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

Abstract

The human ␤-like globin genes (5--G␥-A␥-␦-␤-3) are temporally expressed in sequential order from the 5 to 3 end of the locus, but the nonadult -and ␥-globin genes are autonomously silenced in adult erythroid cells. Two cis elements have been proposed to regulate definitive erythroid ␥-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 ␥-globin silencing. To evaluate the contribution of these hypothetical silencers to ␥-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 ␤-globin yeast artificial chromosomes, which were then used to generate ␥-globin mutant transgenic mice. While DR element mutation led to a dramatic increase in A␥-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 ␥-globin gene silencing.

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site

“…Recent data demonstrate that two direct repeat (DR1) elements located in the proximal ε-globin promoter bind a novel repressor activity in definitive erythroid cells called direct repeat erythroid-definitive protein (DRED) (71). A similar DR1 sequence exists 5Ј to the A ␥-globin gene (49). The nondeletional Ϫ117 Greek hereditary persistence of fetal hemoglobin (HPFH) point mutation (25) alters this sequence, resulting in continued expression of A ␥-globin in adult humans and human ␤-globin locus yeast artificial chromosome (␤-YAC) transgenic mice bearing this mutation (51).…”

“…These two proteins (and others) collaboratively repress transcription of the ε-and ␥-globin genes. Recent evidence demonstrates that the DR sequence element autonomously mediates definitive stage-specific ␥-globin gene silencing (49,69). COUP-TFII (NF-E3) is an orphan receptor that has both repressor and activator prop-erties and may be involved in globin gene switching by repressing ε-globin expression in fetal erythroid cells (21).…”

Fetal hemoglobin chemical inducers for treatment of hemoglobinopathies

“…The DR element, if located around four CCTTG motifs (two upstream and two downstream), and the mutation of the DR motif, but not of the CCTTG motifs, reactivate γ-globin gene expression in adult erythroid cells [62].…”

Persistent Fetal γ-Globin Expression in Adult Transgenic Mice following Deletion of Two Silencer Elements Located 3′ to the Human Aγ-Globin Gene

“…Furthermore, elements of the proximal and the distal Aγ-gene promoter (44), combined with a competition by the other globin genes, are important to downregulate γ-globin gene expression in the adult stage (45,46). Additionally, it is well documented that the distance or gene order relative to the LCR is an essential determinant for the type of globin gene to be activated selectively during the primitive stage of erythropoiesis (47)(48)(49)(50)(51).…”