Email to a Friend

Posts tagged 'Epigenetics'

RSS Feed

Epigenetics (White Paper)

By Bioline 13 April 2016

Epigenetics

Epigenetics can be thought of in broad terms as the range of phenomena that bring phenotype into being without changing the underlying DNA sequence. Consider that the vast majority of cells within multicellular organisms have identical genotypes yet exhibit distinct cellular functions with different gene expression profiles that are stable and, in many instances, heritable. Thus the epigenetic landscape of an organism is an important function of cellular differentiation and much remains to be learned about these processes. Bioline offers a suite of molecular tools, proteomics reagents, assay panels, and complete assay design services to assist research efforts in the field of epigenetics.

Epigenetics – dynamic and global regulatory mechanisms
Our genome is not in a static state, and although our genotype strongly influences resulting phenotype, there are many differences in gene function and expression that cannot be attributed to changes or differences in DNA sequence alone. A range of epigenetic processes have been identified that post-synthetically modify DNA or interact with the proteins intimately associated with DNA, controlling the journey of transcription and ultimate translation of protein to subsequent phenotype. These mechanisms include a range of histone modifications that affect chromatin structure, subsequent DNA packing and chromosome formation, thus controlling the availability of genes to transcription mechanisms.

Methylation patterns of DNA have also been identified as strong drivers of gene activation/inactivation. Nearly all DNA methylation in mammalian genomes occur at cytosine residues of CpG dinucleotides, and high density CpG regions (or islands) that are methylated correlate with transcriptional repression. DNA methylation has been shown to play a role in important cellular processes including X chromosome inactivation, plus changes in methylated state have been identified in diseases such as cancer. DNA methylation patterns can change in response to environmental factors such as diet or exposure to toxins, and it has been shown that some of these adaptive changes are passed on to future generations. There are a range of methods to detect and quantify DNA methylation including PCR, Real-Time PCR, or High Resolution Melt (HRM) analysis, and Bioline provides reagents and kits to support research in these areas.

The role of RNA, in particular non-coding RNA (ncRNA), has been established as a major controlling element of multiple epigenetic events. In addition to the established functional RNAs (mRNA, tRNA, and rRNA) involved with processing of the coding RNA, and associated small nuclear and nucleolar (snRNA and snoRNA) RNAs that splice and modify RNA nucleotides, other small ncRNAs such as microRNAs (miRNA) and short interfering RNA (siRNA) have been identified in epigenetic mechanisms - involved in regulation of target mRNA and chromatin. Long non-coding RNAs have also been implicated in gene regulation, thus there is a complete network of ncRNAs intimately involved in epigenetic processes. These elements can be studied through deep genome sequencing approaches, as well as targeted PCR or Real-Time PCR assays to detect, identify, or quantify the various ncRNA elements, and Bioline offers a range of molecular tools for these analyses.

Epigenetics – the role of miRNAs
There are over 2,000 different short, single stranded RNA molecules currently identified as unique human mature miRNAs in the miRBase sequence database. After binding with the RNA-induced silencing complex (RISC), these miRNA complexes interact with target mRNA to induce degradation or cleavage, or to block translation – thus miRNAs are key epigenetic regulators of post-transcriptional gene expression. With involvement in many key cellular functions such differentiation, proliferation and apoptosis, the study of miRNAs and their mechanisms is key to understanding normal cellular development as well as aberrant disease states.

In addition to the full range of physiological and pathophysiological functions regulated by miRNA activity, they have also shown to be important regulators of other epigenetic mechanisms such as DNA methyltransferases and histone deacetylases. Conversely, DNA methylation and histone modification have been linked to regulation of expression of certain miRNAs, and there is also evidence of miRNAs regulating other miRNAs – thus an epigenetics-miRNA regulatory circuit exists, creating a complex and intricate gene expression process involving feedback mechanisms and elements of self-regulation. The regulatory landscape of miRNAs is far from straight forward and much remains to be understood regarding the expression of miRNAs and their subsequent regulatory roles. In addition to individual molecular tools and kits for isolation, amplification and identification of miRNA targets, Bioline in conjunction with MiRXES, a Singapore-based life science research company, have developed the EPIKTM range of miRNA assay panels representing key miRNA targets identified using extensive bioinformatics mega-studies and linked to important disease states such as cancer.

