Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro methylated genomic DNA

<p>Abstract</p> <p>Background</p> <p>DNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP) assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of <it>in vitro </it>methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples.</p> <p>Findings</p> <p>We performed MeDIP assays using <it>in vitro </it>methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using <it>in vitro </it>methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.</p> <p>Conclusion</p> <p>We illustrate the use of <it>in vitro </it>methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.</p

Characterisation of Genome-Wide PLZF/RARA Target Genes

The PLZF/RARA fusion protein generated by the t(11;17)(q23;q21) translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear

Diversification and Molecular Evolution of ATOH8, a Gene Encoding a bHLH Transcription Factor

ATOH8 is a bHLH domain transcription factor implicated in the development of the nervous system, kidney, pancreas, retina and muscle. In the present study, we collected sequence of ATOH8 orthologues from 18 vertebrate species and 24 invertebrate species. The reconstruction of ATOH8 phylogeny and sequence analysis showed that this gene underwent notable divergences during evolution. For those vertebrate species investigated, we analyzed the gene structure and regulatory elements of ATOH8. We found that the bHLH domain of vertebrate ATOH8 was highly conserved. Mammals retained some specific amino acids in contrast to the non-mammalian orthologues. Mammals also developed another potential isoform, verified by a human expressed sequence tag (EST). Comparative genomic analyses of the regulatory elements revealed a replacement of the ancestral TATA box by CpG-islands in the eutherian mammals and an evolutionary tendency for TATA box reduction in vertebrates in general. We furthermore identified the region of the effective promoter of human ATOH8 which could drive the expression of EGFP reporter in the chicken embryo. In the opossum, both the coding region and regulatory elements of ATOH8 have some special features, such as the unique extended C-terminus encoded by the third exon and absence of both CpG islands and TATA elements in the regulatory region. Our gene mapping data showed that in human, ATOH8 was hosted in one chromosome which is a fusion product of two orthologous chromosomes in non-human primates. This unique chromosomal environment of human ATOH8 probably subjects its expression to the regulation at chromosomal level. We deduce that the great interspecific differences found in both ATOH8 gene sequence and its regulatory elements might be significant for the fine regulation of its spatiotemporal expression and roles of ATOH8, thus orchestrating its function in different tissues and organisms

Uncovering Enhancer Functions Using the α-Globin Locus

Over the last three decades, studies of the α- and β-globin genes clusters have led to elucidation of the general principles of mammalian gene regulation, such as RNA stability, termination of transcription, and, more importantly, the identification of remote regulatory elements. More recently, detailed studies of α-globin regulation, using both mouse and human loci, allowed the dissection of the sequential order in which transcription factors are recruited to the locus during lineage specification. These studies demonstrated the importance of the remote regulatory elements in the recruitment of RNA polymerase II (PolII) together with their role in the generation of intrachromosomal loops within the locus and the removal of polycomb complexes during differentiation. The multiple roles attributed to remote regulatory elements that have emerged from these studies will be discussed

An update on recent methods applied for deciphering the diversity of the noncoding RNA genome structure and function

International audienceThe explosion of high throughput sequencing technologies marked a turn in our way of understanding the complexity and diversity of the transcriptome, including noncoding transcription dependent on RNA polymerase II. Many new ncRNA populations were described in recent years, including for example TSS RNAs, lincRNAs, eRNAs, PROMPTS and several others. Besides the advances in the average depth coverage of RNA-seq experiments, various additional protocols are now available that can be used to address qualitative and quantitative aspects of the noncoding transcriptome complexity and function. In this review, we will focus on methods allowing isolation and characterization of complex RNA populations using sequencing based approaches, including conventional strategies already used for coding genome and more specific developments allowing, for example, the study of nascent strand transcription, protein-bound or structured RNAs

Potassium permanganate as a probe to map DNA-protein interactions in vivo.

Item does not contain fulltextPotassium permanganate (KMnO4) has widely been used in genomic footprinting assays to map unusual gene structures, including the melting DNA block in transcriptional elongation that results from promoter-proximal pausing of RNA polymerase (Pol) II complexes. Although it has been assumed that DNA-bound proteins do not protect underlying nucleic acids from KMnO4 modifications, we provide evidence herein that this chemical can readily be used to detect nuclear factor loading at a promoter when using optimized conditions. Moreover, by comparing parallel KMnO4 and dimethylsulfate (DMS) in vivo footprintings, we show that the utilization of KMnO4 in combination with another chemical probe maximizes the detection of factor occupancy at a DNA regulatory region, thus providing a better opportunity to define the actual profiles of DNA-protein contacts at given genomic sites in living cells

[Functions of lncRNA in development and diseases]

International audienceThe transcription of essentially the entire eukaryotic genome generates a myriad of non-coding RNA species that show complex overlapping patterns of expression and regulation. In the last decade, several large scale genomic analyses have shed light on the widespread existence of long non-coding RNAs (lncRNAs) in mammals. Although the function of most lncRNAs remains unknown, many of them have been suggested to play important roles in the regulation of gene expression during normal development and diseases, including cancers. Indeed, functional studies have demonstrated that lncRNAs participate in various biological processes, including reprogramming of pluripotent stem cells, oncogenic progression and cell cycle regulation. In this review, we summarize recent findings about the biology of lncRNAs and their functions in normal and pathological development in mammals

On load balancing: a mix-aware algorithm for heterogeneous Systems

Today’s web services are commonly hosted on clusters of servers that are often located within computing clouds, whose computational and storage resources can be highly heterogeneous. The workload served typically exhibits disparate computation patterns (e.g., CPU-intensive or IO-intensive), that fluctuate both in terms of volume and mix. The system heterogeneity together with workload diversity further exacerbates the challenge of effective distribution of load within a computing cloud. This paper presents a novel, mixaware load-balancing algorithm, which aims to distribute requests sent by multiple applications in heterogeneous servers such that the application response times are minimized and system resources (e.g., CPU and IO) are equally utilized. To this end, the presented algorithm tries to not only balance the total number of requests seen by each server, but also to shape the requests received by each server into a certain “mix", that is analytically shown to be optimal for response time minimization. Our experimental results— based both on simulation and on a prototype implementation— show that the mix-aware algorithm achieves robust performance in most workload mixes as well as a consistent performance improvement in comparison with one of the most robust load-balancing schemes of the Apache server