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

<p>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 <i>UBE3A</i>, which is maternally expressed exclusively in neurons. <i>UBE3A</i> 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 <i>UBE3A</i> 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.</p

Scoliosis in transgenic founder zebrafish expressing <i>lbx1b</i> using the <i>GATA2</i> minimal promoter and the <i>lbx1b</i> enhancer.

<p>(A) The transgene construct is shown. The <i>GATA2</i> minimal promoter and <i>lbx1b</i> enhancer cooperatively drive <i>lbx1b</i> expression. (B) Dorsal views of live <i>GATA2-1b</i>:<i>MCS</i> (ctrl) or <i>GATA2-1b</i>:<i>lbx1b</i> (<i>lbx1b</i>) injected embryos with 10–13 somites. Somite arrangement is bilateral and symmetric in control embryos, but asymmetrical in <i>lbx1b</i> embryos. The yellow arrows indicate the mediolateral length of somites. (C) Dorsal views of 6 dpf larvae. The red and blue dotted lines indicate the boundaries of the dorsal melanophore stripes and the trunk, respectively. A displaced dorsal melanophore stripe was observed in <i>lbx1b</i> larvae. (D) Lateral views of alizarin red-stained larvae at 21 dpf. The white and yellow arrows indicate the hemivertebrae and block vertebra that developed from the deformed notochord in <i>lbx1b</i> larva, respectively. (E) Dorsal views and micro-computed tomography (μCT) analysis of adult zebrafish. Scoliosis was observed in adult fish grown from embryos with mild notochord deformities. The coronal and sagittal planes are reconstructed from μCT images of the fish in the upper panels, each showing a gross appearance. The red arrows indicate vertebral malformation. (F) Diagram of zebrafish growth stages. Continuous observation of one <i>GATA2</i>-<i>1b</i>:<i>lbx1b</i>-injected larva with a displaced dorsal melanophore stripe, but without notochord deformation, from 6 to 90 dpf. The red and green dotted lines indicate the dorsal middle lines and the right upper boundary of the lateral stripe, respectively. Progressive scoliosis with rotation of the longitudinal axis of the body was observed after 55 dpf. E, embryo; L, larva; J, juvenile; A, adult. (G) μCT analysis of the three-dimensional structure of the spine of the same zebrafish in (F) showing scoliosis. Cobb’s angle was measured in the coronal plane. Scale bars in (B): 200 μm; (C): 1 mm or 100 μm; (D): 500 μm; and (F): 1 mm.</p

Body curvature induced by <i>lbx1b</i> overexpression under the control of the heat shock-inducible <i>hsp70I</i> promoter.

<p>(A) The constructs in the Gal4/UAS—based bidirectional expression system. The F0 driver transgenic carriers were crossed with the F0 responder transgenic carriers to produce <i>Tg(hsp</i>:<i>Gal-VP</i>;<i>EGFP</i>:<i>UAS</i>:<i>lbx1b</i>) F1. Heat shock (HS) treatment activated the <i>hsp70I</i> promoter in the transgene driver (Driver), which expresses <i>Gal4-VP16</i>. Gal4-VP16 protein bound to UAS on the transgene responder (Responder) and activates the expression of <i>lbx1b</i> and <i>EGFP</i> via E1b minimal promoters. (B) Expression of lbx1 protein in 48 hpf transgenic zebrafish with (GFP⁺) or without HS (GFP^-). (C) Body curvature of transgenic 48 hpf zebrafish that received HS at 4 hpf. The severity of body curvature was correlated with GFP intensity. (D) Incidence of body curvature at 48 hpf in embryos with (HS+EGFP⁺, n = 85) and without (HS+EGFP^-, n = 129) EGFP expression and without HS (n = 79). The incidence of curvature was significantly increased in the HS+EGFP⁺ embryos (*<i>p</i> < 0.01). (E) <i>lbx1b</i> expression level and body curvature. A linear regression line was obtained between the relative fluorescence intensity of GFP and the severity of body curvature quantified by the angle <b>θ</b>: [<b>θ</b>/degree] = 21.70 × [relative fluorescence intensity]– 3.37, with the slope coefficient different from 0 (<i>p</i> < 0.01). This model accounted for 92.2% of the <b>θ</b> variance.</p

Defective convergent extension in <i>lbx1b</i> overexpressing zebrafish embryos.