Epigenetics – Applications and Challenges
With epigenetic drivers involved in so many cellular processes, there are any number of applications in which to study the various mechanisms of DNA methylation, histone modification or non-coding RNA control of gene expression. Whether you’re sequencing an entire genome, isolating nucleic acids, amplifying target sequences or studying the resulting proteins, Bioline has the individual reagent, complete kit, individually designed assay service, or ready to use panel to support all of your research efforts.

Epigenetics and cell differentiation mechanisms
Understanding how a single cell grows into a complete multicellular organism still remains somewhat of a puzzle, how are the tissue specific patterns of gene expression established and maintained throughout the life of an organism? How do cells interpret signalling cues, and how do stem cells both maintain their stem cell properties and differentiate progeny into various cell lineages? Given that the majority of cells within this system contain genetically identical material it is clear that epigenetic factors play a key role during organism development, as well as in stem cell renewal and differentiation throughout life. There is still much to be understood about the drivers behind embryogenesis, as well as the signals that fine-tune cell functions and gene expression changes throughout the lifespan of an organism. Although key genes and regulators of those genes have been identified in these processes, any number of epigenetic factors are involved, simultaneously interacting with each other and the local and external environment to bring about the expression of the unique set of genes that define each and every one of us.

Understanding epigenetic inheritance
Many epigenetic changes within cells act as one-way barriers, ensuring that cell-type specification and activity is stable and maintained, thus there are clear examples for mitotic inheritance of epigenetic regulation. Mechanisms exist for propagation of DNA methylation patterns, however little is known about how epigenetic flexibility is maintained during cell development and differentiation, and non-DNA based epigenetic inheritance is still poorly understood. Meiotic inheritance is even more controversial. During mammalian sexual reproduction, the epigenome must be reset back to a totipotent state in preparation for the development of the next generation. Given this erasure of somatic epigenetic signatures, it is difficult to understand how epigenetic changes could be transgenerationally, or meiotically inheritable. Intriguing multi-generational studies have reportedly identified specific epigenetic markers and associated phenotypic characteristics in offspring linked to environmental exposures such as diet restriction or toxin exposure in grand-parents, suggesting transgenerational epigenetic effects may indeed exist. However many studies in this area have been contested, and a mechanism for such inheritance has not yet been defined. There is certainly a wide gap in our knowledge and research efforts persist with mixture of excitement and caution.

Epigenetics in cancer and disease states
Given their key role in so many important cellular functions it stands to reason that aberrant epigenetic processes have been identified in many cancers as well as other disease states. Hyper-methylation of CpG islands has been identified in a range of cancers resulting in subsequent silencing of tumour suppressor genes, DNA repair genes or other processes important to normal cell growth. Epigenetic factors have also been linked to inherited disorders associated with mental retardation such as Fragile X syndrome, Prader-Willi, and Rett syndrome. Epigenetic changes have been observed in response to external environmental factors, and as such, epigenetic links are being investigated for conditions such as obesity, diabetes or heart disease. Although there are many clear correlations between epigenetic states and disease, there are many more confounding studies and results that have been difficult to replicate. There is vast variation in epigenetic alterations across cell populations within an individual, let alone across populations, meaning that many epigenetic studies of disease bring up more questions than they answer. In addition, it is often difficult to determine whether epigenetic changes are a cause or consequence of disease state. Nevertheless, epigenetic profiling continues, and epigenetic therapeutics are also being developed, as efforts continue towards a greater understanding of these important regulatory elements and their roles in disease.

