DNA methylation plays a key role in regulating eukaryotic gene expression. Although mitotically heritable and stable over time, patterns of DNA methylation frequently change in response to cell differentiation, disease and environmental influences. Several methods have been developed to map DNA methylation on a genomic scale. Here, we benchmark four of these approaches by analyzing two human embryonic stem cell lines derived from genetically unrelated embryos and a matched pair of colon tumor and adjacent normal colon tissue obtained from the same donor. Our analysis reveals that methylated DNA immunoprecipitation sequencing (MeDIP-seq), methylated DNA capture by affinity purification (MethylCap-seq), reduced representation bisulfite sequencing (RRBS) and the Infinium HumanMethylation27 assay all produce accurate DNA methylation data. However, these methods differ in their ability to detect differentially methylated regions between pairs of samples. We highlight strengths and weaknesses of the four methods and give practical recommendations for the design of epigenomic case-control studies.

/pdf/quantitative-comparison-of-genome-wide-dna-methylation-2qstztwqub.pdf

Quantitative comparison of genome-wide DNA methylation mapping technologies

http://digital.csic.es/bitstream/10261/124679/1/PharTher%282015%29.pdf

Sp1 transcription factor: A long-standing target in cancer chemotherapy.

SP1 is a ubiquitous transcription factor that is involved in the regulation of various house-keeping genes. It is known that it acts by binding to a double-stranded consensus motif. Here, we have discovered that SP1 binds also to a non-canonical DNA structure, a G-quadruplex, with high affinity. In particular, we have studied the SP1 binding site within the promoter region of the c-KIT oncogene and found that this site can fold into an anti-parallel two-tetrad G-quadruplex. SP1 pull-down experiments from cellular extracts, together with biophysical binding assays revealed that SP1 has a comparable binding affinity for this G-quadruplex structure and the canonical SP1 duplex sequence. Using SP1 ChIP-on-chip data sets, we have also found that 87% of SP1 binding sites overlap with G-quadruplex forming sequences. Furthermore, while many of these immuoprecipitated sequences (36%) even lack the minimal SP1 consensus motif, 5'-GGGCGG-3', we have shown that 77% of them are putative G-quadruplexes. Collectively, these data suggest that SP1 is able to bind both, canonical SP1 duplex DNA as well as G-quadruplex structures in vitro and we hypothesize that both types of interactions may occur in cells.

/pdf/a-non-canonical-dna-structure-is-a-binding-motif-for-the-v2gl8auq8s.pdf

A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro

Transposable elements (TEs) are insertional mutagens that contribute greatly to the plasticity of eukaryotic genomes, influencing the evolution and adaptation of species as well as physiology or disease in individuals Measuring TE expression helps to understand not only when and where TE mobilization can occur but also how this process alters gene expression, chromatin accessibility or cellular signalling pathways Although genome-wide gene expression assays such as RNA sequencing include transposon-derived transcripts, most computational analytical tools discard or misinterpret TE-derived reads Emerging approaches are improving the identification of expressed TE loci and helping to discriminate TE transcripts that permit TE mobilization from chimeric gene–TE transcripts or pervasive transcription Here we review the main challenges associated with the detection of TE expression, including mappability, insertional and internal sequence polymorphisms, and the diversity of the TE transcriptional landscape, as well as the different experimental and computational strategies to solve them Computational tools to analyse RNA sequencing data often disregard or even misinterpret reads derived from transposable elements This Review highlights the main challenges associated with the detection of transposable element expression, including mappability, sequence polymorphisms and transcript diversity, and discusses the experimental and computational strategies to overcome them

https://www.hal.inserm.fr/inserm-03261002/document

Measuring and interpreting transposable element expression.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2011.08.013

MiR-140 is co-expressed with Wwp2-C transcript and activated by Sox9 to target Sp1 in maintaining the chondrocyte proliferation

BACKGROUND: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. METHODOLOGY AND PRINCIPAL FINDINGS: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. CONCLUSION: This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

/pdf/overexpression-of-transcription-factor-sp1-leads-to-gene-1fet0964ux.pdf

Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition

Half of the human genome consists of repetitive DNA sequences. Recent studies in various organisms highlight the role of chromatin regulation of repetitive DNA in gene regulation as well as in maintainance of chromosomes and genome integrity. Hence, repetitive DNA sequences might be potential "sensors" for chromatin changes associated with pathogenesis. Therefore, we developed a new genomic tool called RepArray. RepArray is a repeat-specific microarray composed of a representative set of human repeated sequences including transposon-derived repeats, simple sequences repeats, tandemly repeated sequences such as centromeres and telomeres. We showed that combined to anti-methylcytosine immunoprecipitation assay, the RepArray can be used to generate repeat-specific methylation maps. Using cell lines impaired chemically or genetically for DNA methyltransferases activities, we were able to distinguish different epigenomes demonstrating that repeats can be used as markers of genome-wide methylation changes. Besides, using a well-documented system model, the thermal stress, we demonstrated that RepArray is also a fast and reliable tool to obtain an overview of overall transcriptional activity on whole repetitive compartment in a given cell type. Thus, the RepArray represents the first valuable tool for systematic and genome-wide analyses of the methylation and transcriptional status of the repetitive counterpart of the human genome.

https://hal.inria.fr/hal-00851248/document

Aurélie Laugraud

Papers

Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition

Global analysis of DNA methylation and transcription of human repetitive sequences.