An x-ray micrograph of a yeast cell, Saccharomyces cerevisiae, as it buds before dividing. Courtesy of Carolyn Larabell, UC San Francisco, Lawrence Berkeley National Laboratory and the National Institute of General Medical Sciences.
In Epigenomics, Location is Everything
Researchers exploit gene position to test “histone code”
In a novel use of gene knockout technology, researchers at the University of California, San Diego School of Medicine tested the same gene inserted into 90 different locations in a yeast chromosome – and discovered that while the inserted gene never altered its surrounding chromatin landscape, differences in that immediate landscape measurably affected gene activity.
The findings, published online in the Jan. 3 issue of Cell Reports, demonstrate that regulation of chromatin – the combination of DNA and proteins that comprise a cell’s nucleus – is not governed by a uniform “histone code” but by specific interactions between chromatin and genetic factors.
“One of the main challenges of epigenetics has been to get a handle on how the position of a gene in chromatin affects its expression,” said senior author Trey Ideker, PhD, chief of the Division of Genetics in the School of Medicine and professor of bioengineering in UC San Diego’s Jacobs School of Engineering. “And one of the major elements of that research has been to look for a histone code, a general set of rules by which histones (proteins that fold and structure DNA inside the nucleus) bind to and affect genes.”
The Cell Reports findings indicate that there is no singular universal code, according to Ideker. Rather, the effect of epigenetics on gene expression or activity depends not only on the particular mix of histones and other epigenetic material, but also on the identity of the gene being expressed.
To show this, the researchers exploited an overlooked feature of an existing resource. The widely-used gene knockout library for yeast, originally created to see what happens when a particular gene is missing, was built by systematically inserting the same reporter gene into different locations. Ideker and colleagues focused on this reporter gene and observed what happens to gene expression at different locations along yeast chromosome 1.
“If epigenetics didn’t matter – the state of histones and DNA surrounding the gene – the expression of a gene would be the same regardless of where on the chromosome that gene is positioned,” said Ideker. But in every case, gene expression was measurably influenced by interaction with nearby epigenetic players.
It’s not just our DNA that makes us susceptible to disease and influences its impact and outcome. Scientists are beginning to realize more and more that important changes in genes that are unrelated to changes in the DNA sequence itself – a field of study known as epigenetics – are equally influential.
A research team at the University of California, San Diego – led by Gary S. Firestein, MD, professor in the Division of Rheumatology, Allergy and Immunology at UC San Diego School of Medicine – investigated a mechanism usually implicated in cancer and in fetal development, called DNA methylation, in the progression of rheumatoid arthritis (RA). They found that epigenetic changes due to methylation play a key role in altering genes that could potentially contribute to inflammation and joint damage. Their study is currently published in the online edition of the Annals of the Rheumatic Diseases.
“Genomics has rapidly advanced our understanding of susceptibility and severity of rheumatoid arthritis,” said Firestein. “While many genetic associations have been described in this disease, we also know that if one identical twin develops RA that the other twin only has a 12 to 15 percent chance of also getting the disease. This suggests that other factors are at play – epigenetic influences.”
DNA methylation is one example of epigenetic change, in which a strand of DNA is modified after it is duplicated by adding a methyl to any cytosine molecule (C) – one of the 4 main bases of DNA. This is one of the methods used to regulate gene expression, and is often abnormal in cancers and plays a role in organ development.
While DNA methylation of individual genes has been explored in autoimmune diseases, this study represents a genome-wide evaluation of the process in fibroblast-like synoviocytes (FLS), isolated from the site of the disease in RA. FLS are cells that interact with the immune cells in RA, an inflammatory disease in the joints that damages cartilage, bone and soft tissues of the joint.
