Regulation of the beta globin gene is important for better understanding hemoglobin disorders such as sickle cell anemia and beta thalassemia, yet this regulation is poorly understood. We are currently studying the effect of cis acting DNA sequences and trans acting proteins on beta globin gene expression to begin to approach this question. The area upstream of the beta globin gene can be subdivided into several regions. The first 100 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 bonds to both silencers, making it a candidate for a repressor protein. BP1 binds to one silencer at -530 bp, which is also the site of a mutation found in several independent silent carriers of beta thalassemia. We looked at the binding of BP1 to the mutant DNA and found that it binds more tightly than to the reference sequence, consistent with the hypothesis that BP1 is a repressor since these patients make less beta protein than normal. In addition, there are five well characterized haplotypes associated with sickle cell anemia and each one has a different DNA sequence at -530 bp. We have now analyzed the binding of BP1 to each of the haplotypes' DNA and discovered that there is a hierarchy of binding strengths which correlates with what is known about the amount of HbS present for the haplotype. For example, the DNA of the Indian haplotype binds BP1 the tightest, these patients make less beta sickle protein than usual, and exhibit clinically mild symptoms. On the other hand, BP1 binds the most weakly to DNA of the Bantu haplotype and these patients exhibit more severe symptoms than those with the Indian haplotype. Thus binding of BP1 may be a predictor, along with other factors such as the alpha gene status and level of fetal hemoglobin, of the severity of sickle cell anemia.