<p>(A) A diagram of convergent extension during zebrafish gastrulation. A, P, D, and V indicate anterior, posterior, dorsal, and ventral sides, respectively. The green and red arrows indicate the directions of convergence and extension movements, respectively. (B) <i>In situ</i> hybridization for germ layer markers at the early stages of development. Dorsal views of embryos injected with buffer as control (ctrl) or <i>lbx1b</i> mRNA (<i>lbx1b</i>). Upper panels show marker expression of <i>hgg1</i> (rostral mesoderm), <i>ntl</i> (notochord), and <i>dlx3b</i> (border between neural and non-neural ectoderm, black arrowheads) at the tail bud stage. Middle panels show the expression of <i>papc</i> (paraxial mesoderm) at 11 hpf. Lower panels show the expression of <i>uncx4</i> (somite) at 13 hpf. The scale bar represents 200 μm. (C) Incidence of defective expression patterns. Significant differences (*<i>p</i> < 0.01) were observed for the germ layer markers. The numbers of control and <i>lbx1b</i> zebrafish embryos were 36 and 30 for <i>hgg1</i>/<i>ntl</i>/<i>dlx3b</i>, 32 and 28 for <i>papc</i>, and 28 and 27 for <i>uncx4</i>, respectively. (D) Lateral views of 16 hpf normal sibling (ctrl) and <i>Tg(hsp</i>:<i>Gal4-VP16</i>:<i>EGFP</i>:<i>UAS</i>:<i>lbx1b)</i> (<i>lbx1b</i>) embryos upon heat shock at 4 hpf. The red and black arrowheads indicate the border between the yolk and the rostral part and the yolk and the caudal part, respectively. The yellow lines indicate the developing eyes. (E) Quantitative analysis of extension movement. Progression of the movement was quantified by the angle <b>θ</b> at 16 hpf for sibling control (ctrl; n = 13) and transgenic (<i>lbx1b</i>; n = 11) embryos. Convergent extension movement was significantly delayed in <i>lbx1b</i> embryos (<i>p</i> < 0.01).</p

Time-dependent induction of somite mediolateral elongation and body curvature by <i>lbx1b</i> overexpression.

<p>(A) A schematic diagram of zebrafish early developmental stages. Convergent extension (CE) is mainly involved in gastrulation. (B) Comparison of somite mediolateral length (16 hpf) between <i>Tg(hsp</i>:<i>Gal-VP; UAS</i>:<i>EGFP)</i> with heat shock (HS) treatment at 4 hpf (ctrl 4 hpf), <i>Tg(hsp</i>:<i>Gal-VP; EGFP</i>:<i>UAS</i>:<i>lbx1b)</i> with HS at 4 hpf (<i>lbx1b</i> 4 hpf), and <i>Tg(hsp</i>:<i>Gal4-VP; EGFP</i>:<i>UAS</i>:<i>lbx1b)</i> with HS at 12 hpf (<i>lbx1b</i> 12 hpf). A significant elongation of somite mediolateral length (yellow arrow in the upper panels) was observed in the <i>lbx1b</i> embryos at 4 hpf. The scale bar represents 100 μm. (C) Quantitative analysis of somite mediolateral length in embryos at 16 hpf. A significant difference was observed between ctrl at 4 hpf (n = 16) and <i>lbx1b</i> at 4 hpf (n = 13), and <i>lbx1b</i> at 4 hpf (n = 13) and <i>lbx1b</i> at 12 hpf (n = 14), *<i>p</i> < 0.01. (D) Comparison of body curvature (48 hpf) between <i>Tg(hsp</i>:<i>Gal-VP; UAS</i>:<i>EGFP)</i> with HS treatment at 4 hpf (ctrl 4 hpf), <i>Tg(hsp</i>:<i>Gal-VP</i>:<i>EGFP</i>:<i>UAS</i>:<i>lbx1b)</i> with HS at 4 hpf (<i>lbx1b</i> 4 hpf), and <i>Tg(hsp</i>:<i>Gal-VP</i>:<i>EGFP</i>:<i>UAS</i>:<i>lbx1b)</i> with HS at 12 hpf (<i>lbx1b</i> 12 hpf). More severe curvature of the body axis was induced in <i>lbx1b</i> embryos at 4 hpf than in <i>lbx1b</i> embryos at 12 hpf. The scale bar represents 1 mm (E) Quantitative analysis of body curvature at 48 hpf. A significant difference was observed between ctrl 4 hpf (n = 13) and <i>lbx1b</i> 4 hpf (n = 10), and between <i>lbx1b</i> 4 hpf (n = 10) and <i>lbx1b</i> 12 hpf (n = 11), *<i>p</i> < 0.05. The severity of body curvature was quantified by the angle described in the legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005802#pgen.1005802.g003" target="_blank">Fig 3</a>. Both driver transgenic and responder transgenic fish were F2 lines.</p

Body curvature induced by injection of <i>lbx</i> mRNAs.

