TET1 – the first DNA demethylase

Graph DM

Fig. 1: Example of a DNA methyltransferase assay: DNMT1 enzyme activity, measured using the DNMT1 Assay Kit.

Methylation of DNA plays a crucial role in transcriptional regulation, imprinting, and chromatin structure. DNA methyltransferases add a methyl group to cytosine or adenine DNA nucleotides. DNA methylation is a dynamic and reversible process and alters the expression of genes during cell division and differentiation from embryonic stem cells to specific tissues. The process is usually permanent, preventing cells e.g. from re-differentiation. During zygote formation, methyl groups are removed and subsequently re-established during development.

The reducing effect of gene expression is mediated by methylation at the 5 position of cytosine and it is present on all vertebrates. In adult somatic cells methylation is seen in a CgG context, while non-CpG methylation is current in embryogenic stem cells. Enzymes that catalyze DNA methylation are well characterized and already used as target enzymes for drug discovery screenings. BPS Biosciences offers a range of active DNA methyltransferases and DNA methyltransferase assays for inhibitor screenings (see Fig.1). [Read more…]

Which Antibodies for Epigenetics?

The nucleosome core particle is the fundamental structural unit of the eukaryotic genome. It consists of a histone octamer composed of two H2A-H2B dimers and a H3-H4 tetramer wrapped by ~146 base pairs of DNA. A linker histone (i.e., H1) associates with the nucleosomal dyad as well as with linker DNA on either side of the nucleosome, resulting in the formation of the chromatosome. All the core and linker histones are posttranslationally decorated, with at least 160 total modifications described to date including acetylation, methylation, phosphorylation, propionylation, citrullination, formylation, proline isomerization, butyrylation, ADP ribosylation, ubiquitylation, sumoylation, and the more recently identified glycosylation and crotonylation.

These modifications are thought to impact chromatin functions by either altering chromatin packaging or through the recruitment/inhibition of specific chromatin binding factors. Thus, the combined signal from a particular collection of histone marks constitutes a “histone code” that affects gene expression or other chromatin-based functions. [Read more…]

Buccal cells: best surrogate tissue for brain DNA methylation studies

A recent report published in the American Journal of Medical Genetics (Smith et al. 2014) describes a methodical comparison of buccal cells and blood as potential surrogates for brain tissue in epigenomic studies aimed at assessing the methylation status of DNA. The methylation patterns of specific genes are thought to be biomarkers for a variety of psychological disorders and may be the result of such factors such as childhood abuse, malnutrition, and traumatic stress. The authors find that DNA methylation patterns in buccal cells are more similar than blood to those of brain tissue. Certainly it is an attractive notion that permanent changes in the methylation patterns of the genetic code are at least partially responsible for some chronic psychiatric and behavioral traits, but the idea that a simple cheek swab can help unravel these mysteries even better than a blood sample is the part we find the most fascinating. [Read more…]

Meeting Report: RNA and Cancer – Toulouse, 14 Nov. 2014

“Toulouse may be the mRNA translation capital of France” declared third keynote speaker Jerry Pelletier at the symposium organized by Yvan Martineau, Stéphane Pyronnet et Julie Guillermet-Guibert of the CRCT ( Le Centre de recherche en Cancérologie de Toulouse) within the beautiful and modern Oncopole in Toulouse, France.

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8 new JARID1 – JMJD2 HMT assays

A lot of efforts are made to identify potent and selective inhibitors for the demethylation reactions catalyzed by members of the JMJD2 and JARID1 protein families (1, 2). Both epigenetic eraser enzyme families have been discussed as potential cancer therapeutic targets. In this post, I will review the last releases regarding JMJD2 and JARID1 protein families for assay development and drug discovery applications.

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Check the specificity of modified Histone antibodies

A lot of antibodies that have been raised against modified Histones, e.g. methylated or acetylated histones, do not seem to have been tested for their specificity. Antibodies against mono-methylated lysines should, for instance, not recognize di- or tri-methylated sites. Or totally different sites which might be methylated as well. Eglehofer et al. reported about the specificity of > 200 antibodies against 57 different Histone modifications and found that more than 25% failed in specificity tests (Nat Struct Mol Biol 18.1, 91-3 [2011]). In their paper they advised rigorous testing and provided an Antibody Validation Database.

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Writing – Erasing – Reading Epigenetics: Targeting Erasers in Drug Discovery

DNA and Histone proteins are targets for epigenetic modifications.

One of the roles of Epigenetics is to drive cellular differentiation from totipotent Stem cells to fully differentiated cell types, by regulating gene expression through patterns of modifications that differ according to the cell type and the cell status. This means that the patterns may significantly differ between stem cells, germ cells, and differentiated cells.

It has been found that cancer cells often differ in regard to specific Epigenetic modification sites too. Thus, epigenetic enzymes are considered as potential pharmaceutical drug targets or biomarkers, and quite some effort is made to define drugable targets.

In a previous post I focused on histone modifying enzmes (writers) . Today I will concentrate on those enzymes which erase modification from histones (erasers). Later on, in a further post we’ll be focusing on proteins which recognize specific modifications (readers). [Read more…]

H3K4me3 Histone Demethylase Fbxl10 Activity Assay

The Histone Demethylase FBXL10 (also known as NDY1, KDM2B, or JHDM1B) has been reported to demethylate histone H3K4me3.  By removing active Methyl Epigenetic marks on the chromatin, this “Epigenetic Eraser” thus inactivates gene transcription.

[Read more…]

Epigenetics Writers – Readers – Erasers: Targeting Writers in Drug Discovery

Epigenetic modifications target DNA and Histone proteins.

One of the roles of Epigenetics is to drive cellular differentiation from totipotent Stem cells to fully differentiated cell types, by regulating gene expression through patterns of modifications that differ according to the cell type and the cell status. This means that the patterns may significantly differ between stem cells, germ cells, and differentiated cells.

Writers, Readers & Erasers in Epigenetics and chromatin structure. Source: EpiCypher

Writers, Readers & Erasers in Epigenetics and chromatin structure. Source: EpiCypher

It has been found that cancer cells often differ in regard to specific Epigenetic modification sites too. Thus, epigenetic enzymes are considered as potential pharmaceutical drug targets or biomarkers, and quite some effort is made to define drugable targets.

In this post, I’d like to take a look at Histone modifications, and especially histone modifying enzmes (writers). Later on, in further posts I’ll be focusing on enzymes which recognize specific modifications (readers), and those that erase modification from histones (erasers). [Read more…]

Site-specific modified-histone substrates

Recombinant Histone proteins are popular research solutions in Drug discovery and Epigenetics studies. They can be used as in vitro positive controls in the analysis of post-translational Histone modifications, as substrates for Histone modification enzymes and chromatin studies.

[Read more…]