The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of HBG Promoter DNA Methylation
2018
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 domains1) as a repressor of HbF production. UHRF1 binds hemi-methylated DNA and recruit
DNA methyltransferase1 (
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-
globinrepression. We used
CRISPR/
Cas9and RNA interference to validate UHRF1 as a HbF regulator. Compared to non-targeting gRNA UHRF1 disruption using
Cas9+ 2 separate gRNAs increased the γ-
globin/γ+β-
globinRNA ratio from 1.9 to 25.8/27.1% (P UHRF1 knockout induced genome-wide
demethylationincluding 6
CpG siteslocated at positions -162, -53, -50, +6, +17, +50 positions relative to the γ-
globin(
HBG1and
HBG2) transcription start site.
Demethylationof these sites is thought to be required for γ-
globinde-repression. However, forced
demethylationof these cytosines in HUDEP-2 cells using specific gRNAs + dead (d)
Cas9-TET1 was not sufficient to activate γ-
globinexpression when UHRF1 was present. Additionally, dCas9-DNMT3a-mediated methylation of the HBG promoter CpG residues in UHRF1 knockdown HUDEP-2 cells did not inhibit γ-
globinexpression in UHRF1 knockout HUDEP-2 cells. Based on these studies, we conclude that: 1) UHRF1 regulates γ-
globintranscription; 2)
demethylationof
CpG sitesat the HBG gene promoters is neither necessary or sufficient for γ-
globininduction; 3) UHRF1 regulates γ-to-β
globingene switching either by methylating DNA regions other than those present around the HBG promoter or through non-canonical activities. Distinguishing these mechanisms will elucidate further our understanding of
globingene switching and could identify new pathways for pharmacological induction of HbF. Disclosures No relevant conflicts of interest to declare.
Keywords:
-
Correction
-
Source
-
Cite
-
Save
0
References
0
Citations
NaN
KQI