Posted in: BiolineLab Hints & Tips Tags: Epigenetics

Bioline Scholar - Human Genes, Genetics, Genomes (Special ASHG 2012 Edition)

By Bioline: The PCR Company 20 December 2012 No comments

ASHG 2012 American Society of Human Genetics 2012 Annual Meeting

Bioline was once again an exhibitor (#Booth 1324-1326) at the American Society of Human Genetics (ASHG) annual meeting held in San Francisco in November. The largest human genetics meeting in the world, ASHG 2012 attracted over 7000 delegates to discuss the very latest advances in basic and clinical research.

Sessions included topics such as comparative epigenomics, early exome sequencing in complex traits, large-scale identification of regulators of translation and integrated genetic and functional studies of disease-causing variants.

Bioline USA at ASHG2012 Bioline USA at ASHG 2012

Bioline unveiled its new format exhibition stand at the conference, where our representatives met with scientists and discussed solutions to their molecular biology challenges. Bioline PCR Specialist, Dr. Sven Bocklandt gave a series of well received and thought-provoking talks on some of our latest products, including SensiFAST™, MyFi™ and MyTaq™ DNA polymerases. Exciting results from both published papers and some unpublished experiments from the Bioline scientific community were presented, including results from the Bioline PCR Challenge.

In keeping with ASHG 2012, this month's Bioline scholar is dedicated to Human Genetics. We are pleased to highlight a selection of recent papers featuring Bioline products, perfectly suited for both small- and large-scale genetic and genomic studies in humans.

BIO-X-ACT™ Long DNA Polymerase

Mutations in MTM1 (myotubularin 1) cause X-linked myotubular myopathy that affects 1 in 50,000 males. In neonates or infants, movement and breathing are severely affected. The authors developed a MTM1-specific database of genetic variants, with 474 identified mutations from 472 patients. Next-Gen sequencing (Illumina HiSeq) and cDNA analysis revealed the presence of a novel MTM1/MAMLD1 fusion transcript in one case.

Oliveira, J. et al. (2012). Expanding the MTM1 mutational spectrum: novel variants including the first multi-exonic duplication and development of a locus-specific database. Eur. J. Hum. Genet. doi: 10.1038/ejhg.2012.201

cDNA synthesis kit

Children with X-linked retinitis pigmentosa (XLRP) often go blind by the second decade of life. A severe form of XLRP, RP23, was previously mapped to a 10.71Mb interval on Xp22.31-22.13, containing 62 genes. Using targeted NGS (Illumina), the authors identified a deep intronic mutation in OFD1 as the most likely cause of RP23. Insertion of a cryptic exon produces an aberrant transcript and reduced levels of correctly spliced transcript. This is predicted to result in a severely truncated protein and decreased levels of normal protein.

Webb, T.R. et al. (2012). Deep intronic mutation in OFD1, identified by targeted genomic next-generation sequencing, causes a severe form of X-linked retinitis pigmentosa (RP23). Hum. Mol. Genet. 21(16):3647-3654.

BIOTAQ™ DNA Polymerase

The genetic basis for susceptibility to malaria in a Vietnamese population was investigated by SNP genotyping by researchers from Oxford University and the MalariaGen consortium. Data from 65 SNPs in 42 malarial candidate genes in 956 severe malaria cases and 2350 controls from Vietnam was reported. Variants in six genes (ICAM1, IL1A, IL17RC, IL13, LTA and TNF) encoding adhesion and pro-inflammatory molecules were associated with severe malaria.

Dunstan, S.J. et al. (2012). Variation in human genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Genes Immun. 13(6):503-508.

BioMix™ Red

Facioscapulohumeral muscular dystrophy (FSHD), characterized by muscle weakness and wasting, is associated with a shortened telomeric chr 4q35 due to deletions of the D4Z4 tandem repeat (FSHD1) or DNA methylation changes of D4Z4 (FSHD2). Using exome sequencing (Illumina NGS) the authors identified two known pathogenic mutations in CAPN3, indicating a case of limb-girdle muscular dystrophy type 2A (LGMD2A) rather than FSHD2. The authors conclude that ‘diagnosis by sequencing’ of FSHD may be more commonly adopted in clinical genetics laboratories around the world.