<p>(A) Protein structure of <i>Lbx</i> family members with an engrailed homology domain (Eh) and homeobox domain (Hd). (B) Dorsal views of live embryos (48 hpf) injected with zebrafish <i>lbx1a</i>, <i>lbx1b</i>, or <i>lbx2b</i> mRNA, as well as human <i>LBX1</i> or <i>FLJ41350</i> mRNA, in comparison with those injected with <i>lbx1aΔhd</i> mRNA which lacks the homeodomain, and <i>lbx1aΔeh</i>, <i>lbx1bΔeh</i>, and <i>lbx2Δeh</i> mRNA which lacks the engrailed domain. Buffer was injected as a control (ctrl). Severe curvature was observed in embryos injected with <i>lbx1a</i>, <i>lbx1b</i>, and <i>LBX1</i> mRNA, while mild body curvature was observed in those injected with <i>lbx2</i> mRNA, but no curvature was observed in those injected with <i>lbx</i> genes lacking the functional domains. Bending of the body axis was not observed in embryos injected with <i>FLJ41350</i>. (C) Quantitative analysis of the phenotype of embryos injected with different doses of mRNA. Body curvature occurred in <i>lbx1b</i>, <i>LBX1</i>, <i>lbx1a</i>, <i>and lbx2</i> embryos in a dose-dependent manner. <i>lbx1b</i> embryos presented with the highest incidence of curvature (45% for 50 pg, 59% for 100 pg, and 82% for 150 pg), and then <i>LBX1</i> (29% for 50 pg, 46% for 100 pg, and 64% for 150 pg), <i>lbx1a</i> (24% for 50 pg, 39% for 100 pg, and 51% for 150 pg), <i>and lbx2</i> (16% for 50 pg, 25% for 100 pg, and 38% for 150 pg). The incidence of curvature was less than 10% in <i>lbx1a</i>Δhd, <i>lbx1a</i>Δeh, <i>lbx1b</i>Δeh, <i>lbx2</i>Δeh, and <i>FLJ41350</i> embryos regardless of dose. The data represent the mean ± standard error of three independent injections (n = 27–37). (D) Dorsal views of live larva are shown. <i>lbx1b</i>^<i>+/-</i> and <i>lbx1b</i>^<i>-/-</i> mutants as well as <i>lbx2</i>^<i>+/-</i> and <i>lbx2</i>^<i>-/-</i> mutants are comparable to WT with a straight body axis. The scale bar represents 500 μm.</p

Downregulation of <i>wnt5b</i> during gastrulation by <i>lbx1b</i> overexpression.

<p>(A) <i>In situ</i> hybridization for non-canonical Wnt/PCP ligands (<i>wnt5b</i> and <i>wnt11</i>) in control and <i>lbx1b</i>-overexpressing embryos at 90% epiboly. Dorsal views (anterior to the top) for <i>wnt5b</i> and <i>wnt11</i> of embryos injected with buffer (ctrl) or <i>lbx1b</i> mRNA (<i>lbx1b</i>). (B) Incidence of the defective expression patterns observed in the embryos shown in panel A. Significant differences (*<i>p</i> < 0.01) were observed for <i>wnt5b</i>. The numbers of control and <i>lbx1b</i> zebrafish embryos were 22 and 21 for <i>wnt5b</i> and 23 and 22 for <i>wnt11</i>, respectively. NS: not significant. (C) Decreased <i>wnt5b</i> expression in 90% epiboly embryos injected with <i>lbx1b</i> mRNA by quantitative RT-PCR assays. *<i>p</i> < 0.01. (D) <i>In vivo</i> luciferase assay in 90% epiboly embryos injected with control vector (<i>luc</i>) or the putative promoter regions (P1 and P2) of zebrafish <i>wnt5b</i>. P2 showed higher transcriptional activity than P1. Co-injection of <i>lbx1b</i> mRNA in P2 significantly repressed the transcriptional activity. *<i>p</i> < 0.01. AU, arbitrary unit.</p

Characterization of rs11190870.

<p>(A) A schematic representation of the most significantly associated SNP, rs11190870, and the surrounding genome region with the chromatin fragments obtained by the 3C assay. Cross-linked chromatin was digested with <i>Sau</i>3AI (vertical red lines). The green broken lines indicate primer positions. The blue lines indicate vertebrate conservation. The arrowheads indicate PCR primers. (B) Electrophoretic mobility shift assay of rs11190870 using 17-bp DIG-labeled probes for risk (T) and non-risk (C) alleles. Excess unlabeled probes were used as competitors. The arrow indicates that some nuclear factor(s) bound with higher affinity to the risk allele. (C) Physical interaction of rs11190870 and the nearby genomic region with the promoter region of <i>LBX1-FLJ41350</i> in the 3C assay (lane 1: rhabdomyosarcoma cells, lane 2: A172 cells, lane 3: HeLa cells). The arrow indicates specific PCR products of the expected sizes derived from the indicated primer sets in (A). (D) Transcriptional activity of the <i>LBX1-FLJ41350</i> promoter region. Luciferase activity of the promoter fragment (-917 to +153; direction of <i>LBX1</i> transcription) and its reverse fragment (direction of <i>FLJ41350</i> transcription) was measured in A172 cells. (E) The effect of the 590-bp conserved region containing rs11190870 on the <i>LBX1</i> promoter in HEK 293T cells. Higher activity was observed for the risk allele (T) construct than for the non-risk allele construct (C). Luciferase activity was normalized to the internal control and expressed as a ratio relative to the promoter-less construct (ctrl). The data in (D) and (E) represent the mean ± standard error of two independent experiments. *<i>p</i> < 0.05.</p