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The LRF/ZBTB7A Transcription Factor Is a BCL11A-Independent Repressor of Fetal Hemoglobin

Takeshi Masuda, Xin Wang, Manami Maeda, Matthew C. Canver, Falak Sher, Alister P.W. Funnell, Chris Fisher, Maria Suciu, Gabriella E. Martyn, Laura J. Norton, Ruijia Zhu, Ryo Kurita, Yukio Nakamura, Jian Xu, Douglas R. Higgs, Merlin Crossley, Daniel E. Bauer, Stuart H. Orkin, Peter V. Kharchenko and Takahiro Maeda

Abstract

Induction of fetal-type hemoglobin (HbF: α2γ2) is a promising means to treat hemoglobinopathies; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Such knowledge is essential to develop mechanism-based, targeted approaches to reactivate HbF production. Here, we show that Leukemia/lymphoma Related Factor (LRF), encoded by the ZBTB7A gene, is a novel and potent repressor of HbF production.

To assess the effects of LRF loss on the mouse erythroid transcriptome, we performed RNA-Seq analysis using splenic erythroblasts from control and LRF conditional knockout (Zbtb7aF/F Mx1-Cre+) mice. LRF-deficient adult erythroblasts showed significant induction of Hbb-bh1, but not Hbb-y. The results were validated at the protein levels via isoelectric focusing of peripheral blood (PB) hemolysates and MALDI-TOFMS analysis. LRF loss also reactivated human fetal globin expression in vivo in LRF conditional KO mice harboring the human β-globin gene cluster as a yeast artificial chromosome transgene (β-YAC).

To determine whether LRF loss could induce HbF in human erythroid cells, we employed human CD34+ hematopoietic stem and progenitor (HSPC)-derived primary erythroblasts and determined γ-globin expression levels upon shRNA-mediated LRF knockdown (KD). HbF levels in LRF KD cells (49-70%) were much greater than those seen in parental or scrambled-shRNA transduced cells. We next employed a novel human immortalized erythroid line (HUDEP-2). This line possesses an advantage over lines currently used for globin switching studies because it expresses predominantly adult hemoglobin (HbA: α2β2), with very low background HbF expression. Using CRISPR/cas9 gene modification, we knocked out ZBTB7A in HUDEP-2 cells and performed RNA-Seq analysis. As expected, γ-globin (HBG1 and HBG2) transcripts, but not those of embryonic ε-globin (HBE1), were markedly induced in ZBTB7A KO (ZBTB7AΔ/Δ) HUDEP-2 cells. ZBTB7AΔ/Δ cells exhibited HbF levels greater than 60%, while that of parental cells was less than 3%. Notably, the HbF reactivation occurred without changes in levels of transcripts encoding known HbF repressors, including BCL11A, the principal known switching factor.

We next performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) with an anti-LRF antibody using HSPC-derived proerythroblasts and HUDEP-2 cells. The most enriched motif identified in either was concordant with that previously identified in vitro using CAST analysis (Maeda et. al. Nature 2005), confirming antibody specificity. Supporting a direct role of LRF at the β-globin cluster, we observed several significant enrichment of LRF-ChIP binding signals at adult (HBB), fetal (HBG1) globin loci and the upstream hypersensitivity (HS) sites within the locus control region (LCR). ATAC-Seq (for assay for transposase-accessible chromatin with high-throughput sequencing) analysis revealed strong chromatin accessibility at the γ-globin locus in ZBTB7AΔ/Δ cells. Strikingly, differential enrichment of ATAC-signals in ZBTB7AΔ/Δ cells was evident only at the γ-locus. Thus, while LRF binds to the HBB locus and HS sites as well as to the HBG1 locus, LRF depletion specifically opens chromatin at the γ-globin locus.

Finally, to determine whether LRF and BCL11A suppress γ-globin expression via distinct mechanisms, we established LRF/BCL11A double knockout HUDEP-2 cells. Strikingly, HUDEP-2 lines lacking both LRF and BCL11A exhibited almost a complete switch in expression from adult- to fetal-type globin, suggesting that these two factors cumulatively represent the near entirety of γ-globin repressive activity in adult erythroid cells. Our findings reveal a novel molecular mechanism regulating γ-globin silencing and may open a new window for therapeutic targeting in the treatment of hemoglobinopathies.

Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Orkin: Editas Inc.: Consultancy.

  • * Asterisk with author names denotes non-ASH members.