A New Edge in Bisulfite Conversion

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Aberrant methylation events have significant impacts in terms of incidence of cancer and development disregulation. Researchers studying DNA methylation are often working with DNA from “difficult” tissues such as formalin-fixed, paraffin embedded tissues, which characteristically yield DNA that is more fragmented than that purified from fresh tissue. Traditional methods for bisulfite conversion involve a long protocol, harsh chemicals, and generally yield highly fragmented DNA. The DNA fragmentation may significantly impact the utility of the converted DNA in downstream applications such as bisulfite-specific PCR or bisulfite sequencing.

An ideal bisulfite conversion system enables complete conversion of a DNA sample in a short period of time, provides high yield of DNA, minimally fragments the DNA, works on a wide range of input DNA amounts (from a wide variety of sample types), and, while we’re at it, is easy to use and to store. Whew! That’s quite the list.

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Decorating Histones and Their Tails

There are at least two tail stories associated with big scientific discoveries. One is Darwin’s story about the tail loss during human evolution process. The other story is associated with discovery of benzene ring structure. In his creative dream Kekule saw the snake (the linear carbon chain) eating its own tail. Even better representation of benzene structure is the comic image of six monkeys holding each other hands and tails. Nowadays, the most popular scientific story in the field of epigenetics is the story of histones and their tails. This time instead of monkey or snake, an elephant is the animal whose characteristics allegorically represent epigenetics.

elephants_web

We all know how the histone octamer wrapped with DNA represents a nucleosome – the first unit of chromatin formation. Histones, which are basic due to numerous arginines and lysines, easily attract negatively charged DNA and in that way facilitate formation of nucleosome. The nature of two materials is important but not sufficient for such complex biological function like efficient packaging of DNA and regulation of gene expression. For that reason both the DNA and histones are decorated by numerous chemical groups.

Post-translational modifications (PTMs) of histones and histone variants themselves can cause alternation of net charge, changes histone dynamics and interaction with other chromatin proteins. The extreme complexity of interactions that can be achieved by histone modifications inspired Jenuwein and Allis to launch an idea of “histone or epigenetic code”. Core histones consist of a N- terminal tail, the globular portion and a C terminus. PMTs were discovered first on the N-terminal tail of core histones. However, the logical question was: Are only the tails decorated or are there more?” Continue reading “Decorating Histones and Their Tails”

Hypomethylation in the Hippocampus: Can Age-Related Cognitive Decline in Mice Be Reversed by the Activity of One Gene?

Partial ribbon structure of DNMT3a Source: Protein Database
Buried in the middle of the August issue of Nature Neuroscience is an article (1) by Oliveira, Hemstedt and Bading that caught my eye. It isn’t often that I see a paper about gene rescue in a neuroscience journal, especially in a study about cognitive decline.

I looked for a News and Views summary of the article, thinking that if the conclusions of the article were anything like what the title and abstract indicated, there must be an editorial summary. I wasn’t disappointed. Su and Tsai provided a nice summary of the paper and discussed some of the potential implications of the work (2). Continue reading “Hypomethylation in the Hippocampus: Can Age-Related Cognitive Decline in Mice Be Reversed by the Activity of One Gene?”

Methylation of Your Genome Decreases as You Age

If asked what are the differences between a grandfather and his newborn granddaughter, I would reply with the obvious ones: size (the grandfather is larger than his granddaughter), condition of the skin (babies have soft, smooth skin and elders have age spots and wrinkles) and life expectancy. Other visual cues may seem more similar than different. For example, grandfather and granddaughter may both lack hair on their heads or need assistance to move from one place to another. However, both baby and elder are a product of the genes expressed from their genome even if the exact sequence is not identical between them. Because genes are expressed differently over a human’s lifetime, Heyn et al. decided to examine the methylation profile in the genomes of newborns and individuals 89 years old or older. Continue reading “Methylation of Your Genome Decreases as You Age”

Dark Matter, Rosetta Stones and Hordes of Beasties

Key regulatory roles are being identified for non-coding DNA sequences, once considered "junk".
The more you know, the more you find out about how much you still don’t know. So goes the old saying. A recent New York Times article nicely illustrated the practical outworking of this phenomenon in the context of cancer research. The article highlighted several recent papers and reviews showing how much progress has been made over the last ten years, and illustrating how the focus has changed to incorporate not only research on protein-coding sequences, but also the “dark matter” of noncoding RNAs and the potential contributions of genes from the millions of bacteria that colonize the human body.

In 2000, a review describing six key traits of cancer cells was published in the journal Cell, it is one of the most cited papers from that journal. In March of this year, the same authors published an update entitled Hallmarks of Cancer: The Next Generation describing current knowledge of the mechanisms underlying the same six traits, and adding two new ones. Continue reading “Dark Matter, Rosetta Stones and Hordes of Beasties”

The Link Between Childhood Adversity and Cellular Aging

Neglected childAdversity and stress are known risk factors for psychiatric disorders, cardiovascular and immune disease, cognitive decline and other health problems. The long-term negative effects of adversity seem to be greatest if the traumatic events were experienced during childhood, when the brain and other biological systems are developing and maturing. Researchers are working to identify the mechanisms involved and have identified telomere shortening as one possible mechanism by which adversity increases morbidity and mortality. Continue reading “The Link Between Childhood Adversity and Cellular Aging”

Use of Cell-Free Protein Expression for Epigenetics-Related Applications

Epigenetics is the study of the processes involved in the genetic development of an organism, especially the activation and deactivation of genes. One way that genes are regulated is through the remodeling of chromatin. Chromatin is the complex of DNA and the histone proteins with which it associates. The conformation of chromatin is profoundly influenced by the post-translational modification of the histone proteins. These modifications include acetylation, methylation, ubiquitylation, phosphorylation and sumolyation. The following references illustrate the use of cell-free expression to characterize this process.

