Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly

The human beta -globin genes are regulated by the locus control region (LCR ), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5' to the genes. Various functional studies indicate that the LCR c onfers high-level, position-independent, and copy number dependent expressi on to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconst ituted in an erythroid cell-specific manner on chromatin-assembled LCR temp lates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid pr oteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The ge neration of LCR HS sites in the absence of chromatin assembly leads to the formation of S1- and KMnO4-sensitive regions in HS2 and HS3, These sites ar e also sensitive to SP nuclease in erythroid cells in vivo, suggesting a di storted DNA structure in the LCR core enhancer elements, Finally, we show t hat RNA polymerase II initiates transcription in the HS2 and HS3 core enhan cer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidi rectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and re cruit polymerase II transcription complexes. These results further our unde rstanding of the structural basis for LCR: function and provide an explanat ion for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.