Kabuki syndrome stem cell models reveal locus specificity of histone methyltransferase 2D (KMT2D/MLL4)

Abstract

Kabuki syndrome is frequently caused by loss-of-function mutations in one allele of histone 3 lysine 4 (H3K4) methyltransferase KMT2D and is associated with problems in neurological, immunological and skeletal system development. We generated heterozygous KMT2D knockout and Kabuki patient-derived cell models to investigate the role of reduced dosage of KMT2D in stem cells. We discovered chromosomal locus-specific alterations in gene expression, specifically a 110 Kb region containing Synaptotagmin 3 (SYT3), C-Type Lectin Domain Containing 11A (CLEC11A), Chromosome 19 Open Reading Frame 81 (C19ORF81) and SH3 And Multiple Ankyrin Repeat Domains 1 (SHANK1), suggesting locus-specific targeting of KMT2D. Using whole genome histone methylation mapping, we confirmed locus-specific changes in H3K4 methylation patterning coincident with regional decreases in gene expression in Kabuki cell models. Significantly reduced H3K4 peaks aligned with regions of stem cell maps of H3K27 and H3K4 methylation suggesting KMT2D haploinsufficiency impact bivalent enhancers in stem cells. Preparing the genome for subsequent differentiation cues may be of significant importance for Kabuki-related genes. This work provides a new insight into the mechanism of action of an important gene in bone and brain development and may increase our understanding of a specific function of a human disease-relevant H3K4 methyltransferase family member.

Figure 1

Generation of CRISPR-Cas9 engineered KMT2D mutation iPSC lines. (A) Immunostaining of pluripotency markers of the control and engineered KMT2D KO iPSCs used in the study. Scale bars indicate 50 μm. (B) Illustration of the editing location and size in exon 2 of KMT2D in the two mutation lines generated. (C) qPCR analysis of KMT2D mRNA expression showing absence of nonsense RNA-mediated decay in the lines carrying mutations. There were four independent samples in the analysis (Control1, Control2, KMT2D^ex2-del and KMT2D^ex2-ins). The two independent isogenic control lines were derived from the same fibroblast line used for the generation of the two KMT2D KO lines, reprogrammed and isolated from two different iPSC clones. Each KMT2D^ex2-del and KMT2D^ex2-ins KO line were derived from different gene-edited iPSC colonies expanded from single cells. Each iPSC sample underwent independent RNA extraction, cDNA synthesis and qPCR analysis. (D) The alignment and identification of RNAseq reads of exon2 of KMT2D of KMT2D^ex2-del iPSC lines. (E) The RPKM of KMT2D expression of the KMT2D KO cells compared to control cells. (F) Western blot analysis of KMT2D expression in mutation cells in comparison to control cells. ^***P ≤ 0.001.

Figure 2

Identification of locus-specific gene downregulation in KMT2D deficient cells. (A) List of most significant downregulated genes in the RNAseq analysis. There were four independent samples in the analysis (Control1, Control2, KMT2D^ex2-del and KMT2D^ex2-ins). The two independent isogenic control lines were derived from the same fibroblast line used for the generation of the two KMT2D KO lines, reprogrammed and isolated from two different iPSC clones. Each KMT2D^ex2-del and KMT2D^ex2-ins KO line were derived from different gene-edited iPSC colonies expanded from single cells. Each iPSC sample underwent independent RNA extraction, cDNA synthesis and sequencing. (B) (top) UCSC database snapshot of the downregulated genes locus located at 19q13.33 (hg38) exhibiting the adjacency of the affected genes; (bottom) Differential expression analysis of the RNAseq result indicating the downregulation of the four genes located within the same locus.^**P ≤ 0.01, ^***P ≤ 0.001. (C) (top) UCSC database snapshot of the downregulated genes locus located at 19q13.42 (hg38) exhibiting the adjacency of the affected genes; (bottom) Differential expression analysis of the RNAseq result indicating the downregulation of the four genes located within the same locus. ^**P ≤ 0.01, ^***P ≤ 0.001. (D) (top) UCSC database snapshot of the downregulated genes locus located at 14q32.33 (hg38) exhibiting the adjacency of the affected genes; (bottom) Differential expression analysis of the RNAseq result indicating the downregulation of the four genes located within the same locus. ^**P ≤ 0.01, ^***P ≤ 0.001.