Shao, Y. et al. (2010) Nucl. Acid. Res. 38, 2813–24.
Carbonic anhydrase IX (CAIX) plays an important role in the growth and survival of tumor cells.The MORC proteins contain a CW-type zinc finger domain and are predicted to have the function of regulating transcription, but no MORC2 target genes have been identified. CAIX mRNA to be down-regulated 8-fold when MORC2 was overexpressed. Moreover, MORC2 decreased the acetylation level of histone H3 at the CAIX promoter. Among the six HDACs tested, histone deacetylase 4 (HDAC4) had a much more prominent effect on CAIX repression. Assays showed that MORC2 and HDAC4 were assembled on the same region of the CAIX promoter. Interaction between MORC2 and HDAC 4 were confirmed by using cell free expression of MORC2 and GST-HDAC (GST pull-downs). Cell-free expression was also used to express MORC2 proteins to determine through gel shifts the binding location on the CAIX promoter region (gel shift experiments)

Denis, H. et al. (2009) Mol. Cell. Biol. 29, 4982–93.
The recent identification of enzymes that antagonize or remove histone methylation offers new opportunities to appreciate histone methylation plasticity in the regulation of epigenetic pathways. PAD4 was the first enzyme shown to antagonize histone methylation. Very little is known as to how PADI4 silences gene expression. Through the use of cell-free expression to express both PAD4 and HDAC1 proteins and E. coli expression of GST fusions of PAD4 and HDAC1, pulldown experiments confirmed by in vivo experiments that PADI4 associates with the histone deacetylase 1 (HDAC1), and the corresponding activities, associate cyclically and coordinately with the pS2 promoter during repression phases.

Brackertz, M. et al. (2006) Nucl. Acid. Res. 34, 397-406.
The Mi-2/NuRD complex is a multi-subunit protein complex with enzymatic activities involving chromatin remodeling and histone deacetylation. The function of p66α and of p66β within the multiple subunits has not been addressed. GST-fused histone tails of H2A, H2B, H3 and H4 were expressed in E. coli used in an in vitro pull-down assay with radioactively labeled p66-constructs expressed using cell free systems. Deletions at the C terminus noted reduced binding of p66 where as deletions at the N terminus did not affect binding. Also observed was that acetylation of histone tails reduces the association with both p66-proteins in vitro.

Zhou, R. et al. (2009) Nucl. Acids. Res. 37, 5183–96.
Lymphoid specific helicase (Lsh) belongs to the family of SNF2/helicases. Disruption of Lsh leads to developmental growth retardation and premature aging in mice. However, the specific effect of Lsh on human cellular senescence remains unknown. In vivo results noted that Lsh requires histone deacetylase (HDAC) activity to repress p16INK4a. Moreover, overexpression of Lsh is correlated with deacetylation of histone H3 at the p16 promoter. In vitro pull-downs using cell free expression and GST fusions from E. coli were used to collaborate interactions between Lsh, histone deacetylase 1 (HDAC1) and HDAC2 observed in vivo.

An Epigenetic Mountain for Ovarian Cancer Research

Fluorescent Stained ovarian carcinoma cells. Note the visible nuclei vimentin, also known as fibroblast intermediate filament, it is the major intermediate filament found in non-muscle cells and is present in over 40% of ovarian cancer cases. Mag: unknown.

It’s tempting to look at a field like cancer research and conclude that all of the big breakthroughs have been made; that what remains are the tiny discoveries that come from throwing the contents of the Sigma chemical catalog at cells and looking for effects, screening for even the most minute “hit” that might show some promise against a tumor. Indeed the “mountains”, single genes that are found to be disrupted in a majority of occurrences of a given cancer type, are becoming rarer and rarer finds. However, in a recent study of ovarian clear cell carcinoma (OCCC), Jones and colleagues report a discovery of such a mountain. Continue reading “An Epigenetic Mountain for Ovarian Cancer Research”

The Ongoing Legacy of the Human Genome Sequence

When the first draft sequence of the human genome was announced, I was a research assistant for a lab that was part of the Genome Center of Wisconsin where I created shotgun libraries of bacterial genomes for sequencing. Of course, the local news organizations were all abuzz with the news and sought opinions on what this meant for the future, including that of the lab’s PI and oddly enough, my own. While I do not recall the exact words I offered on camera, I believe they were something along the lines of this is only the first step toward the future of human genetics. Ten years later, we have not fulfilled the potential of the grandiose words used to report the first draft sequence but have gained enough knowledge of what our genome holds to only intrigue scientists even more.

Continue reading “The Ongoing Legacy of the Human Genome Sequence”