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

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.     

Linkage disequilibrium at the Angelman syndrome gene UBE3A in autism families.

Autistic disorder is a neurodevelopmental disorder with a complex genetic etiology. Observations of maternal duplications affecting chromosome 15q11-q13 in patients with autism and evidence for linkage and linkage disequilibrium to markers in this region in chromosomally normal autism families indicate the existence of a susceptibility locus. We have screened the families of the Collaborative Linkage Study of Autism for several markers spanning a candidate region covering approximately 2 Mb and including the Angelman syndrome gene (UBE3A) and a cluster of gamma-aminobutyric acid (GABA(A)) receptor subunit genes (GABRB3, GABRA5, and GABRG3). We found significant evidence for linkage disequilibrium at marker D15S122, located at the 5' end of UBE3A. This is the first report, to our knowledge, of linkage disequilibrium at UBE3A in autism families. Characterization of null alleles detected at D15S822 in the course of genetic studies of this region showed a small (approximately 5-kb) genomic deletion, which was present at somewhat higher frequencies in autism families than in controls.     

Angelman syndrome and severe infections in a patient with de novo 15q11.2–q13.1 deletion and maternally inherited 2q21.3 microdeletion

Angelman syndrome is a neurodevelopmental disorder characterized by mental retardation, severe speech disorder, facial dysmorphism, secondary microcephaly, ataxia, seizures, and abnormal behaviors such as easily provoked laughter. It is most frequently caused by a de novo maternal deletion of chromosome 15q11–q13 (about 70–90%), but can also be caused by paternal uniparental disomy of chromosome 15q11–q13 (3–7%), an imprinting defect (2–4%) or in mutations in the ubiquitin protein ligase E3A gene UBE3A mostly leading to frame shift mutation. In addition, for patients with overlapping clinical features (Angelman-like syndrome), mutations in methyl-CpG binding protein 2 gene MECP2 and cyclin-dependent kinase-like 5 gene CDKL5 as well as a microdeletion of 2q23.1 including the methyl-CpG binding domain protein 5 gene MBD5 have been described. Here, we describe a patient who carries a de novo 5 Mb-deletion of chromosome 15q11.2–q13.1 known to be associated with Angelman syndrome and a further, maternally inherited deletion 2q21.3 (~ 364 kb) of unknown significance. In addition to classic features of Angelman syndrome, she presented with severe infections in the first year of life, a symptom that has not been described in patients with Angelman syndrome. The 15q11.2–q13.1 deletion contains genes critical for Prader–Willi syndrome, the Angelman syndrome causing genes UBE3A and ATP10A/C, and several non-imprinted genes: GABRB3 and GABRA5 (both encoding subunits of GABA A receptor), GOLGA6L2, HERC2 and OCA2 (associated with oculocutaneous albinism II). The deletion 2q21.3 includes exons of the genes RAB3GAP1 (associated with Warburg Micro syndrome) and ZRANB3 (not disease-associated). Despite the normal phenotype of the mother, the relevance of the 2q21.3 microdeletion for the phenotype of the patient cannot be excluded, and further case reports will need to address this point.     

Prader-Willi-Syndrom und Angelman-Syndrom

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic disorders caused by the loss of function of imprinted genes in the chromosomal region 15q11q13. An approximately 2 Mb region inside 15q11q13 is subject to genomic imprinting. As a consequence the maternal and paternal copies in this region are different in DNA methylation and gene expression. The most frequent genetic lesions in both disorders are an interstitial de novo deletion of the chromosomal region 15q11q13, uniparental disomy 15, an imprinting defect or, in the case of AS, a mutation of the UBE3A gene. Microdeletions in a small number of patients with PWS and AS with an imprinting defect have led to the identification of the chromosome 15 imprinting centre (IC) upstream of the SNURF-SNRPN gene, which acts in cis to regulate imprinting in the whole 15q imprinted domain. The IC consists of two critical elements: one in the more centromeric part which is deleted in patients with AS and which is thought to be responsible for the establishment of imprinting in the female germ line, and one in the more telomeric part which is deleted in patients with PWS and which is required to maintain the paternal imprint.     

Recent assembly of an imprinted domain from non-imprinted components

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how—and especially why—epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105–180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B′. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.     

Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes

Perturbations in activity and dosage of the UBE3A ubiquitin-ligase have been linked to Angelman syndrome and autism spectrum disorders. UBE3A was initially identified as the cellular protein hijacked by the human papillomavirus E6 protein to mediate the ubiquitylation of p53, a function critical to the oncogenic potential of these viruses. Although a number of substrates have been identified, the normal cellular functions and pathways affected by UBE3A are largely unknown. Previously, we showed that UBE3A associates with HERC2, NEURL4, and MAPK6/ERK3 in a high-molecular-weight complex of unknown function that we refer to as the HUN complex (HERC2, UBE3A, and NEURL4). In this study, the combination of two complementary proteomic approaches with a rigorous network analysis revealed cellular functions and pathways in which UBE3A and the HUN complex are involved. In addition to finding new UBE3A-associated proteins, such as MCM6, SUGT1, EIF3C, and ASPP2, network analysis revealed that UBE3A-associated proteins are connected to several fundamental cellular processes including translation, DNA replication, intracellular trafficking, and centrosome regulation. Our analysis suggests that UBE3A could be involved in the control and/or integration of these cellular processes, in some cases as a component of the HUN complex, and also provides evidence for crosstalk between the HUN complex and CAMKII interaction networks. This study contributes to a deeper understanding of the cellular functions of UBE3A and its potential role in pathways that may be affected in Angelman syndrome, UBE3A-associated autism spectrum disorders, and human papillomavirus-associated cancers.     

Paternal expression of a novel imprinted gene, Peg12/Frat3, in the mouse 7C region homologous to the Prader-Willi syndrome region.

Paternally expressed imprinted genes (Pegs) were systematically screened by comparing gene expression profiles of parthenogenetic and normal fertilized embryos using an oligonucleotide array. A novel imprinted gene, Peg12/Frat3, was identified along with 10 previously known Pegs. Peg12/Frat3 is expressed primarily in embryonic stages and might be a positive regulator of the Wnt signaling pathway. It locates next to the Zfp127 imprinted gene in the mouse 7C region, which has syntenic homology to the human Prader-Willi syndrome region on chromosome 15q11-q13, indicating that this imprinted region extends to the telomeric side in the mouse.     

Denaturing high-performance liquid chromatography for the detection of mutations and polymorphisms in UBE3A

Angelman syndrome (AS) is caused by maternal deficiency of UBE3A, the gene encoding E6-AP ubiquitin-protein ligase. Our objectives were to develop conditions for denaturing high-performance liquid chromatography (dHPLC) analysis of UBE3A and to compare the sensitivity to direct genomic sequencing. Genomic DNA was obtained from 17 Angelman patients with known mutations and from 120 normal controls. DNA was amplified for the 10 coding exons and 6 upstream noncoding exons of UBE3A. Using dHPLC, the mutations previously identified in 17 Angelman patients were all easily detected using a single dHPLC condition for most exon-containing fragments. An analysis of all 16 exons in 120 normal controls identified 15 other DNA alterations of varying frequency, all of which are assumed to be benign. We conclude that dHPLC is a reliable and convenient method for detecting mutations in UBE3A causing Angelman syndrome. No disease-causing mutations were found in the noncoding exons.     

Imprinting mutations suggested by abnormal DNA methylation patterns in familial Angelman and Prader-Willi syndromes.

The D15S9 and D15S63 loci in the Prader-Willi/Angelman syndrome region on chromosome 15 are subject to parent-of-origin-specific DNA methylation. We have found two Prader-Willi syndrome families in which the patients carry a maternal methylation imprint on the paternal chromosome. In one of these families, the patients have a small deletion encompassing the gene for the small nuclear ribonucleoprotein polypeptide N, which maps 130 kb telomeric to D15S63. Furthermore, we have identified a pair of nondeletion Angelman syndrome sibs and two isolated Angelman syndrome patients who carry a paternal methylation imprint on the maternal chromosome. These Angelman and Prader-Willi syndrome patients may have a defect in the imprinting process in 15q11-13. We propose a model in which a cis-acting mutation prevents the resetting of the imprinting signal in the germ line and thus disturbs the expression of imprinted genes in this region.     

Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.     

Clinical,cytogenetical and molecular analyses of Angelman syndrome

A total of 95 patients suspected with the clinical diagnosis of AS were evaluated and 37 cases (39%) were confirmed by cytogenetic or molecular studies as affected by Angelman syndrome. The clinical analysis was performed according to a specific clinical protocol for the diagnosis of AS. Cytogenetical analysis was used to detect chromosome rearrangements by determining the karyotype in lymphocytes by GTG banding and revealed an abnormal karyotype in two cases (5.4%), both of them presenting a new pericentromeric inversion in chromosome 15. Molecular analyses included determination of DNA methylation within the 15q11-13 region by Southern blotting and microsattelite analysis within the 15q11-13 region by PCR and the UBE3A gene was also studied by mutational screening. In 16 cases (43.2%) a de novo deletion was detected in the maternal chromosome 15:3 cases (8.1%) presented imprinting defect at the 15q11-13 region; one case is due to a paternal uniparental dissomy (2.7%) and another two cases showed a inherited mutation at the UBE3A gene (5.4%). Thirteen cases (35.1%) showed no deletion, no UPD, no imprinting defect, no UBE3A mutation and the diagnosis of AS could be ruled out in 58 patients. The objective of the present work was to describe the clinical and laboratory protocols employed at our laboratory in order to establish the AS study. We conclude that the protocols employed here were efficient for the diagnosis of AS, a frequently underdiagnosed pathology.