Figure 3

Generation of KS case-derived KMT2D-deficient iPSCs. (A) Illustration of the location of 4 bp deletion in exon 53 of KMT2D in the KS case cell. (B) Representative images of immunostaining of pluripotency markers expression in generated using the KS case and sex-matched family control cells. Scale bars indicate 50 μm. (C) qPCR analysis and validation of absence of non-sense mediated decay in KMT2D mRNA expression in KS case cells. There were two independent samples in the analysis (Mom and KS case). Each sample was extracted with three replicates for RNA samples, where replicates were the same iPSC line grown in separate wells and underwent independent RNA extraction, cDNA synthesis and qPCR analysis. (D) Western blotting result showing reduced expression of KMT2D expression in KS case cells. (E) qPCR analysis of SYT3, SHANK1, CLEC11A and C19orf81 in engineered KMT2D KO iPSCs. There were four independent samples in the analysis (Control1, Control2, KMT2D^ex2-del and KMT2D^ex2-ins). The two independent isogenic control lines in the control group were derived from the same fibroblast line used for the generation of the two KMT2D KO lines, reprogrammed and isolated from two different iPSC clones. The two KMT2D KO lines consist of KMT2D^ex2-del and KMT2D^ex2-ins; each KO line were derived from different gene-edited iPSC colonies expanded from single cells. Each sample was extracted with 3 replicates for RNA samples, where replicates were the same iPSC line grown in separate wells and underwent independent RNA extraction, cDNA synthesis and qPCR analysis.^***P ≤ 0.001. (F) qPCR analysis of SYT3, SHANK1, CLEC11A and C19orf81 in KS case iPSCs compared to the healthy sex-matched control family iPSCs. There were two independent samples in the analysis (Mom and KS case). Each sample was extracted with three replicates for RNA samples, where replicates were the same iPSC line grown in separate wells and underwent independent RNA extraction, cDNA synthesis and qPCR analysis. ^***P ≤ 0.001.

Figure 4

KMT2D deficiency leads to locus-specific reduction of H3K4me1 levels. (A) Illustration of the proposed mechanism of action on how KMT2D regulates the affected locus. (B) ChIP-PCR analysis of KMT2D binding in comparison to IgG control antibody with two different control iPSCs. There were two independent samples in the analysis (Mom and KS1). Each sample was extracted with three replicates for DNA samples, where replicates were the same iPSC line grown in separate dishes with different passage number, and underwent independent chromatin immunoprecipitation and PCR analysis. Each replicate signal was normalized to its own input samples. ^*P ≤ 0.05, ^***P ≤ 0.001. (C) Mass spectrometry analysis result showing different H3K4me1/me2/me3 level in control cells. There were three independent iPSC samples in the analysis (Control1, Control2 and Control3). Control1 and Control2 were reprogrammed from two different healthy control renal epithelial cells, and Control3 were reprogrammed from a healthy control fibroblast line. Each sample was extracted with three replicates, where replicates were the same iPSC line grown in separate dishes with different passage number, and underwent independent histone extraction and mass spectrometry analysis. (D) Integrative Genomics Viewer (IGV) map snapshot of ChIPseq peaks identified at the 19q13.33 locus (hg38) in control and KS case iPSCs. There were two independent samples in the analysis (Mom and KS1). Each sample was extracted with three replicates for DNA samples, where replicates were the same iPSC line grown in separate dishes with different passage number, and underwent independent chromatin immunoprecipitation and PCR analysis. Each replicate signal was normalized to its own input samples. (E) IGV map snapshot of ChIPseq peaks identified at the 19q13.42 locus (hg38) in control and KS case iPSCs. (F) IGV map snapshot of ChIPseq peaks identified at the 14q32.33 locus (hg38) in control and KS case iPSCs.