Leidenroth, A. et al. (2012). Diagnosis by sequencing: correction of misdiagnosis from FSHD2 to LGMD2A by whole-exome analysis. Eur. J. Hum. Genet. 20(9):999-1003

An arrayed human genomic library comprising 115,000 PAC clones was constructed. Functional studies with a p53-containing PAC clone in p53-null human osteosarcoma cells showed the utility of individual library members in human cell culture models. The library can be used to validate candidate genes identified by GWAS and for gene therapy in different recessive disorders.

Fuesler, J. et al. (2012). An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy. Gene. 496(2):103-109

Immomix™

Charcot-Marie-Tooth disease (CMT) is an inherited disorder of peripheral nerve dysfunction resulting in numbness and weakness. Mutations in four genes encoding an aminoacyl-tRNA synthetase (ARS) have been associated with CMT. This study from the University of Michigan found that the p.Arg329 AARS mutation is a recurrent, loss-of-function mutation that arises due to methylation-mediated deamination of a CpG dinucleotide.

McLaughlin, H.M. et al. (2012). A Recurrent loss-of-function alanyl-tRNA synthetase (AARS) mutation in patients with charcot-marie-tooth disease type 2N (CMT2N) Hum. Mutat. 33(1):244-253

Introducing the Bioline Scholar Publications Database

Introducing Bioline Scholar - from Bioline: The PCR Company Bioline Scholar - from Bioline: The PCR Company

If you're interested in knowing more about the fields and areas of research in which Bioline products have been used to further knowledge, we've added a Bioline Scholar page to our web site listing the papers and publications in which our molecular biology reagents are featured.

You can search for publications by author, title, topic and product and the archive contains papers from 1994 through to the present.  Currently the Bioline Scholar database lists and provides links to publications where the research was conducted using the following Bioline products:

That's it for the Bioline blog in 2012—season's greetings to all our customers and readers alike, from all at Bioline—see you in 2013!

MyTaq™ - How to Take Your PCR From 11 Hours to TWO

By Bioline: The PCR Company 14 June 2012 No comments

Fides LayWe caught Fides Lay in the lab on the day of a basketball game between UCLA and crosstown arch-rival USC. As a former UCLA undergrad now working on her PhD at USC, she never wants to miss a game between the two. Unfortunately, the long hours in the lab don’t always allow her to be home in time to watch. At least, that’s how it used to be for her. Thanks to Bioline’s MyTaq HS Mix, now she doesn’t miss a game.

In Peter Jones’ lab at USC, Fides is part of a team studying the epigenetic regulation of cancer. And as anyone who ever worked on epigenetics knows, you need to be a master of PCR to get any results at all. When you need to amplify bisulfite converted DNA to measure methylation status, you work with small samples that often have been digested with several enzymes, with DNA that is fragmented and damaged.

For the longest time the lab amplified bisulfite converted DNA with a tedious protocol; tedious because of the long set-up with using Taq from a previous supplier, DMSO, and other components added one at a time, and extremely slow cycling with a PCR protocol that was 7-11 hours long.

Anything could go wrong at any time, and the result was impossible to predict. Simply using a different thermocycler with a different ramping speed could cause the reaction to fail. And once you found out, it would be too late to fix because every new attempt takes a whole other day. Since the lab usually clones and sequences the PCR fragments, there was always still the risk that no clones would have inserts. Hard to predict, and that meant starting over.

All that changed when Fides first tried Bioline’s MyTaq HS Mix, an easy, all-in-one mix that contains the enzyme, dNTPs, buffer and all optimizers. There’s no need to add any DMSO, it works right away on almost all templates. And it's fast! Reactions are done in less than two hours, even on bisulfite converted DNA, with highly consistent results, and always with nice bands. The PCR products are much easier to clone, and on the rare occasion that something does go wrong, there’s still time to redo the experiment AND get the samples off to pyrosequencing the same day.

Now Fides has time to run multiple experiments and redo anything that goes wrong, all in time to get home, kick up her feet and watch the game.

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.

5-hydroxymethyl-dCTP

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

SensiFAST™ SYBR Lo-ROX Kit

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

TRIsure

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

^ Back to Top