bp, or proximal promoter, is known to contain sequences important for both the correct initiation of transcription and for the amount of mRNA synthesized. The next 100 bp, from -100 to -200, is called the distal promoter because it seems to contain sequences required for erythroid specific transcription of the gene. We have found, by deletion analysis, that DNA between -233 and - 185 is necessary for gene expression. In addition, we have defined two regions that act as silencers or negative regulatory elements, one between -610 and -490 bp, the other between -338 and -233 bp. We have also identified a protein! (BP1) which binds to both silencers, making it a candidate for a represser protein. Consistent with this possibility, BP1 binds more tightly to the DNA of sickle cell anemia patients with the Indian haplotype, who produce less beta protein than normal and are relatively healthy. Conversely, BP1 binds more weakly to the DNA from patients with the sickle cell Bantu haplotype, who exhibit much more severe clinical symptoms. Further studies are underway examining other haplotypes. In vitro experiments have shown that IVS2 is required for expression of the beta globin gene, although its function is not known. There is also a class of thalassemic patients who have a single base, mutation in IVS2 and who make no beta protein. We have identified binding sites for at least two known proteins in this region, NFI (nuclear factor I) and eryf1 (or GF1), an erythroid specific protein. Using purified factors, we see much stronger binding of eryf1 to this region in the normal DNA than the mutant. eryf1 is known to be a positive acting transcription factor, so decreased binding might cause decreased transcription. Our data suggest that NFI and eryf1 may be interacting to cause this effect.
