We are focusing on the role of the distal promoter (up to -500 nucleotides) in the regulation of the human beta globin gene. Previous experiments (project #ZOI DK 25045-05 LCB), were based on transient expression assays, in which recombinant plasmids, containing a series of deletion mutants of the 5' flanking region of the beta globin, linked to the CAT reporter gene and to the termination signal of the SV40, were transfected into K562 erythroid cells, followed by CAT assays. These experiments suggested that the region from -233 to -185 (the -200 region) stimulates the beta globin expression. Of note is that this region contains a consensus sequence for binding of nuclear factor I (NFI), a protein which was found to be necessary for the replication of the DNA of adenovirus, and appears to be indistinguishable from the CAAT box transcription factors (CTF). Another, although partial and hence weaker NFI site is located in the -150 region. The results, so far, can be summarized as follows: 1) There is a protein-DNA binding, and mapped to the NF1 site (by both gel shift competition assays and DNase I footprinting assay). 2) Purified NFl resulted in a similar gel shift pattern to K562. 3) Antibody anti-NFI confirmed the involvement of the NFI in this binding. 4) Initial data suggest that NF1 interacts with other factors in potential regulation of the human beta-globin gene. In the current project, we are focusing on a detailed structural analysis of the above region. We are looking for putative cis-regulatory sequences, recognized by potential regulatory proteins (trans-acting factors), as well as trying to identify these factors.
