Epigenetics Tools

Epigenetics Tools Epigenetics refers to all that heritable yet extragenetic information that so profoundly influences gene expression. Generally speaking, researchers study three epigenetic control layers — DNA methylation, histone modification, and non-coding RNAs – and there exist multiple types of tools to address each one.

DNA methylation

In eukaryotes DNA generally is methylated on cytosines in the context of CpG dinucleotides – a modification called 5-methylcytosine (5-mC). More recently, researchers have discovered that cytosines can also be hydromethylated (5-hmC), formylated (5-fC) and carboxylated (5-caC).

Researchers can purify methylated DNA using either antibodies or recombinant methyl-binding proteins (MBDs), methods that sometimes are called methyl-DNA immunoprecipitation (MeDIP) and the Methylated DNA Island Recover Assay (MIRA), respectively.

To differentiate standard cytosine from its various modified forms, researchers have several options. Some restriction enzymes can distinguish methylated and unmethylated CpGs. Or, they can use sodium bisulfite to convert cytosine (but not 5-mC or 5-hmC) to uracil and sequence the resulting nucleic acids (bisulfite sequencing). More recently, tools have been developed to differentiate 5-mC- and 5-hmC-containing nucleic acids (which bisulfite cannot do), such as the next-gen sequencing methods, TAB-Seq and OxBS-Seq.

Histone modification

Histone proteins are often heavily modified post-translation with methyl, acetyl, phosphate, and other groups. The most common method for detecting those modifications is ChIP (chromatin immunoprecipitation), in which an antibody to the specific modification is used to pull down associated nucleic acids. These are then detected using PCR, microarrays (ChIP-chip) or next-gen DNA sequencing (ChIP-Seq).

Researchers studying the enzymes that create these histone modifications sometimes use peptide microarrays of differently modified histone tails to assess substrate specificity.

Noncoding RNAs

Increasingly, researchers have become aware of the role noncoding RNAs play in gene regulation. These include short RNAs, especially microRNAs, which control gene expression by complementarity between the miRNA and transcript, and long noncoding RNAs, which operate by multiple different mechanisms, including enzyme recruitment.

Noncoding RNAs can be studied using the traditional suite of transcriptome tools: qPCR, microrrays and NGS. Function can be addressed using small RNA-mediated knockdown assays, such as RNA interference, while location can be determined using FISH. There also are assays to identify proteins that bind specific RNAs (eg, ChIRP, CHART and RAP) and RNAs that bind specific proteins (eg, RNA immunoprecipitation).

Recommended Reading:

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