Identifying Novel Regulators of γ-Globin Expression Using a Genome-Scale CRISPR Activation Screen

β-hemoglobinopathies are the most common monogenic disorders worldwide, and are defined based on whether patients have quantitative (β-thalassemia) or qualitative (Sickle Cell Disease (SCD)) defects in β-globin synthesis. Unfortunately, there are limited treatment options for β-hemoglobinopathies, and the majority of patients continue to develop life-threatening complications from their diseases. An alternative β-like subunit, γ-globin, has been shown to inhibit pathogenic hemoglobin polymerization in SCD and to functionally replace β-globin in β-thalassemia. The discovery of novel strategies to upregulate γ-globin expression may lead to effective therapies for β-hemoglobinopathies.

To identify novel regulators of γ-globin expression, we performed a genome-wide pooled CRISPR activation (CRISPRa) screen, using the Synergistic Activation Mediator (SAM) system. The screen was performed in the HUDEP-2 cell line, which expresses adult β-globin.

We generated a clonal HUDEP-2-MPH cell line, which stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1). This cell line was transduced with a viral library that delivers a VP64 activation domain fused to a catalytically dead Cas9 (dCas9-VP64), a blasticidin resistance cassette, and one of 3 unique sgRNAs targeting virtually every coding gene in the human genome (the library consists of 70,290 sgRNAs). At day 8 of erythroid differentiation, the top and bottom 10% γ-expressing cells were sorted, and integrated sgRNAs were sequenced using NextGen sequencing.

As expected, sgRNAs targeting BCL11A and ZBTB7A (known negative regulators of γ-globin) were enriched in the bottom 10% γ-expressing cells, while sgRNAs targeting HIF1A (known positive regulator of γ-globin) were enriched in the top 10% γ-expressing cells. Notably, this screen identified several novel candidate positive regulators of γ-globin expression, including the transcriptional repressor Hypermethylated in Cancer 1 (HIC1).

In preliminary validation studies, we used 2 independent sgRNAs to activate HIC1 in HUDEP-2-MPH cells and measured the percentage of γ-globin expressing cells (F-cells) by flow cytometry. Compared to cells transduced with control sgRNAs, increased HIC1 transcription (>200-fold) resulted in a profound increase in the proportion of F-cells, from a baseline of ~6% up to ~76%. Similarly, HIC1 overexpression in HUDEP-2 cells resulted in a ~13-fold increase in γ-globin mRNA levels and reduction in BCL11A mRNA level to ~30% of normal. These results suggest that the increased γ-globin expression resulting from HIC1 overexpression may result, at least in part, from reduced BCL11A levels (which we are currently validating). Recently, increased expression of HIC2, a paralogous protein for HIC1, was reported to result in increased γ-globin expression. To exclude the possibility that HIC1 targeting sgRNAs may also target HIC2, we measured the HIC2 mRNA level in HUDEP-2 cells targeted with sgRNAs aimed at increasing HIC1 expression. HIC2 mRNA was not increased in the latter cells.

We next overexpressed HIC1 cDNA in erythroid cells differentiated from primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). In early preliminary results, we found that HIC1 overexpression resulted in increased γ-globin expression, validating the HUDEP-2 data. Additional studies are ongoing to define the role of HIC1 in the regulation of γ-globin expression, and to define the impact of HIC1 overexpression on erythroid differentiation.

In summary, our screen uncovered HIC1, and other potential novel regulators of γ-globin expression, which we are currently validating. These findings may result in future therapeutic approaches for β-hemoglobinopathies.