Highly Parallel SNP Genotyping Reveals High-resolution Landscape of Mono-allelic Ube3a Expression Associated with Locus-wide Antisense Transcription
ABSTRACT: Genomic imprinting is a mammalian-specific gene expression regulation system that distinguishes two parental alleles and yields parent-origin-specific gene expression. It has been identified approximately 90 imprinted gene loci in the mouse genome thus far. One of the molecular bases that establish genomic imprinting is through endogenous antisense transcription. In several imprinted loci, antisense transcription is observed in a repressive allele, probably contributing to the parent-origin-specific gene expression establishment. We investigated the allele- and strand-specific transcriptional dynamics of a megabase-wide genomic region of mouse Ube3a (ubiquitin protein ligase E3A), which is maternally expressed in a tissue-specific manner, by means of a highly parallel SNP genotyping platform that targets the tissue transcriptome. We successfully observed higher resolution transcriptional activity in the vicinity, including brain-specific widespread antisense transcription. We have listed up SNP sites within Ube3a-Snurf/Snrpn region between C57BL/6J and MSM/Ms. SNP sites were loaded onto Illumina GoldenGate Assay platform, and were assayed by targeting total RNA came from brain and liver of F1 hybrid mice.
Project description:We carried out the analyses of chromosome variations between low-grade and high-grade gliomas in Chinese population. We found out the differences in chromosomes, cytobands, genes, pathways and GO functions. To identify the glioma tissue-specific genomic alterations and compare the genomic variations between low-grade and high-grade gliomas.
Project description:We investigated the allele- and strand-specific transcriptional landscape of a megabase-wide genomic region of mouse Ube3a (ubiquitin protein ligase E3A) by means of a highly parallel SNP genotyping platform. We have successfully identified maternal-specific expression of Ube3a and its antisense counterpart (Ube3a-ATS) in brain, but not in liver. Because of the use of inter-subspecies hybrid mice, this megabase-wide analysis provided high-resolution picture of the transcriptional patterns of this region. First, we showed that brain-specific maternal expression of Ube3a is restricted to the second half part of the locus, but is absent from the first half part. Balance of allelic expression is altered in the middle of the locus. Second, we showed that expression of the brain-specific Ube3a-ATS appeared to be terminated in the region upstream to the Ube3a transcription start site. The present study highlights the importance of locus-wide competition between sense and antisense transcripts. Overall design: We have listed up SNP sites within Ube3a-Snurf/Snrpn region between C57BL/6J and MSM/Ms. SNP sites were loaded onto Illumina GoldenGate Assay platform, and were assayed by targeting total RNA came from brain and liver of F1 hybrid mice.
Project description:This strand-specific array is performed to characterize expression features of Ube3a-ATS, including its imprinting status, its exon-intron structure, its transcriptional initiation and termination site as well as its polyadenylation status. The array contains reverse-complementary probes detecting transcripts from both strands and therefore we are able to pick up signal from both Ube3a sense and antisense. By comparing wild-type with various mutants, and total RNA with polyA RNA, we concluded that Ube3a-ATS is a paternally imprinted gene covering the whole gene body of Ube3a in the antisense orientation. It does not have an obvious exon-intron structure. Its transcription initiates at Snrpn major promoter and terminates ~40kb upstream of Ube3a transcriptional start site. Total RNA from Snrpn-Ube3a maternal deletion mutant (del s-u/+), its wild-type littermate, paternal deletion mutant and its wildtype littermate were analyzed. Mutant mice with S-U maternal deletion and Snrpn promoter paternal deleiton which leads to depletion of Ube3a-ATS (del s-u/0.9 and del s-u/4.8) were also analyzed. polyA RNA was purified from the sample 2 and sample 4. All eight samples were hybridized to the custom 8X60k Agilent CGH array.
Project description:There were four groups:control A/J mice;control B6 mice;SS2-infected A/J mice;SS2-infected B6 mice.The RNA samples of four groups were sent to Biostar Genechip Inc. (Shanghai, China) for microarray hybridization. The pooled RNA sample from each group was hybridized to one Illumina mouse Genome Beadchip Array (catalog number 5022612022, Mouse WG-6_V2, Illumina). Therefore, four BeadChips were used in total, one for each of the A/J and B6 infected and control mice groups. Biotin-labeled cRNA preparation and hybridization were performed as described previously. The arrays were scanned on an Illumina BeadStation 500 System and the hybridization data analyzed using Illumina BeadStudio software. The following filtering criteria were used for selection of differentially expressed genes: positive gene in either test or control, and test DiffScore e +20 or dc20. The differentially expressed genes were selected by comparing the following groups: SS2-infected A/J vs. SS2-infected B6; control A/J vs. control B6; SS2-infected A/J vs. control A/J; SS2-infected B6 vs. control B6. Four RNA samples were hybridized with four BeadChips of Illumina. They were respectively control A/J mice, control B6 mice, SS2-infected A/J mice, and SS2-infected B6 mice.The differentially expressed genes were selected by comparing the following groups: SS2-infected A/J vs. SS2-infected B6; control A/J vs. control B6; SS2-infected A/J vs. control A/J; SS2-infected B6 vs. control B6.In the raw and normalized date, A/J1 represents control A/J, A/J2 represents SS2-infected A/J, B61 represents control B6, B62 represents SS2-infected B62.
