Histone and DNA methylation defects at Hox genes in mice expressing a SET domain-truncated form of Mll

Abstract

The Mll gene is a member of the mammalian trithorax group, involved with the antagonistic Polycomb group in epigenetic regulation of homeotic genes. MLL contains a highly conserved SET domain also found in various chromatin proteins. In this study, we report that mice in which this domain was deleted by homologous recombination in ES cells (DeltaSET) exhibit skeletal defects and altered transcription of particular Hox genes during development. Chromatin immunoprecipitation and bisulfite sequencing analysis on developing embryo tissues demonstrate that this change in gene expression is associated with a dramatic reduction in histone H3 Lysine 4 monomethylation and DNA methylation defects at the same Hox loci. These results establish in vivo that the major function of Mll is to act at the chromatin level to sustain the expression of selected target Hox genes during embryonic development. These observations provide previously undescribed evidence for the in vivo relationship and SET domain dependence between histone methylation and DNA methylation on MLL target genes during embryonic development.

Fig. 1.

Generation of the MllΔSET allele. (A) Mll-SET domain targeting vector with homology arms, LoxP sites (triangles) flanking the neomycin resistance cassette. Partial restriction map of the Mll locus (A, ApaI; B, BamHI; H, HindIII) is shown before and after targeted replacement of the SET domain encoding region by the neomycin resistance cassette. Southern strategy to detect homologous recombination (BamHI digestion and “ext5” probe) and CRE deletion of the neomycin resistance cassette (HindIII digestion and “int3” probe) is indicated. RT-PCR primers encompassing the SET domain region are indicated by arrows. (B) The expression of the WT and MLL SET truncated alleles was evaluated by RT-PCR analysis in embryonic fibroblasts generated from 12.5 dpc embryos. Primers overlap the SET domain region, distinguishing the WT and truncated alleles. Equivalent loading of RNA was controlled by using GAPDH primers. (C) Western blot analysis of selected MLL-associated factors: MLLc, Menin, and WDR5 in WT and MLLΔSET MEFs. Molecular sizes of marker proteins are shown on the right. (D) Coimmunoprecipitation of MLL with associated factors. Nuclear extracts of WT and mutant MEFs (lane 1 input) were subjected to immunoprecipitation (IP) by using an antibody specific for MLL^N. The immunoprecipitates were fractionated in SDS/PAGE and immunoblotted with the antibodies indicated to the left of the panels (anti-MLL^C and anti-Menin)

Fig. 2.

Skeletal abnormalities in ΔSET mice. Views are shown of the thoracic (A–D) and cervical (E and F) regions of cleared skeletons of WT (A and E) and ΔSET mutant mice (B–D and F). Typical sternal abnormalities were detected in mutant mice. The additional ossification center is indicated by an arrow in B. Two different fusions between ribs are shown (C and D, indicated by arrows). Lateral view of the cervical region of a WT (E) and ΔSET mouse (F) is shown. ΔSET mice present bone abnormalities in the cervical region. The C2 vertebra is broadened (C2∗). The anterior arch of the atlas is severely reduced (aaa∗). C6 (black arrow) show posterior transformation to C7.

Fig. 3.

Altered expression of Hox genes in ΔSET mice. (A) Hoxd4, Hoxc8, and Hoxc9 whole-mount in situ hybridization on 9.5 dpc, somite-matched, ΔSET and WT embryos. Red arrows mark the anterior limit of neurectoderm expression, and black arrows mark the anterior limit of mesodermal expression. The numbers of ΔSET −/− embryos presenting decreased levels of Hox gene expression are indicated. (B) Quantitative PCR analysis of Hoxc8 expression in 9.5 dpc embryos (a) and thymocytes (b). (C) Semiquantitative RT-PCR analysis of Hox gene expression in thymocytes (Hoxd4, Hoxa7, and Hoxa5). Actin was used as a control to standardize RNA input and serial dilutions (1/20 and 1/80) of RNA used to compare expression levels.

Fig. 4.

H3 lysine-4 methylation pattern alteration at Hox loci in ΔSET mice. (A) Western blots confirming the steady levels of H3K4me2 and H3K9me2 in the ΔSET mutant MEFs. (B) Chromatin immunoprecipitation analysis in which the abundance of histone modifications (H3K4me1, H3K4me2, H3K9ac, and H3K9me2) were compared at Hoxd4 P1 and Hoxc8 promoters in the head and trunk of WT (black histograms) and ΔSET (gray histograms) 9.5 dpc embryos. Values shown are the mean of five independent experiments with average deviation. (C) Immunofluorescence (a) and Western blots (b) confirming the steady levels of H3K4me1 in the ΔSET mutant MEFs. Whole-cell extracts were prepared from MEFs cultures, and histone H1 was used as a control to standardize protein loading.

Fig. 5.

DNA methylation changes at Hox loci in ΔSET mice. (A) A partial map of the mouse Hoxd4 gene is shown. Methylation of cytosines was assessed by bisulfite sequencing upstream of exon 6, a CpG rich region of the Hoxd4 gene. A summary of methylation data from MEFs is shown where each line represents a separate clone. Methylated CpGs are represented by filled beads, and unmethylated CpGs are represented by open beads. (B) Southern blot analysis of DNA methylation changes at Hoxd4 in ΔSET embryonic cells (trunk from embryonic day 9.5 embryos) by using the methylation-sensitive enzyme HpaII.