Adult Stage γ-Globin Silencing Is Mediated by a Promoter Direct Repeat Element

Abstract

The human β-like globin genes (5′-ε-Gγ-Aγ-δ-β-3′) are temporally expressed in sequential order from the 5′ to 3′ end of the locus, but the nonadult ε- and γ-globin genes are autonomously silenced in adult erythroid cells. Two cis elements have been proposed to regulate definitive erythroid γ-globin repression: the DR (direct repeat) and CCTTG elements. Since these two elements partially overlap, and since a well-characterized HPFH point mutation maps to an overlapping nucleotide, it is not clear if both or only one of the two participate in γ-globin silencing. To evaluate the contribution of these hypothetical silencers to γ-globin regulation, we generated point mutations that individually disrupted either the single DR or all four CCTTG elements. These two were separately incorporated into human β-globin yeast artificial chromosomes, which were then used to generate γ-globin mutant transgenic mice. While DR element mutation led to a dramatic increase in Aγ-globin expression only during definitive erythropoiesis, the CCTTG mutation did not affect adult stage transcription. These results demonstrate that the DR sequence element autonomously mediates definitive stage-specific γ-globin gene silencing.

ACKNOWLEDGMENTS

We thank Y. Tanimoto for technical assistance in generating transgenic mice, K.-C. Lim for creative efforts, and our laboratory members for helpful discussions and encouragement.

This work was supported by research grants from the NIH (HL24415 and HL73465 to J.D.E. and O.T.), the Cooley's Anemia Foundation (K.T. and O.T.), the Inamori Foundation (K.T.), the Naito Foundation (K.T.), the 21st Century COE Program (A.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and Grants-in-Aid for Scientific Research (A, MEXT; A.F.), Scientific Research on Priority Areas (MEXT; K.T.), and Young Scientists (A, MEXT; K.T.)