The long-term goals of this grant are to identify and characterize DNA sequences and trans-acting factors that regulate the expression of the human beta globin gene complex, especially those involved in the switch from gamma to beta synthesis in late fetal life. Several different specific DNA sequences and trans- acting factors have been identified that act at the beta globin gene complex, both at the locus-control region (LCR) and close to individual globin genes which are required for either their optimal erythroid-specific or developmental stage-specific regulation, or both. However, to date, no single factor has been implicated in the switch from human gamma to beta synthesis. We have described a trans-acting protein complex (PYR complex) present primarily in adult hematopoietic cells which binds to pyrimidine-rich sequences, including one located 1 kb upstream of the human delta globin gene (delta PYR binding site) which may function in hemoglobin switching. Recently, we have discovered that PYR complex is a specialized human SWI/SNF-like complex. SWI/SNF complexes are known to disrupt chromatin structure and permit transcription factor binding and gene activation. PYR complex is the first SWI/SNF complex with a DNA sequence- dependent binding site. We have also demonstrated a functional role for the delta PYR binding site in enhancing human gamma to beta switching. In these studies in transgenic mice, we have shown that deletion of this sequence leads to delayed switching.
The specific aims of this grant are to: (1) characterize the protein subunits of PYR complex by purification and sequencing; (2) define the structure and configuration of the PYR complex binding site more precisely; (3) determine the general function effects of the complex on chromatin structure; (4) localize the minimal DNA sequence required for the functional effects of the complex on globin switching; (5) search for gene targets of PYR complex action other than those at the human beta globin gene locus in erythroid cells, and in other adult hematopoietic cells that express the complex; and (6) define other SWI/SNF complexes, particularly a putative one at the human alpha globin locus. These studies should provide new insights into the mechanisms controlling the hemoglobin switching. They may result in new approaches to the treatment of the beta thalassemias and sickle cell disease since these diseases are due to abnormal beta globin synthesis and could theoretically be cured by allowing optimal gamma globin synthesis to persist into adult life.
