Protein-DNA interactions in vivo of an erythroid-specific, human beta-globin locus enhancer.

作者：P M Reddy, C.‐K. James Shen · 发表于：Proceedings of the National Academy of Sciences · 年份：1991 · DOI：10.1073/pnas.88.19.8676 · 被引用次数：71 · 研究领域：Cancer-related gene regulation、RNA modifications and cancer、Genomics and Chromatin Dynamics

The 5' DNase I-hypersensitive site 2 (5' HS-2) is an erythroid-specific enhancer located 11 kilobases (kb) upstream of the human beta-globin gene cluster. Presence in cis of 5' HS-2 confers a high level of erythroid cell-specific and developmentally regulated promoter activities of human globin genes in transfected cell cultures and in transgenic mice. Combining the use of the methylation protection assay and polymerase chain reaction, we have studied nuclear factor-DNA interactions of the 5' HS-2 enhancer in vitro and in vivo. The data from analyses of three different sequence motifs within 5' HS-2 represent three different modes of protein-DNA interaction with respect to cell-type specificities and in vivo vs. in vitro differences. First, a GATA-1 motif was found to bind nuclear factor(s), presumably the GATA-1 factor, present in K-562 cell extracts and in living K-562 cells. No such binding was seen in nonerythroid HeLa cells or extract. A second motif, NF-E2/AP1 (nuclear factor-erythroid 2/activator protein 1), consists of tandemly arranged dimers of AP1 binding consensus. The presence of either HeLa extract or K-562 extract protects the NF-E2/AP1 motif from methylation, but the footprints are different. This is most likely due to different protein-DNA contacts of the AP1-DNA complex formed in HeLa extract and the NF-E2-DNA complex in K-562 extract. In vivo methylation protection patterns of this motif parallel those observed in vitro, suggesting that it is also bound by ...