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Stanford Team Detects Poised Enhancers in Embryonic Stem Cells

By Andrea Anderson

NEW YORK (GenomeWeb News) – Human embryonic stem cells contain both active enhancers and enhancers that remain inactive until early development, according to a study appearing online today in Nature.

Stanford University researchers used chromatin immunoprecipitation coupled with massively parallel sequencing, along with RNA sequencing, to assess enhancer locations and gene activity across the genome in human embryonic stem cells. In so doing, they not only uncovered some 5,000 enhancers associated with active genes, but also more than 2,000 enhancers — which they dubbed 'poised enhancers' — that are found near genes not expressed in the stem cells, but rather in early stages of development.

"At the beginning, we were mainly interested in characterizing active enhancers in general in human [embryonic stem] cells," lead author Alvaro Rada-Iglesias, a post-doctoral researcher in developmental biology and chemical and systems biology researcher Joanna Wysocka's Stanford lab, told GenomeWeb Daily News.

But in the process, the team also uncovered a previously unknown set of dormant enhancers located near genes that are typically inactive in stem cells.

"There were things that looked like enhancers, but not really," Rada-Iglesias said. "They have several of the same [chromatin] marks but they have H3K27 trimethylation instead of acetylation."

To track down enhancers in human embryonic stem cells, Rada-Iglesias and his co-workers did ChIP-Seq with the Illumina Genome Analyzer using antibodies to chromatin regulators such as p300 and to several histone modifications, including H3K4me1, H3K27ac, H3K4me3, and H3K27me3. They subsequently used RNA-Seq to look at the expression of transcripts near enhancer-associated transcription start sites.

In the process, the researchers identified 5,118 so-called class 1 elements, which turned up near genes expressed in the stem cells.

But they also found another 2,287 enhancer-like marks falling near genes that are inactive in stem cells but which are known to become active during early stages of cellular development.

"Some of these sites have been indirectly previously characterized because they bind some of the core transcription factors in [embryonic stem] cells, like OCT4 or NANOG," Rada-Iglesias noted.

But, he added, in the past such interactions were either dismissed or classified as potential repressors. "Our data showed that, in fact, based on the chromatin signature, they actually represent enhancers that are ready to go," he said.

Based on their subsequent computational analyses and experiments, the researchers concluded that these class 2 elements are generally found near genes involved in the early stages of development for the three major cell lineages — the ectoderm, the endoderm, and the mesoderm.

"What we think is happening is that the genes that we see having these [poised] enhancers are involved in earlier steps of development and not so much in later ones," Rada-Iglesias said.

These "class 2" elements or 'poised enhancers' have slightly different chromatin signatures than those observed for class 1 enhancers, the team explained. For instance, Rada-Iglesias explained, active enhancers typically have acetylated histone H3K27 residues, while the poised enhancers show trimethylation at these residues.

Such differences make it relatively straightforward to distinguish between the class 1 and class 2 enhancers, Rada-Iglesias added. Coupled with the transient nature of these enhancers, he explained, that suggests many of these marks could serve as reporters for those interested in identifying and studying cells at specific stages of development.

"This is going to be an enormous resource for researchers interested in tracking cells involved in early human development," Wysocka said in a statement. "It will be very interesting to learn how these enhancers affect gene expression in each cell type."

For their part, the researchers plan to do more research to determine when and how the class 2 enhancers become established in stem cells, Rada-Iglesias said, and to explore the ways in which enhancer activity shifts once differentiation and development are underway.