The role of WDR5 in silencing human fetal globin gene expression

Abstract

Background: Histone H3 lysine 4 (K4) methylation has been linked with transcriptional activity in mammalian cells. The WD40-repeat protein, WDR5, is an essential component of the MLL complex that induces histone H3 K4 methylation, but the role of WDR5 in human globin gene regulation has not yet been established.

Design and methods: To study the role of WDR5 in human globin gene regulation, we performed knockdown experiments in both K562 cells and primary human bone marrow erythroid progenitor cells (BMC). The effects of WDR5 knockdown on γ-globin gene expression were determined. Biochemical approaches were also employed to investigate WDR5 interaction molecules. Chromosomal marks in the globin locus were analyzed by ChIP.

Results: We found that WDR5 interacted with protein arginine methyltransferase 5 (PRMT5), a known repressor of γ-globin gene expression, and was essential for generating tri-methylated H3K4 (H3K4me3) at the γ-globin promoter in K562 cells. Enforced expression of WDR5 in K562 cells reduced γ-globin gene expression, whereas knockdown of WDR5 increased γ-globin gene expression in both K562 cells and primary human bone marrow erythroid progenitor cells. Consistent with this, both histone H3 and H4 acetylation at the γ-globin promoter were increased, while histone H4R3 and H3K9 methylation were decreased, in WDR5 knockdown cells compared to controls. We found that WDR5 interacted with HDAC1 and a PHD domaincontaining protein, ING2 (inhibitor of growth), an H3K4me3 mark reader, to enhance γ-globin gene transcriptional repression. In human BMC, levels of WDR5 were highly enriched on the γ-promoter relative to levels on other globin promoters and compared to the γ-promoter in cord blood erythroid progenitors, suggesting that WDR5 is important in the developmental globin gene expression program.

Conclusions: Our data are consistent with a model in which WDR5 binds the γ-globin promoter in a PRMT5-dependent manner; H3K4me3 induced at the γ-globin promoter by WDR5 may result in the recruitment of the ING2-associated HDAC1 component and consequent silencing of γ-globin gene expression.

Figure 1.

WDR5 associates with NF-E4 and binds the γ-globin promoter. (A) SimplyBlue Safestain of an SDS-PAGE gel of anti-Flag antibody immunoprecipitates from K562 cells transfected with NF-E4-Flag, or vector alone (control), before analysis by mass spectrometry. The bands corresponding to WDR5, PRMT5, NF-E4, polyubiquitinated NF-E4, and the known PRMT5 partner proteins, nucleolin and pICln, are shown. Hatch marks indicate bands that correspond to common background proteins including keratin, tubulins, and ribosomal proteins. Asterisks indicate immunoglobulin chains. The WDR5 peptide sequence identified is shown below. (B) HA antibody immunoprecipitation-Western blot analysis of WDR5 and NF-E4 from 293T cells transfected with both NF-E4-HA and WDR5-FLAG plasmids. Normal rabbit IgG served as the control. (C) Flag antibody immunoprecipitation-Western blot analysis of WDR5 and NF-E4 from 293T cells transfected with WDR5-FLAG plasmids. Normal rabbit IgG served as the control. (D) GST pull-down assay. Purified GST and GST-NF-E4 fusion proteins pre-adsorbed to glutathione-Sepharose beads were incubated with S-labeled in vitro transcribed translated (IVTT) WDR5. Specifically bound protein was eluted from washed beads and visualized by autoradiography after SDS-PAGE. (E) Binding of the endogenous WDR5 and NF-E4 to the γ-promoter or the MyoD promoter analyzed by ChIP assays in K562 cells. No antibody or normal rabbit IgG served as the control.

Figure 2.

WDR5 interacts with NF-E4 through PRMT5. (A) Co-immunoprecipitation and Western blot analysis of PRMT5 and WDR5 from K562 cells. Normal rabbit IgG served as the control. (B) GST pull-down assay. Purified GST and GST-PRMT5 fusion protein pre-adsorbed to glutathione-Sepharose beads were incubated with Slabeled WDR5. Specifically bound protein was eluted from washed beads and visualized by autoradiography after SDS-PAGE. (C) Co-immunoprecipitation and Western blot analysis of endogenous NF-E4, PRMT5, and WDR5 from scrambled control and PRMT5-KD K562 cells with indicated antibodies. Relative quantitation of protein was analyzed with ImageJ software (NIH, USA). (D) Binding of NF-E4, WDR5, and PRMT5 at the γ-promoter was measured by ChIP analyses in scrambled and PRMT5-KD K562 cells. Results are shown as mean ± SD from 3 independent experiments. #P>0.05, **P<0.01 compared to the scrambled control.

Figure 3.

