WD repeat-containing protein 5, a ubiquitously expressed histone methyltransferase adaptor protein, regulates smooth muscle cell-selective gene activation through interaction with pituitary homeobox 2

Abstract

WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMC-marker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMC-marker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.

FIGURE 1.

siRNA-mediated knockdown of WDR5 decreased SMC-marker gene expression in two different SMC differentiation systems. A, expression of WDR5, SMC-marker genes, and control genes was determined by real-time RT-PCR. A404 cells were transfected with control or WDR5 siRNAs and were induced to differentiate into SMCs by RA treatment for 48 h. B, as in A, the protein of WDR5, SMC-marker genes, and control genes was analyzed by Western blot. C, heterozygous knock-out of WDR5 in embryonic stem cells decreased SMC-marker gene expression in an ESC-EB smooth muscle cell differentiation model system at days 7 and 15. Wild-type and heterozygous WDR5 knock-out ES cells were cultured and induced to differentiate into SMCs in the context of embryoid bodies (21, 23), and the level of SMC-marker genes, NeuroD, and WDR5 was determined by real-time PCR at days 7, 15, and 28 EBs. Values represent the mean ± S.E. of three independent experiments.

FIGURE 2.

WDR5 knockdown in X. laevis significantly decreased SMC-marker gene expression. A, WDR5 expression at the blastula stage was greatly decreased by WDR5-specific morpholino. Protein from whole embryos at blastula stage was harvested and analyzed by Western blot. Each lane represents for a group of three embryos. B, the expression of WDR5 gene and SMC-marker genes was determined by real-time PCR at two different stages of X. laevis by real-time PCR. C, expression of the SMC-marker genes SM-MHC, SM22α, and SM α-actin was analyzed by semiquantitative RT-PCR. Pluripotent animal cap cells from two-cell-stage X. laevis embryos injected with WDR5 morpholino (WDR5) or control morpholino (CoMO) were treated with bFGF to enhance SMC differentiation. −, without bFGF treatment; −MO, no morpholino injection; Emb, whole embryo; −RT, no reverse transcriptase control; ODC, ornithine decarboxylase.

FIGURE 3.

Morpholino-mediated knockdown of WDR5 in X. laevis significantly decreased SMC-marker gene expression in vivo. In situ hybridization analyses of SM α-actin (A–D), SM 22α (E–H), and SM-MHC (I–L) mRNA expression in stage 42 X. laevis embryos injected with either control morpholinos or WDR5 morpholinos at the two-cell stage. The location of visceral muscle and the dorsal aorta are indicated with arrows, whereas arrowheads highlight embryonic regions with either reduced visceral muscle (VM) cells or dorsal aortic (DA) cells expressing SMC-marker genes. B, D, F, H, J, and L represent higher magnifications of panels A, C, E, G, I, and K, respectively.

FIGURE 4.

WDR5 complex regulated H3K4me within the promoter loci of SMC-marker genes. A, distinct WDR5-interacting proteins in differentiated A404 versus undifferentiated A404 cells were identified by mass spectroscopy. A TAP-WDR5 expression plasmid or a TAP-empty plasmid was transfected into A404 progenitor cells. RA was added to induce SMC differentiation for 24 h. The distinct bands that only appeared in 24-h RA-treated cells on the silver-stained SDS-PAGE gel were subjected to linear trap quadrupole-Fourier transform mass spectrometry analyses as indicated by asterisks. B, the protein samples from more time point treatment as described in A were analyzed by Western blots using RbBP5, WDR5, and Ash2L antibodies. C, the enrichment of SRF and H3K4me1, H3K4me2, and H3K4me3 was determined using ChIP assays within SM α-actin and SM22α promoter loci in control or WDR5 siRNA-transfected A404 cells. D, enrichment of H3K4me1, H3K4me2, and H3K4me3 was detected by ChIP assays in day-15 EBs using primers targeting SM α-actin and SM22α promoters. Values represent the mean ± S.E. of three independent experiments.

FIGURE 5.

WDR5 bound with the homeodomain protein Pitx2. A, coimmunoprecipitation assays were performed in A404 cells cotransfected with Myc-WDR5 expression plasmid and wild-type FLAG-Pitx2 expression plasmid at different time points of RA treatment. IP, antibody that was used for immunoprecipitation; IB, antibody that was used for Western blot. B, shown is a schematic representation of Pitx2 and its deletion mutants. C, coimmunoprecipitation assays were performed in COS cells cotransfected with Myc-WDR5 expression plasmid and wild-type FLAG-Pitx2 expression plasmid or deletion mutants. The asterisk indicated antibody heavy chain. D, immunoprecipitated proteins from COS cells cotransfected with WDR5 expression plasmid and three homeodomain protein expression plasmids were analyzed by Western blot. They are: Myc-WDR5 and FLAG-Pitx2, Myc-WDR5 and FLAG-Mox1, and NTAT-WDR5 and Myc-Prx1 expression plasmids. Asterisks indicate antibody heavy/light chain.

FIGURE 6.

WDR5 bound to SMC-marker gene promoter loci. A, stable cell lines containing either empty or Myc-WDR5 expression plasmid had comparable differentiation potential to the original A404 cell line. Control stable, stable A404 cell line containing empty pcDNA3.1 myc/his vector. B, WDR5 and Pitx2 bound to the SM α-actin and SM22α gene promoters as determined by ChIP assays. Stable cell lines containing either empty or Myc-WDR5 expression plasmids were processed to ChIP assays using Myc antibody and Pitx2 antibody. C, WDR5 and Pitx2 bound to the same physical chromatin within SM α-actin promoter. Sequential ChIP (1) assays were performed using Pitx2 antibody and Myc antibody in those stable cell lines as described in A.

FIGURE 7.

Mutation of the Pitx2 binding site within the SM α-actin promoter nearly abolished RA-induced activation of the gene and decreased the H3K4me1 and H3K4me3 levels. A, siRNA induced suppression of Pitx2 in A404 cells decreased SRF binding and the level of H3K4me1 and H3K4me3 within the SM α-actin promoter. B, siRNA-induced knockdown of Pitx2 decreased WDR5 binding in stable cell line expressing Myc-WDR5 as described in Fig. 5B. C, the genotype of stable cell lines containing either wild-type or ATTA mutant SM α-actin promoter-enhancer-luciferase transgene was examined by semiquantitative PCR using luciferase primers. D, the induction of the endogenous SM α-actin gene in these two stable cell lines (as described in C was comparable with original A404 as determined by real-time RT-PCR. E, ATTA mutation decreased the H3K4me1 and H3K4me3 levels within the transgene promoter by ChIP assays. The enrichment of SRF and H3K4me1, H3K4me2, and H3K4me3 was determined by ChIP assays using primers specific for the transgene and the endogenous gene. Values represent the mean ± S.E. of three independent experiments. WT, stable cell line containing the wild-type SM α-actin promoter-enhancer-luciferase transgene; ATTA_Mut, stable cell line containing the TAATCT mutant SM α-actin promoter-enhancer-luciferase transgene.