“Birth of DNA (Epigenetics)” by Zdenko Herceg
Deciphering DNA’s hidden code
Reading the genetic “Book of Life” is not easy, an observation scientists learn all of the time. Consider the well-known nucleobases that comprise DNA. There are only four: adenine, thymine, guanine and cytosine (plus uracil, which is found in RNA). It turns out, however, that cytosine comes in two modified forms: 5-methylcytosine (5-mc) and 5-hydroxymethlcytosine (5-hmC). The versions look almost alike, but affect genes in very different ways.
In a paper published in the journal Cell today, researchers at the University of Chicago, the Ludwig Institute for Cancer Research at UC San Diego and Emory University describe a new technique for reading the particular differences in cytosine, an achievement that has ramifications for better understanding fundamental life processes.
These two modifications of cytosine “regulate gene expression that has broad impact on stem cell development, various human diseases such as cancer, and potentially neurodegenerative disease,” said Chuan He, a professor of chemistry at the University of Chicago. “They may even shape the development of the human brain.”
He, with Bing Ren, PhD, head of the Laboratory of Gene Regulation at the Ludwig Institute for Cancer Research at UC San Diego, and colleagues developed a method called TAB-Seq that directly measures 5-hmC and produced the first map of the entire genome of 5-hmC at single-base resolution. Ren applied TAB-Seq to human embryonic stem cells; Peng Jin of Emory applied the method to mouse embryonic stem cells.
The work is expected to have a significant impact upon the field of epigenetics, which looks at changes in gene expression caused by factors other than alterations in the actual DNA. 5-mC and 5-hmC appear to be major epigenetic players. 5-mC is generally found on genes that are turned off; it helps silence genes that aren’t supposed to be turned on. Conversely, 5-hmC appears to be abundant on active genes, especially in brain cells.
“This is a major breakthrough in that TAB-Seq allows precise mapping of all 5-hydroxymethylcytosine sites in a mammalian genome using well-established, next-generation DNA sequencing methods,” said Joseph Ecker, a professor at the Salk Institute for Biological Studies, who was not involved in the Cell study. “The study showed very clearly that deriving useful knowledge about this poorly understood epigenetic regulator requires determination of the exact locations of 5-hmC with base-level accuracy. I expect that their new method will immediately become widely adopted.”
A five-day-old human blastocyst.
Researchers at the Ludwig Institute for Cancer Research, the University of California, San Diego School of Medicine and the Toronto Western Research Institute peel away some of the enduring mystery of how zygotes or fertilized eggs determine which copies of parental genes will be used or ignored.
In developing humans and other mammals, not all genes are created equal – or equally used. The expression of certain genes, known as imprinted genes, is determined by just one copy of the parents’ genetic contribution. In humans, there are at least 80 known imprinted genes. If a copy of an imprinted gene fails to function correctly – or if both copies are expressed – the result can be a variety of heritable conditions, such as Prader-Willi and Angelman syndromes, or diseases like cancer.
In the Cell paper, a team of scientists, led by Bing Ren, PhD, head of the Laboratory of Gene Regulation at the Ludwig Institute for Cancer Research at UC San Diego, describe in greater detail how differential DNA methylation in the two parental genomes set the stage for selective expression of imprinted genes in the mouse. Differential DNA methylation is essential to normal development in humans and other higher organisms. It involves the addition of hydrocarbon compounds called methyls to cytosine, one of the four bases or building blocks of DNA. Such addition alters the expression of different genes, boosting or suppressing them to help direct embryonic growth and development.
The process is sometimes called epigenetic regulation. Epigenetics is the study of factors influencing inheritance beyond the genes themselves. “DNA is just half the story,” said Ren, who also heads the San Diego Epigenome Center, one of four centers established by the National Institutes of Health to focus on epigenetics research.
“Understanding how these limited imprinted regions control regulation can help us better understand how certain diseases happen,” said Ren, a professor of cellular and molecular medicine in the UC San Diego School of Medicine. “That can help us develop better diagnostic tools for detecting genetic abnormalities and perhaps learn how to predict whether something bad will happen.”