Posts Tagged ‘epigenetics’

The fight for buzz: Epigenetics Versus Genome Structure

 :: Posted by American Biotechnologist on 08-01-2011

There is a huge buzz in the molecular biology community around epigenetic factors such DNA methylation and chromosomal orientation and their affect on molecular pathway activity. In two recent posts, we highlighted a new tool for epigenetic analysis which was recognized as one of the most innovative new products of 2011. However, according to a recent study published in Nature Biotechnology, structural variations involving large scale changes in DNA sequences should be just as “buzz-worthy” as epigenetics.

In a review of this article, Wired Science wrote that structural variations in DNA are more specific to individuals than single nucleotide polymorphisms, and may be more responsible than SNPs for genetic difference among people.

Truth be told, epigenetic factors may play a big role in determining which DNA sequences will be modified by our molecular machinery, so perhaps epigenetics does in fact trump (or perhaps even define), structural makeup. One commentator notes that the wired article has nothing to do with epigenetics, however, based on what I’ve noted above, I’m not sure that he is correct.

What are your thoughts? What’s more buzz-worthy? Epigenetics or Genome Structure?

To read more visit Your Genome Structure, Not Genetic Mutations, Makes You Different

EpiQ Chromatin Analysis Kit: Most Innovative New Product Finalist

 :: Posted by American Biotechnologist on 07-28-2011

Epigenetic changes, such as DNA methylation and histone modification, control gene expression by altering chromatin structure. Bio-Rad’s EpiQ kit was recognized at the European Lab Automation conference in Hamburg, Germany, as one of this year’s most innovative new products for its ability to quantify chromatin state and correlate the impact of these epigenetic events on gene expression.

Previously, the only way to quantitatively assess chromatin structure was through the use of homebrew methods that required isolating nuclei from several million cells and took several days to complete. The EpiQ kit is the first commercial tool that can determine the chromatin structure of a few targeted gene regions within six hours of cell harvest. It can work with as few as 50,000 cultured cells from a simple real-time PCR assay.

To learn more visit www.bio-rad.com/epiq.

Be sure also checkout our previous post tools for epigenetic chromatin analysis.

Tools for Epigenetic Chromatin Analysis

 :: Posted by American Biotechnologist on 07-27-2011

In this slideshow, you will learn the latest epigenetic techniques including:

  • discriminating epigenetically inactive chromatin from active chromatin
  • discriminating between aberrant and Monoallelic DNA methylation
  • predicting gene expression levels via chromatin structure assay
  • analyzing how DNA methylation affects promoter activity

First mapping of methylation event in embryonic stem cells

 :: Posted by American Biotechnologist on 07-25-2011

Stem cell researchers at UCLA have generated the first genome-wide mapping of a DNA modification called 5-hydroxymethylcytosine (5hmC) in embryonic stem cells, and discovered that it is predominantly found in genes that are turned on, or active.

According to Steven E. Jacobsen, a professor of molecular, cell and developmental biology in the Life Sciences and a Howard Hughes Medical Institute investigator, 5hmC is formed from the DNA base cytosine by adding a methyl group and then a hydroxy group. The molecule is important in epigenetics because the newly formed hydroxymethyl group on the cytosine can potentially switch a gene on and off.

The molecule 5hmC was only recently discovered, and its function has not been clearly understood, Jacobsen said. Until now, researchers didn’t know where 5hmC was located within the genome.

For more read UCLA scientists complete first mapping of molecule found in human embryonic stem cells

PCR Assay for Chromatin Accessibility

 :: Posted by American Biotechnologist on 06-10-2011

Epigenetics is the study of inherited phenotypic changes caused by mechanisms other than mutations in the underlying DNA sequence. In mammalian cells, most of the chromatin—a complex of DNA, proteins, and histones—exists in a condensed, transcriptionally silent form called heterochromatin. The transcriptionally active form of chromatin is called euchromatin; it exists in a relaxed, less condensed state.

Histone subunits and DNA can be chemically modified as a result of environmental factors. These chemical modifications, called epigenetic markers (or marks), which include DNA methylation and histone tail modification (Figure 1), influence chromatin structure by altering its electrostatic nature or by modulating the affinity of chromatin-binding proteins. By altering chromatin structure, epigenetic changes have a profound effect on the expression of the genes present in the genomic regions affected.

