The Protein Methyltransferase Prmt5 Links Histone H4 Methylation to Dnmt3a-Dependent DNA Methylation and Transcriptional Silencing of the Fetal Globin Genes.

Elevated levels of fetal hemoglobin ameliorate the severity of sickle cell disease and β-thalassemia, fuelling interest in the mechanisms underpinning the fetal (γ) to adult (β) switch in β-like globin chain subtype. We have previously identified a tripartite protein complex consisting of p22 NF-E4, CP2 and ALY, collectively known as the stage selector protein (SSP) that binds to the proximal γ-promoters, and fosters the preferential expression of the γ-genes in fetal erythroid cells. We have also identified a 14 kDa isoform of the NF-E4 protein that plays a role in γ-gene repression by binding CP2 and sequestering it away from the γ-promoter, resulting in disassembly of the activator SSP complex. Despite the loss of SSP binding, we showed by chromatin immunoprecipitation (ChIP) analysis that p22 NF-E4 remained bound to the γ-promoter in this context. To determine whether p22 NF-E4 could serve as the cornerstone for assembly of a larger repressor complex in this setting, we analyzed the proteins that were co-immunoprecipitated with p22 NF-E4 from K562 cell extract by mass spectrometry. One protein identified was PRMT5, an arginine methyltransferase that has been linked to gene silencing by establishing repressive arginine methyl marks including symmetrical dimethylation of arginine 3 on histone H4 (H4R3me2s). We confirmed the interaction between the two endogenous proteins by direct co-immunoprecipitation, and co-localized p22 NF-E4 and PRMT5 to the γ-globin gene promoters by ChIP. In vitro methylation studies using PRMT5 co-immunoprecipitated with p22 NF-E4 confirmed that histone H4 was the major substrate of the enzyme complex in K562 cells. In accord with this, we demonstrated a marked increase in H4R3me2s at the γ-promoter by ChIP in the setting of enforced expression of wild type PRMT5, accompanied by silencing of γ-gene expression. To determine whether additional factors cooperated with PRMT5 in γ-gene repression, we interrogated PRMT5 containing immunoprecipitates with antisera to a range of candidate proteins. We isolated a large repressor complex containing members of the NuRD complex and the methyl domain-binding proteins (MBD2 and MDB3). We also isolated the DNA methyltransferase 3a (Dnmt3a), a finding of considerable interest in view of the links between γ-gene silencing and methylation of CpG dinucleotides. Using bisulfite DNA sequencing, we demonstrated in K562 cells in which PRMT5 expression had been enforced, an increase in the density of methylated CpG dinucleotides clustered around the transcriptional start site. In contrast, cells transfected with an expression vector stably expressing hairpin short interfering RNAs, which induced a 90% reduction in PRMT5 protein levels, showed complete abrogation of DNA methylation at these CpGs, coincident with a five-fold induction of γ-gene expression. ChIP analysis of the human β-globin locus in βYAC transgenic mice revealed a marked enhancement of H4R3me2s at the γ-promoters in adult erythropoietic cells, and absence of this repressive mark at the γ-promoter in the E12.5 fetal liver. This data establishes a direct link between the PRMT5-induced repressive histone mark H4R3me2s and DNA methylation in developmental regulation of γ-gene expression. It also provides impetus for new strategies aimed at reactivation of fetal globin gene expression.