The human beta globin locus control region (LCR) aids in the establishment of an open chromatin configuration thereby facilitating the expression of linked downstream globin genes in a temporal and spatial manner along the developmental ontogeny. The LCR also functions as a classical enhancer of the beta like globin genes. The four DNAase-I hypersensitive regions (HS 1–4) on the LCR are believed to act as nucleation centers for the assembly of multi-protein transcriptional complexes that interact with the distant gene promoters. Present work is focused on characterizing the protein complex assemblies on the HS4 region and delineating their function in the context of goblin gene regulation. Our finding shows an assembly of a 2–5 MDa complex on the HS4 locus that is both erythroid restricted and sequence specific. Electrophoretic mobility shift assays (EMSA) were performed using K562 and HeLa nuclear extracts and ∼35mer double stranded oligonucleotides with 7 bp overlaps tiling the entire core HS4 sequence. We detected prominent K562 specific bands formed with an AT rich 39 bp HS4 sequence. Sequence specificity of DNA-protein complex formation on this AT rich sequence was determined by competitive EMSA assays. Gel supershift assays with a wide range of antibodies and subsequent partial purification of this multiprotein complex followed by the LC-MS/MS analysis reveal the presence of a mini chromosome maintenance (MCM) complex that is associated with the DNA replication, DNA repair associated Rad50-Mre-11-NBS-1 complex, histone acetyl transferase p300 and DNA binding transcription factors ILF2 (NF45) and ILF3 (NF90). The association of these proteins was determined by co-immunoprecipitation experiments. Chromatin immunoprecipitation (ChIP) experiments demonstrate the in vivo recruitment of these proteins on the HS4 region of the LCR. We are currently working on further characterization and functional studies of this complex and its significance on the role of the LCR in DNA replication and repair.
Disclosure: No relevant conflicts of interest to declare.