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  • 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.

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