Abstract

The hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common genetic disorders in the world producing significant morbidity and mortality. Drug-mediated fetal hemoglobin (HbF) induction ameliorates the clinical severity of SCD and improves long-term survival. To define mechanisms of γ-globin regulation, previously we demonstrated the role of the Gγ-globin cAMP response element, in drug-mediated HbF induction (Sangerman et al., Blood 2006). Subsequently, data generated by the ENCODE project led us to investigate a region 4 kb upstream of Gγ-globin, which harbors a DNase hypersensitive site and enrichment of the histone 3 lysine 4, mono-methylated (H3K4Me1) enhancer mark, that we named the Fetal Chromatin Domain (FCD). Recently, a synthetic zinc finger DNA-binding domain targeting this region was shown to reduce γ-globin gene expression in K562 cells (Shen et al., Blood 128:320, 2016). Initially, we performed multispecies sequence alignments which revealed the FCD is located in a genomic region shared by Old World monkeys (humans and Baboons) with a delayed fetal to adult globin switch that occurs after birth. By contrast, the FCD region is absent in New World monkeys where the switch occurs during the fetal developmental stage. These findings support involvement of the FCD region in γ-globin regulation during hemoglobin switching.

Subsequent studies were performed to define the functional role of the FCD during erythropoiesis. ENCODE long RNA-sequencing assay detected PolyA RNA species throughout the FCD region in K562 cells. Based on these findings, we designed two small-interference RNA molecules targeting the core FCD (siFCD). Both siFCD molecules were transfected into KU812 cells and individual γ-globin gene expression measured using RT-qPCR with gene-specific primers. The level of Gγ-globin mRNA increased by 64%, while Aγ-globin transcription was reduced 38%. These results suggest the FCD facilitates differential regulation of the γ-globin genes. To further characterize FCD function, a series of γ-globin promoter luciferase reporters were constructed with the sense or antisense 500-bp core FCD region, cloned downstream of the luciferase gene. In KU812 stable cell lines, the sense FCD construct enhanced Aγ-globin promoter activity 10-fold, whereas the antisense construct repressed promoter activity. By contrast, the FCD in both orientations silenced Gγ-globin promoter activity. These results support unidirectional gene-specific enhancer properties of the FCD. Subsequent pulldown assay with a 34-bp biotinylated FCD probe confirmed NFE2, BHLHE40 and cMyc binding, which was abolished by mutation of the E-box sequence (CACGTG) in the FCD core.

To build on the KU812 cell data, we performed μChIP assay using primary erythroid progenitors generated from adult CD34+ stem cells (Zhu et al., Haematologica 2017). In our system, the γ-globin to β-globin gene switch occurred on day 4 in culture. Initially, we measured levels of acetylated histone H3 (AcH3), H3K4Me1, and TFIID binding in the β-globin locus control region hypersensitivity site 2 (HS2) and FCD. As expected, high AcH3 and H3K4Me1 marks occurred in HS2 consistent with its enhancer function. Similar patterns of histone marks and binding of TFIID, NFE2 and BHLHE40 occurred in HS2 and FCD region. It is known that HS2 participates in DNA looping to accomplish developmentally regulated globin gene expression during hemoglobin switching therefore chromosome conformation capture (3C) assay was performed in primary erythroid progenitors to determine if the FCD facilitates changes in chromatin structure. We observed strong long-range interactions between the FCD and HS2 with 4.4-fold maximal chromatin enrichment on day 5; likewise the FCD showed interactions with Aγ-globin (4.7-fold) and Gγ-globin (3.5-fold) by day 3 before switching occurred. Of note, low level interactions occurred between the FCD and β-globin gene at all time points tested.

Collectively, these data support the FCD as a unidirectional enhancer element involved in γ-globin regulation during erythropoiesis. To further define mechanisms of FCD function, the effects of CrisprCas9-mediated deletions in the FCD on globin gene expression will be explored.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.