The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of HBGPromoter DNA Methylation

Pharmacological or genetic induction of fetal hemoglobin (HbF, α2γ2) in adult red blood cells is a proven strategy to ameliorate the clinical symptoms of sickle cell disease (SCD) and β-thalassemia. Therefore, efforts are underway to better understand mechanisms that mediate the perinatal switch from HbF to adult hemoglobin (HbA, α2β2). We performed a CRISPR-Cas9/guide (g) RNA screen to identify novel proteins that regulate HbF production in HUDEP-2 cells, a human erythroid line that normally expresses HbA. We identified UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a repressor of HbF production. UHRF1 binds hemi-methylated DNA and recruit DNA methyltransferase 1 (DNMT1) to ensure faithful maintenance of DNA methylation during DNA replication. Numerous UHRF1-interacting proteins, including DNMT1, EHMT1/2 and HDAC2 are associated with γ-globin repression. We used CRISPR/Cas9 and RNA interference to validate UHRF1 as a HbF regulator. Compared to non-targeting gRNA UHRF1disruption using Cas9 + 2 separate gRNAs increased the γ-globin/γ+β-globin RNA ratio from 1.9 to 25.8/27.1% (P<0.01), increased the fraction of HbF immunostaining cells (“F-cells”) from 7.5 to 25.1/35.4% and increased HbF protein from 2.10 to 16.3/15.0% (P<0.01) in HUDEP-2 cells. Compared to a control luciferase shRNA, 2 different UHRF1shRNAs increased theγ-globin/γ+β-globin RNA ratio from 9.68% to 21.59/28.93% (P<0.01), increased the F-cell fraction from 37.9 to 49.8/55.6% and increased HbF protein from 9.1 to 16.18/18.5% (P<0.05) in erythroid cells derived from normal adult peripheral blood CD34+cells. UHRF1 deficiency did not alter erythroid maturation or expression of key transcription factor genes that regulate HbF expression in HUDEP-2 or CD34+cells (BCL11A, ZBTB7A, MYBand KLF1). UHRF1 mutant proteins defective in recognizing H3K9me2 (FW237/238AA), binding to hemi-methylated DNA (R491A) or ubiquitination of H3K23 to enhance DNMT1 recruitment (C741A), were unable to repress HBG1/HBG2. These mutations have the most profound effects on maintaining DNA methylation, indicating that UHRF1 represses HBG1/HBG2in HUDEP-2 cells through this mechanism.