Histones are strongly alkaline proteins that package and order DNA into structural units called nucleosomes. They are the major protein component of chromatin and can undergo several covalent chemical modifications, including methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation. A clear correlation has been established between histone acetylation and active transcription. Conversely, many histone methylation events are correlated with transcriptional silencing. Different histone modifications likely function in different ways; acetylation at one position will have a different effect than acetylation at another position.

Multiple modifications exist simultaneously and likely work together to influence chromatin state and gene expression. The concept of multiple dynamic modifications regulating gene expression in a systematic and reproducible fashion is known as the histone code.

In eukaryotes, DNA can be modified by methylation of cytosine bases. The enzymes that carry out this modification are called DNA methyltransferases. Aberrant or increased levels of methylation has been correlated with gene silencing and the development of several cancers. Recently, a second cytosine modification, 5-hydroxymethylcytosine (5-hmC), has been characterized in eukaryotes and research efforts are focused to understand its function.

Methods Used to Study Epigenetics
The most common technique for assessing DNA methylation involves the use of sodium bisulfite to convert unmethylated and methylated cytosine residues to uracil and cytosine, respectively. Methylated cytosines can then be identified through various downstream nucleic acid analysis methods, including PCR, qPCR, HRM, and sequencing. This commonly used technique can both identify individual methylation sites and quantify the level of methylation for a particular genomic region.

Other techniques for methylation analysis include the use of restriction enzymes, which are either resistant or sensitive to DNA methylation, and methylated DNA immunoprecipitation (MeDIP), which utilizes antibodies to isolate methylated DNA fragments for analysis.

The interaction between proteins (for example, histones) and DNA is most often studied using a technique known as chromatin immunoprecipitation (ChIP). ChIP is a preparatory method that uses highly specific antibodies against DNA-binding proteins to isolate DNA fragments that bind transcription factors or other DNA-binding proteins.

The isolated fragment can be characterized by using various nucleic acid analysis techniques, including PCR, qPCR, sequencing, and microarray hybridization. This can help determine whether specific proteins are associated with specific genomic regions. ChIP is also useful for identifying regions of the genome that are associated with specific histone modifications (for example, activating or repressive).

Researchers are interested in understanding the role of epigenetic changes, including DNA methylation and histone modifications, in disease (cancer) development and cell differentiation and reprogramming. The methods described thus far provide detailed molecular information about epigenetic markers that can be correlated to changes in gene-expression levels. However, they do not provide direct information regarding the chromatin state associated with these epigenetic marks.

Furthermore, several of these methods are time-consuming and may require up to five days to complete. New tools that deliver additional information about the functional state of chromatin in a shorter time are needed to accelerate the pace of epigenetics research.

Epigenetics Analysis
The EpiQ™ chromatin analysis kit developed by Bio-Rad Laboratories is a real-time PCR assay for the quantitative assessment of chromatin states in cultured cells. Using this kit, chromatin structure data can be obtained within six hours directly from cultured cells (a few as 5 x 104) without the need for nuclei isolation.

In situ chromatin states can be identified based on how accessible the DNA is to nucleases. The DNA in heterochromatin is inaccessible to outside proteins, including exogenous nucleases, rendering it protected from nuclease digestion and available for subsequent quantitation by qPCR. Analysis of heterochromatin using the EpiQ kit reveals a minimal Cq shift between digested and undigested samples (Figure 2).

In contrast, the DNA in euchromatin is accessible to exogenous nucleases, making it susceptible to nuclease digestion and unavailable for qPCR. Analysis of euchromatin using the EpiQ kit shows a large Cq shift between digested and undigested samples (Figure 3).

By combining in situ chromatin digestion, genomic DNA purification, and real-time PCR, the chromatin state for several gene promoters can be studied simultaneously using the EpiQ chromatin analysis kit. The kit helps quantify the impact of epigenetic events, such as DNA methylation and histone modification, on gene-expression regulation through chromatin state changes.

The EpiQ chromatin analysis kit requires cultured (adherent or suspension) cells as starting material and includes the necessary supplies for performing chromatin assessment. Kit components include buffers for cell permeabilization and in situ chromatin digestion, optimized nuclease, materials for genomic DNA purification, control assays (qPCR primers) for chromatin assessment of a reference (epigenetically silenced) and control (constitutively expressed) gene, and the EpiQ chromatin SYBR® Green supermix, a real-time PCR reagent designed to amplify genomic DNA. Experimental results for user-specified gene targets are analyzed against the reference target to quantify the extent of chromatin accessibility.

Thanks to Viresh Patel, Ph.D. of Bio-Rad Laboratories for the guest post.