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April 2012

Bioline Scholar Monthly: March 2012 Roundup - Epigenetics

By Bioline: The PCR Company 11 April 2012 No comments

Focus on Epigenetics

Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. The term refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such changes are DNA methylation and histone modification, both of which serve to regulate gene expression without altering the underlying DNA sequence.

These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism's genes to behave differently.

Just as in genetic changes, epigenetics is known to be involved in diseases such as cancer, silencing control genes. Epigenetics can also be beneficial by helping control gene expression during development and silence viral insertions, preventing them from promoting viral proliferation. Scientists have only just started scratching the surface of epigenomes, trying to make sense of the patterns of epigenetic marks.

Bioline has a number products designed to help in these studies and this edition of Bioline Scholar Monthly focuses on the use of Bioline products in the developing field of epigenetics research.


Due to their very similar chemical structure, discrimination of the rare hmC against the far more abundant mC is technically challenging and to date no methods for direct sequencing of hmC have been reported. This paper used 5-hydroxymethyl-dCTP to report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves hmC-containing sequences, showing its potential to interrogate hmC patterns in mammalian genomes.

Szwagierczak, A., et al. Nucl. Acids Res. 39(12): 5149-5156. (2011) - Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5–hydroxymethylcytosine

Model substrates were created using 5-hydroxymethyl-dCTP to show its effects, both at the promoter and in the gene body, on in vitro gene transcription. The results suggest that the presence of 5hmC in a promoter prevents the binding of essential transcription factors or recruits factors that repress transcription.

Robertson, J., et al. Biochem. Biophy. Res. Comm. 411(1): 40–43 (2011) - The presence of 5-hydroxymethylcytosine at the gene promoter and not in the gene body negatively regulates gene expression

Bioline’s dCTP and 5-hydroxymethyl-dCTP were used to help determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.

Szwagierczak, A., et al. Nucl. Acids Res. 38(19): e181. (2010) - Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA


RT-qPCR was performed using SensiFAST SYBR Lo-ROX on primary cultures and ovarian cell lines for SFRP4 and its key downstream regulators. The results support a role for SFRP4 as a tumor suppressor gene in ovarian cancers via inhibition of the Wnt signaling pathway. This has not only predictive implications but could also facilitate a therapeutic role using epigenetic targets.

Jacob, F., et al. PLoS ONE 7(2):e31885.doi:10.1371/journal.pone.0031885 (2012) - Loss of Secreted Frizzled-Related Protein 4 Correlates with an Aggressive Phenotype and Predicts Poor Outcome in Ovarian Cancer Patients

IMMOLASE™ DNA polymerase

Radical surgery is the de facto treatment for early rectal cancer. Conservative surgery can achieve high rates of cure but the histopathological measures of outcome used to select local treatment lack precision. In this paper five sites were significantly hypermethylated in cancer compared with adjacent tissue and hypermethylation of two or more of these genes was associated with localized disease.

Leong, K. J., et al. Br. J. Surg, 98: 724–734 (2011) - Methylation profiling of rectal cancer identifies novel markers of early-stage disease

The Sry (sex determining region on Y chromosome) gene is a master gene for sex determination. The Sry gene has tissue-dependent and differentially methylated regions. This study found unique non-CpG methylation in the Sry T-DMR. This non-CpG methylation was detected several mouse strains and has been associated with gene expression in the developmental process. The finding shows that non-CpG methylation has unique characteristic and is still conserved in mammals.

Nishino, K., et al. J. Reprod. Develop. 57(5): 586-593 (2011) - Non-CpG Methylation Occurs in the Regulatory Region of the Sry Gene

MangoTaq™ DNA Polymerase

Orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development, shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

Paterson, S., et al DMD 39(1): 77-82 (2011) - Histone Deacetylase Inhibitors Increase Human Arylamine N-Acetyltransferase-1 (NAT1) Expression in Human Tumor Cells

Over 90% of low risk (LR) neuroblastoma patients survive whereas less than 30% of high risk (HR) patients are long term survivors. Age (children younger than 18 months old) is associated with LR disease. This paper suggests that adaptive immune responses may play an important role in the progression of HR disease whereas innate immune responses may be active in LR patients.

Gowda, M., et al. J. Tran. Med. 9:170 (2011) - Distinct signatures of the immune responses in low risk versus high risk neuroblastoma


Genetic engineering can expand the utility of pigs for modeling human diseases, however the inefficient production of transgenic pigs represents a technological bottleneck. This paper assesses the hyperactive Sleeping Beauty transposon system for transgene integration into the embryonic porcine genome. It demonstrates germ-line transmission, through F1-offspring and insertion into transposon-tagged genomic loci followed by nuclear transfer. This potentially facilitates the development of large animal models for human diseases.

Garrels, W., et al. Genome.PLoS ONE 6(8):e23573 (2011) - Germline Transgenic Pigs by Sleeping Beauty Transposition in Porcine Zygotes and Targeted Integration in the Pig

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