WDR5 represses γ-globin expression. (A) Western blot analyses of cellular extracts from WDR5-OE or vector only K562 cells with indicated antibodies. (B) γ-globin gene expression analysis by Q-RT-PCR of RNA from vector control or WDR5-OE K562 cells. Results are shown as mean±SD from 3 independent experiments. *P<0.05 compared to the vector control. (C) Western blot analyses of cellular extracts from WDR5-KD1 and WDR5-KD2 or scrambled control (Scr) K562 cells with indicated antibodies. (D) γ-globin gene expression analysis by Q-RT-PCR of RNA from WDR5-KD1, WDR5-KD2, and scrambled control (Scr) K562 cells. Results are shown as mean±SD from 3 independent experiments. **P<0.01 compared to the scrambled control. (E) Co-immunoprecipitation and Western blot analysis of WDR5 and MLL2 from K562 cells. Normal rabbit IgG served as the control. (F) Histone H3K4me1, H3K4me2, and H3K4me3 ChIP analyses at the γ-promoter were performed in scrambled control and WDR5-KD K562 cells. Results are shown as mean±SD from 3 independent experiments. #P>0.05, *P<0.05 compared to the scrambled control. (G) Western blot analyses of cellular extracts from vector control, WDR5 wild-type (WT), WDR5-F133A, and WDR5-Y191F K562 cells with indicated antibodies. (H) H3K4me3 ChIP analyses at the γ-promoter were performed in vector control, WDR5 wild-type (WT), WDR5-F133A, and WDR5-Y191F K562 cells. Results are shown as mean±SD from 3 independent experiments. #P>0.05, **P<0.01 compared to the vector control. (I) γ-globin gene expression analysis by Q-RT-PCR of RNA from vector control, WDR5 wild-type (WT), WDR5-F133A, and WDR5-Y191F K562 cells. Results are shown as mean±SD from 3 independent experiments. #P>0.05, **P<0.01 compared to the vector control.

Figure 4.

WDR5 associates with the ING2-HDAC1 complex at the γ-promoter. (A) Histone H3ac, H4ac, H4R3me2s, and H3K9me3 ChIP analyses at the γ-promoter were performed in K562 and WDR5-KD cells. Results are shown as mean ± SD from 3 independent experiments. **P<0.01, *P<0.05 compared to the scrambled control. (B) Binding of WDR5, and PRMT5 at the γ-promoter was measured by ChIP analyses in scrambled and WDR5-KD K562 cells. Results are shown as mean ± SD from 3 independent experiments. #P>0.05, **P<0.01 compared to the scrambled control. (C) WDR5 antibody co-immunoprecipitation and Western blot analysis of endogenous WDR5 and HDAC1 from K562 cells with indicated antibodies. The asterisk indicates IgG heavy chains. Normal rabbit IgG served as the control. (D) ING2 antibody co-immunoprecipitation and Western blot analysis of endogenous WDR5 and ING2 from K562 cells. Normal rabbit IgG served as the control. (E) Western blot analysis of extract from K562 cells expressing Flag-tagged PRMT5 (PRMT5-f) fractionated by Superose 12 gel filtration. Column fractions were concentrated and analyzed using the antibodies indicated. (F) ING2 and HDAC1 ChIP analyses at the γ-promoter from WDR5 over-expressing or vector control K562 cells. Results are shown as mean ± SD from 3 independent experiments. **P<0.01, *P<0.05 compared to the vector control. (G) ING2 gene expression analysis by Q-RT-PCR of RNA from ING2 knockdown (ING2-KD) and scrambled control (Scr) K562 cells. Results are shown as mean ± SD from 3 independent experiments. **P<0.01 compared to the scrambled control. (H) γ-globin gene expression analysis by Q-RT-PCR of RNA from ING2 knockdown (ING2-KD) and Scr K562 cells. Results are shown as mean ± SD from 3 independent experiments. **P<0.01 compared to the scrambled control.

Figure 5.

Role of WDR5 in developmental globin gene silencing. (A) WDR5 ChIP analysis at the γ-promoter in erythroid progenitor cells from cord blood (CB) and adult bone marrow (BM). Results are shown as mean±SD from 3 independent experiments. **P<0.01 compared to CB. (B) Localization of WDR5 across the β-globin locus measured by ChIP in chromatin fractions from erythroid progenitors from adult bone marrow. The precipitated DNA was amplified with primers specific for the indicated regions of the β-globin locus. HS: hypersensitive site; pro:promoter; ^G/Aγ: intergenic region between ^Gγ- and ^Aγ-globin genes; OR51B4: Olfactory receptor 51B4 gene. Results are shown as mean±SD from 3 independent experiments. #P>0.05 compared to the IgG control, **P<0.01 compared to OR51B4-pro, and ***P<0.05 compared to all other loci. (C) Expression of WDR5 and (D) ING2 was analyzed in erythroid progenitors from BM expressing specific shRNAs or scrambled control (Scr). Results are shown as mean±SD from 3 independent experiments. **P<0.01 compared to the scrambled control. (E) Q-RT-PCR analysis of γ-globin and β-globin gene expression in WDR5-KD, ING2-KD, and Scr BM cells. Results are shown as mean±SD from 3 independent experiments. #P>0.05, *P<0.05, and **P<0.01 compared to the scrambled control.