We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to these disorders, and then implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories 1. A total of 1,941 unique genetic variants in 37 genes, encoding globins (HBA2, HBA1, HBG2, HBG1, HBD, HBB) and other erythroid proteins (ALOX5AP, AQP9, ARG2, ASS1, ATRX, BCL11A, CNTNAP2, CSNK2A1, EPAS1, ERCC2, FLT1, GATA1, GPM6B, HAO2, HBS1L, KDR, KL, KLF1, MAP2K1, MAP3K5, MAP3K7, MYB, NOS1, NOS2, NOS3, NOX3, NUP133, PDE7B, SMAD3, SMAD6, and TOX) are currently documented in these databases with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants and now provides a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The large repository of previously reported data, together with more recent data, acquired by microattribution, demonstrates how the comprehensive documentation of human variation will provide key insights into normal biological processes and how these are perturbed in human genetic disease. Using the microattribution process set out here, datasets which took decades to accumulate for the globin genes could be assembled rapidly for other genes and disease systems. The principles established here for the globin gene system will serve as a model for other systems and the analysis of other common and/or complex human genetic diseases.

Autonomous silencing of ␥-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the A ␥-globin gene was identified between ؊730 and ؊378 relative to the mRNA start site. A marked copy of the A ␥-globin gene inserted between locus control region 5 DNase I-hypersensitive site 1 and the -globin gene was transcriptionally silenced in adult ␤-globin locus yeast artificial chromosome (␤-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the ؊566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low ␥-globin levels, whereas only a weak signal was detected when ␥-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from ␤-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the A ␥-globin ؊566 or G ␥-globin ؊567 GATA site when ␥-globin expression was low (day 18) but not when ␥-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, ␥-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the ؊566/؊567 GATA sites of the proximal ␥-globin promoters.

One mode of ␥-globin gene silencing involves a GATA-1⅐FOG-1⅐Mi2␤ repressor complex that binds to the ؊566 GATA site relative to the A ␥-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the ؊566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the A ␥-globin promoter at the ؊566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in ␤-globin locus yeast artificial chromosome (␤-YAC) bone marrow cells. When WT ␤-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2␤ at the A ␥-globin promoter is increased. In addition, OGT and Mi2␤ recruitment is increased at the A ␥-globin promoter when ␥-globin becomes repressed in postconception day E18 human ␤-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2␤ is modified with O-GlcNAc, and both OGT and OGA interact with Mi2␤, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of ␥-globin gene regulation mediated by modulating the assembly of the GATA-1⅐FOG-1⅐Mi2␤ repressor complex at the ؊566 GATA motif within the promoter.

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

Sickle cell disease (SCD) and β-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH) mutations that result in increased levels of fetal hemoglobin (HbF, γ-globin chains) in adulthood. Thus, research has focused on manipulating the reactivation of γ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the −117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene and in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients. Herein, we report that gg1-VP64 increased γ-globin gene expression in vivo, in peripheral blood samples from gg1-VP64 β-YAC double-transgenic (bigenic) mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.