December 30, 2010

Genetic Regions Identified That Are Key to Embryonic Stem Cell Development

(Ivanhoe Newswire) "“ More than 2,000 genetic regions involved in early human development have been identified by researchers at the Stanford University School of Medicine. These regions, called enhancers, are responsible for triggering the expression of distant genes, when embryonic stem cells begin to divide, to form the plethora of tissues of a growing embryo.

"This is going to be an enormous resource for researchers interested in tracking cells involved in early human development," Joanna Wysocka, PhD, assistant professor of developmental biology and of chemical and systems biology and senior author of the study, was quoted as saying. "It will be very interesting to learn how these enhancers affect gene expression in each cell type."

The researchers also discovered that embryonic stem cells aren't bad at planning ahead. The cells prepare for the demands of future embryonic development by priming a subset of enhancers for activation with proteins and chemical tags. These "poised" enhancers are simultaneously kept in check by other modifications that keep them inactive. When the modifications (also known as epigenetic changes) are removed, the enhancers can quickly trigger the expression of genes needed to toggle from a mere stem cell to a developing embryo.

Dr. Wysocka and Dr. Rada-Iglesias, first author of the study, didn't start out trying to identify enhancers involved in development. Instead, they were looking for regions that activated genes involved in the maintenance of the embryonic stem cell state.
"We are interested in understanding how genomic information is integrated with epigenetic changes to produce cell-type-specific regulation "” in this case in the embryonic stem cells," said Dr. Wysocka. "Often this regulation is accomplished via gene activation mediated by a distant enhancer."

But enhancers can be difficult to identify because they trigger the activation of genes tens to hundreds of kilobases away; it's rarely clear if and where enhancers of that gene might be without conducting laborious genetic studies. Recent studies identifying specific protein and DNA modifications associated with active enhancers are making the process easier, though.

Dr. Rada-Iglesias mixed antibodies that specifically recognize epigenetic changes associated with active enhancers with extracts from human embryonic stem cells. He then used a technique called chromatin immunoprecipitation to remove the antibodies from the solution and catalogued the snippets of DNA that were included in the antibody complexes.

As expected, he found about 5,000 candidate active enhancers, which he termed class-1 elements, but 2,200 additional regions were more perplexing. These had two types of modifications "” both activating and inactivating. Some of these dually tagged DNAs, he noticed, were near genes known to be involved in early embryonic development. Rada-Iglesias called these regions class-2 elements.

Wysocka and Rada-Iglesias used a software program called GREAT (for Genomic Regions Enrichment of Annotation Tools) developed in the laboratory of Stanford researcher Gill Bejerano, PhD, assistant professor of developmental biology and of computer science, to analyze the categories of genes the two classes of enhancers might be controlling. They confirmed that the class-1 enhancers regulate genes active in embryonic stem cells, while the class-2 enhancers are associated with genes involved in processes such as gastrulation and the formation of germ layers "” events that occur very early in development.

"When we compared the expression of the genes controlled by class-1 and class-2 elements, we found that the class-1 genes were active, as you would expect in human embryonic stem cells, and the class-2 genes were inactive," said Dr. Wysocka.

When the researchers triggered the differentiation of the embryonic stem cells into a cell type called neuroectoderm, however, about 200 of the class-2 enhancers began to display a class-1, or activated, signature, and their associated genes were turned on. The researchers expect that specific sets of class-2 elements are activated during the development of various tissue types during embryogenesis. "These class-2 elements are clearly poised to orchestrate development in a cell-type-specific manner," said Wysocka.

Finally, the researchers attached individual enhancers to a reporter gene that would glow green when expressed. When they introduced the enhancer-reporter constructs into one-celled zebrafish embryos and allowed the embryos to develop, they saw a pattern of expression that was developmentally specific in location and timing and mimicked the expression of nearby developmental genes involved in normal fish embryogenesis.

"It's clear that these enhancers are becoming active at specific times during development," said Dr. Wysocka. "Now we have over 2,000 elements that can be used to study development and isolate transient cell populations."

Furthermore, Dr. Wysocka and Dr. Rada-Iglesias and their colleagues are now interested in identifying the mechanism that triggers the switch of the class-2 elements from an inactive to an active state, as well as how the poised state is initially set up at these elements in the embryonic stem cells.

SOURCE: Stanford University Press Release, released December 2010