Showing papers by "Alexander Meissner published in 2011"

lincRNAs act in the circuitry controlling pluripotency and differentiation

Abstract: Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

Abstract: Genome-wide mapping of 5-methylcytosine is of broad interest to many fields of biology and medicine. A variety of methods have been developed, and several have recently been advanced to genome-wide scale using arrays and next-generation sequencing approaches. We have previously reported reduced representation bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich parts of the genome, thereby reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. The approach provides single-nucleotide resolution, is highly sensitive and provides quantitative DNA methylation measurements. This protocol should enable any standard molecular biology laboratory to generate RRBS libraries of high quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 bp) are size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome Analyzer. Note that alignment and analysis of RRBS sequencing reads are not covered in this protocol. The extremely low input requirements (10-300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the technique's single-nucleotide resolution extends RRBS to a wide range of biological and clinical samples and research applications. The entire process of RRBS library construction takes ∼9 d.

Genomic distribution and inter-sample variation of non-CpG methylation across human cell types.

Abstract: DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.

Global DNA Demethylation During Mouse Erythropoiesis in Vivo

Abstract: In the mammalian genome, 5'-CpG-3' dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific β-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease.

Functional genomics assay for characterizing pluripotent stem cell utility and safety

Abstract: The present invention generally relates set of reference data or "scorecard" for a pluripotent stem cell, and methods, systems and kits to generate a scorecard for predicting the functionality and suitability of a pluripotent stem cell line for a desired use. In some aspects, a method for generating a scorecard comprises using at least 2 stem cell assays selected from: epigenetic profiling, differentiation assay and gene expression assay to predict the functionality and suitability of a pluripotent stem cell line for a desired use. In some embodiments, the scorecard reference data can be compared with the pluripotent stem cells data to effectively and accurately predict the utility of the pluripotent stem cell for a given application, as well as any to identify specific characteristics of the pluripotent stem cell line to determine their suitability for downstream applications, such as for example, their suitability for therapeutic use, drug screening and toxicity assays, differentiation into a desired cell lineage, and the like.

lincRNAs act in the circuitry controlling pluripotency and differentiation

Abstract: Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

Analyse de génomique fonctionnelle pour caractérisation de l'utilité et de l'innocuité de cellules souches pluripotentes

Abstract: La presente invention concerne generalement un ensemble de donnees de reference ou une « carte de pointage » d'une cellule souche pluripotente, ainsi que des procedes, des systemes et des necessaires pour generer une carte de pointage pour la prediction de la fonctionnalite et du caractere approprie d'une lignee de cellules souches pluripotentes pour l'utilisation voulue. Sous certains aspects, un procede de generation d'une carte de pointage consiste a utiliser au moins 2 analyses de cellules souches, choisies parmi : le profilage epigenetique, l'analyse de differenciation et l'analyse d'expression genique pour predire la fonctionnalite et le caractere approprie d'une lignee de cellules souches pluripotentes pour l'utilisation voulue. Dans certains modes de realisation, les donnees de reference de carte de pointage peuvent etre comparees avec les donnees de cellules souches pluripotentes afin de predire efficacement et avec precision l'utilite de la cellule souche pluripotente pour une application donnee, ainsi que pour identifier des caracteristiques specifiques de la lignee de cellules souches pluripotentes pour determiner leur caractere approprie dans le cadre d'applications ulterieures, telles que par exemple leur caractere approprie a une utilisation therapeutique, a des essais de criblage pour la recherche de medicament et de toxicite, a une differenciation en une lignee cellulaire voulue et autres.