UBE3A-mediated regulation of imprinted genes and epigenome-wide marks in human neurons - PubMed (original) (raw)

UBE3A-mediated regulation of imprinted genes and epigenome-wide marks in human neurons

S Jesse Lopez et al. Epigenetics. 2017.

Abstract

The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition.

Keywords: DNA methylation; autism; chromatin; epigenetic; histone modification; imprinting.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Multi-layered genomics analysis of SH-SY5Y and SH(15M) UBE3A siRNA knockdown reveals how altered UBE3A levels impact epigenomic patterns. (A) UBE3A protein levels quantified from Western blot in SH(15M) and SH-SY5Y for siRNA control and UBE3A knockdown relative to GAPDH (blot in Fig. S1). Labeled are the four comparison groups used for differential genomic analyses. Error bars represent the mean ± SEM of three replicates. Significance by 2-way ANOVA P < 0.0001. (B) Study design: following UBE3A siRNA knockdown and control treatment, each triplicate culture was harvested for DNA, RNA, or chromatin to assay DNA methylation, differentially expressed genes, or histone peaks, respectively. (C) Table outlining results of the genomic assays for each comparison group by UBE3A levels. Each of the four comparisons generated independent lists of genes that overlapped with differential marks from each of the genomic assays.

Figure 2.

Figure 2.

Overlap of dataset gene lists shows effect of altered UBE3A levels on the epigenome. Size-dependent Venn diagrams show a graphical representation of the gene lists generated by each genomic assay for each comparison group. Circles indicate the relative number of genes for each dataset and show the level of overlap between datasets within each comparison group.

Figure 3.

Figure 3.

Gene Ontology enrichment reveals that UBE3A effects epigenetic regulation of neuronal development and imprinted genes. (A) GO enrichment heatmap for DMR-associated gene lists for each comparison group. Significance of term enrichment by FDR q-value for each group is indicated by colored key. (B) GO enrichment heatmap for differential H3K4me3-associated gene lists for chr15M, KDSH, and chr15M-minus comparison groups. Significance of term enrichment by FDR q-value for each group is indicated by colored key. (C) GO enrichment heatmap for differentially expressed genes for each comparison group. Significance of term enrichment by Bonferroni P value for each group is indicated by colored key. (D) GO enrichment for differential H2A.Z-associated genes in the chr15M comparison group. Significance of term enrichment by FDR q-value is represented by -log10 bar values. (E) GO enrichment for differential H2A.Z-associated genes in the KDSH comparison group. Significance of term enrichment by FDR q-value is represented by -log10 bar values.

Figure 4.

Figure 4.

UBE3A knockdown in SH-SY5Y and SH(15M) changes H2A.Z promoter and gene body levels. (A) Percent of genes with high or low promoter H2A.Z and high or low gene body H2A.Z from quadrants in Figure S5. Percent of total genes and percent of genes with differential H2A.Z peaks are shown for each cell type and siRNA condition. Comparisons indicate significance of the change in number of genes following UBE3A knockdown by Fisher's exact test. (* P < 0.01, ** P < 0.001, **** P ≅ 0) (B) Representative UCSC Genome Browser snapshot of IGF2 showing tracks of each genomic dataset and highlighting increase of H2A.Z at imprinted genes.

References

    1. Christensen DL. Prevalence and characteristics of autism spectrum disorder among children aged 8 years—autism and developmental disabilities monitoring network, 11 sites, United States, 2012. MMWR. Surveill. Summ. 2016;65:1-23. doi: 10.15585/mmwr.ss6503a1. -DOI -PMC -PubMed
    1. Bourgeron T. From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat Rev Neurosci. 2015;16:551-563. doi: 10.1038/nrn3992. -DOI -PubMed
    1. Hallmayer J, Cleveland S, Torres A. et. al. Genetic heritability and shared environmental factors among twin pairs with autism. Arch. Gen. Psychiatry. 2011;68:1095-1102. doi: 10.1001/archgenpsychiatry.2011.76. -DOI -PMC -PubMed
    1. Krumm N, O'Roak BJ, Shendure J, Eichler EE. A de novo convergence of autism genetics and molecular neuroscience. Trends Neurosci. 2014;37:95-105. doi: 10.1016/j.tins.2013.11.005. -DOI -PMC -PubMed
    1. De Rubeis S, He X, Goldberg AP, Poultney CS, Samocha K, Cicek AE, Kou Y, Liu L, Fromer M, Walker S, et al.. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature. 2014;515:209-215. doi: 10.1038/nature13772. -DOI -PMC -PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources