Epigenetics

Epigenetics Overview

Epigenetics is a term coined to describe changes that are not mutation based but can still be passed on from generation to generation. Genes that are activated or repressed without any change in DNA sequence are epigenetically controlled. Epigenetic modifications are stable, but potentially reversible alterations in gene expression that occur without permanent changes in DNA sequence.

Many types of epigenetic processes have been identified--they include methylation, acetylation, phosphorylation, ubiquitylation, and sumolyation of histones as well as DNA methylation. These modifications change with the environmental conditions. More recently researchers have shown that these changes alter gene expression and phenotypes (1-3). The best-known epigenetic process, in part because it has been easiest to study with existing technology, is DNA methylation. This is the addition or removal of a methyl group (CH3), predominantly where cytosine bases occur consecutively. The current model of epigenetic regulation starts with DNA wound around a set of completely acetylated histones; this represents an activated, fully transcribed gene. Transcriptional repression can be initiated near the promoter by deacetylating lysine residues of nearby histone proteins. Subsequently, the lysines can be methylated up to three times per lysine, each time locking in gene shutdown. Finally, the cytosine in the gene can be methylated at their 5’ carbon. Each modification in this chain from acetylation to DNA methylation is associated with a compaction of the gene into dense, untranscribable chromatin.

Cancer, often described as a disease of regulation, is known to have hypo-methylated (under methylated) DNA (5). Histone deacetylace (HDAC) and DNA methylase inhibitors are now being used in the treatment of cancer (6). Other epigenetic targets have been identified for psychiatric disorders (7) and drug addiction (8). One recent and fascinating study (4) shows correlations between DNA methylation and the age (and environmental differentiation) in twins. Adding to this is a steady stream of epigenetic papers describing new histone modifying enzymes and their effects (9, 10). Epigenetics is a very active topic in current research.

There are several common ways to determine whether a gene contains methylated DNA. Since all methylation occurs at cytosines, researchers take advantage of the fact that methylated cytosine (meC) is stable to bisulfite treatment but unmethylated cytosine is transformed to uracil under the same conditions. Bisulfite treatment, manifested as the MOD50 kit at Sigma, mutates unmethylated genes to contain uracil. These treated genes can then be sequenced to determine the methylation state of the original sample; this process is termed bisulfite sequencing. The kit can also be used to perform methylation specific PCR, which exploits the C to U change and uses primers that will anneal differentially. MOD50 was the fastest and most sensitive kit when placed on the market in March 2007.

The overall degree of methylation of a genome can be a useful measure of global regulatory changes. Measurement of this parameter is usually performed after complete digestion to the single base and then use of HPLC or mass spec instrumentation. The MDQ1 kit, released in October 2008, allows the researcher to monitor DNA methylation using a format similar to a sandwich ELISA. This is a format more friendly to biological researchers, and is the first of it’s kind on the market to open global meC quantitation to biological research scientists.

Another significant epigenetic process is chromatin modification. Chromatin is the complex of proteins (histones) and DNA that is tightly bundled to fit into the nucleus. Histone modifications change chromatin structure, and are an early indicator of epigenetic regulation. One way to study this phenomenon is via chromatin immunoprecipitation (ChIP). This technique starts with cells, and uses a cross linking agent to chemically link the DNA and it’s interacting proteins. The resulting DNA is isolated, sheared, a precipitated from the bulk using a protein specific antibody (e.g. acetylated histone). The cross links are reversed or broken, and the precipitated DNA, now enriched for sequences that interact with the protein of interest, is examined to determine which sequences are present. Detection can be via PCR when looking for a few genes, or can be done using microarrays (ChIP-chip) or parallel (deep) sequencing (ChIP-seq). The Sigma CHP1 kit was targeted toward histone modifications, and is the only kit on the market using a plate format. The resulting kit affords a high throughput system, ideal for researchers eager to screen multiple samples for histone modifications. Application data presented at AACR 2008.

ChIP-chip requires amplification of the enriched DNA sample, as immunoprecipitation does not supply the up to 1ug of DNA required for microarray analysis. The GenomePlex Whole Genome Amplification kit (WGA2, WGA4) has been very successfully applied to ChIP DNA amplification, and is the method of choice for generating more from a fragmented DNA sample.

While these kits are important to studying epigenetics, antibodies are an equally important aspect to this field of research. ChIP cannot be accomplished without specific antibodies to the histone modifications. Furthermore, not all antibodies work in ChIP, as the antigen epitope may be blocked or altered once cross-linked. Sigma has a growing collection of validated antibodies, and we are working to improve this list.

References


  1. Lyko F, et al. (1999).  Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila. Nat Genet. 23(3): 363-6.
  2. Lee JH, Hart SR and Skalnik DG. (2004). Histone deacetylase activity is required for embryonic stem cell differentiation. Genesis 38(1): 32-8.
  3. Mutskov V, Felsenfeld G. (2004).  Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9. EMBO J. 23(1): 138-49 
  4. Fraga MF et al. (2005). Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci U S A. 102(30): 10604-9.
  5. Feinberg AP and Vogelstein B. (1983). Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301(5895): 89-92.
  6. Witt O, et al. (2008).  HDAC family: What are the cancer relevant targets? Cancer Lett. Sep 26.
  7. Abel T, and Zukin RS. (2008). Epigenetic targets of HDAC inhibition in neurodegenerative and psychiatric disorders. Curr Opin Pharmacol. 8(1): 57-64.
  8. Romieu P et al. (2008).  Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats. J Neurosci. 28(38): 9342-8.
  9. Bártová E et al. (2008).  Histone modifications and nuclear architecture: a review. J Histochem Cytochem. 56(8): 711-21.
  10. Sims, RJ III et al. (2007).  Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing. Mol Cell 28(4): 665-76.