Project description:We profiled genome-wide gene expression of 170 individual mid-gestation (embryonic day 11.5) whole mouse embryos derived from a 2-generation interspecies mouse cross and asked to what extent genetic variation drives four important parameters of regulatory architecture: allele-specific expression (ASE), imprinting, trans-regulatory effects, and maternal effect. The inbred strain C57BL/6J and wild-derived inbred strain CAST/EiJ were used in reciprocal crosses to generate F1 embryos. F1 progeny were backcrossed to C57BL/6J in reciprocal crosses to generate 154 N2 embryos. We employed a backcross design, in which N2 offspring have genotypically distinct parents, to enable comparison of gene expression for offspring from each side of the reciprocal cross. Our findings demonstrate that genetic variation contributes to widespread gene expression differences during mammalian embryogenesis. Transcriptome analysis of E11.5 mouse embryos: 16 F1 embryos from reciprocally crossed C57BL/6J and CastEi/J parents; and 154 N2 embryos from reciprocal backcross of F1s to the C57BL/6J parent.
Project description:High throughput RNA Sequencing has revealed that the human genome is widely transcribed. However, the extent of natural antisense transcription, the molecular mechanisms by which natural antisense transcripts (NATs) might affect their cognate sense genes, and the role of NATs in cancer are less well understood. Here, we use strand-specific paired-end RNA sequencing (ssRNASeq) on a cohort of 376 cancer patients covering 9 tissue types to comprehensively characterize the landscape of antisense expression. Our results reveal that greater than 60% of annotated transcripts have measureable antisense expression and the expression of sense and antisense transcript pairs is in general positively correlated. Furthermore, by studying the expression of sense/antisense pairs across tissues we identify lineage-specific, ubiquitous and cancer-specific antisense loci. Our results raise the possibility that NATs participate in the regulation of well-known tumor suppressors... (for more see dbGaP study page.)
Project description:This SuperSeries is composed of the following subset Series: GSE20989: Mesothelioma integrative genomics: DNA methylation GSE21057: Copy number alterations in pleural mesothelioma Refer to individual Series
Project description: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. Overall design: triplicate siRNA knockdowns of UBE3A (siUBE3A) or control (siCTL) in a human neuronal cell line (SH-SY5Y) and a chromosome duplication model of Dup15q syndrome (SH(15M)). From these cell cultures, we isolated DNA for WGBS, RNA for RNA-seq, and chromatin for ChIP-seq of one histone mark of active promoters (H3K4me3) and one histone variant (H2A.Z) of active promoters and poised genes.
Project description:Small ubiquitin-like modifiers (SUMOs) are post-translational modifications that play crucial roles in most cellular processes. While methods exist to study exogenous SUMOylation, large-scale characterization of endogenous SUMO has remained technically daunting. Here, we describe a proteomics approach facilitating system-wide and in vivo identification of lysines modified by endogenous and native SUMO2/3. We identified 14,869 endogenous SUMO sites in human cells during heat stress and proteasomal inhibition, and mapped 1,963 SUMO sites across eight mouse tissues; brain, heart, kidney, lung, liver, muscle, spleen, and testis. Quantification of the SUMO equilibrium highlighted striking differences in SUMO metabolism, between cells and tissues. Targeting preferences of SUMO varied across different organ types, coinciding with markedly differential SUMOylation states of all enzymes involved in the SUMO conjugation cascade. Collectively, our systemic investigation details the SUMOylation architecture across species and organs and provides a resource of endogenous SUMOylation sites on factors important in organ-specific functions and disease.
Project description:Chronic antibody-mediated rejection (CAMR) represents the main cause of kidney graft loss, but its pathogenesis is unclear. In order to uncover the molecular mechanisms underlying this condition, we characterized the molecular signature of circulating peripheral blood mononuclear cells and, separately, of CD4+ T lymphocytes isolated from CAMR patients compared to kidney transplant recipients with normal graft function and histology. In total peripheral lympho-monocytes, forty-five genes resulted differentially expressed between the two groups, most of them were up-regulated in CAMR and were involved in type I interferon signaling. In addition, in the same set of patients, 16 microRNAs resulted down-regulated in CAMR subjects compared to controls: 4 were predicted modulators of 6 mRNAs identified in the transcriptional analysis. In silico functional analysis supported the involvement of type I interferon signaling. To further confirm this hypothesis, we investigated the transcriptomic profiles of CD4+ T lymphocytes in an independent group of patients and we observed that the activation of type I interferon signaling was a specific hallmark of CAMR. In addition, in CAMR patients we detected a reduction of circulating BDCA2+ dendritic cells, the natural type I interferon- producing cells and their recruitment into the graft along with an increased expression of MXA, a type I interferon-induced protein, at tubulointerstitial and vascular level. In conclusion, our data suggest that type I interferon signaling may represent the molecular signature of CAMR. For microarray analysis, we studied 8 patients included into the control-group and 10 included into the study group. The control group was represented by renal transplant recipients undergoing protocol graft biopsies, with normal renal function and histology, in the absence of circulating anti-HLA antibodies. Study group patients showed clinical and histological evidence of CAMR according to Banff 2011 criteria.