NEW YORK (GenomeWeb Daily News) – High-throughput sequencing is helping scientists unravel the transcriptional network governing mouse embryonic stem cells — and understand how it interacts with external signaling pathways.
In a paper published in the latest issue of Cell, researchers from the Genome Institute of Singapore and the National University of Singapore investigated 13 transcription factors and two transcription regulators in mouse embryonic stem cells using ChIP-Seq, or chromatin immunoprecipitation followed by sequencing. The team found that specific sets of transcription factors flock to hotspots in the genome. And by analyzing the groups of transcription factors at each site, they were able to gain a better understanding of how signaling pathways contribute to transcriptional control.
“We think that these ‘stemness’ hotspots are the most critical points in the genetic blueprint of [embryonic stem] cells,” senior author Huck-Hui Ng, senior group leader at the Genome Institute of Singapore, said in a statement. “By targeting these hotspots, we may be able to reconnect the wiring in non-stem cells and jump-start the stem cell program in them.”
Embryonic stem cells depend on specific transcription factors, in combination with external signals, to maintain their characteristic undifferentiated state and their abilities to both renew themselves and become any cell type. More than a dozen transcription factors seem to play a role in one aspect of stem cell maintenance or another — from maintaining pluripotency to controlling cell cycle progression, as do the transcription regulators p300 and Suz12.
“This particular group of factors is responsible for maintaining the self-renewal and pluripotency of the embryonic stem cells,” explained Singapore Stem Cell Consortium Executive Director Alan Colman in a statement.
Still, there is a dearth of information about the genes that are actually regulated by these transcription factors. In 2006, Ng and his team began mapping the transcriptional control of two of the transcription factors, Oct4 and Nanog. That work appeared in Nature Genetics. Similarly, in a paper published earlier this year in Cell, another group of investigators assessed regulation by nine transcription factors in mouse stem cells.
For the latest work, Ng and his colleagues expanded their view to get more in-depth information about transcriptional regulation in mouse stem cells using ChIP-Seq. They combined chromatin immunoprecipitation with massively parallel short-tag-based sequencing, done using the Illumina Genome Analyzer, to map transcription factor and regulator binding sites across the genome in E14 mouse embryonic stem cells.
For the 13 transcription factors tested, they found between roughly 1,100 and 39,600 binding sites in the mouse stem cell genome. When the team mapped the binding sites, they found nearly 3,600 hotspots in the genome — areas that are prone to binding by several of the transcription factors.
Subsequent mapping and gene expression analyses revealed that certain transcription factors tend to work together, producing distinct gene expression effects on the groups of genes they regulate.
In particular, the researchers identified sets of transcription factors in parts of the genome that were akin to enhanceosomes — areas in which specific transcription factors collaboratively bind enhancer DNA and help recruit other activators. For instance, the researchers discovered that three transcription factors — called Nanog, Oct4, and Sox2 — seem to recruit the transcription regulator p300.
The researchers also started untangling the complex interplay between signaling pathways and transcriptional networks. They noted, for example, that the Nanog-Oct4-Sox2 group of transcription factors, in collaboration with the transcription factors Smad1 and STAT3, seem to unite two main signaling pathways influencing embryonic stem cells.
The researchers cautioned that many of the binding sites could be non-functional or indirect interactions. But, they added, looking at the binding profiles of several transcription factors at once helped cut through some of this noise, pinpointing parts of the genome that were most likely to be related to stem cell biology.
“This blueprint that we obtained is like a treasure map, pointing us to specific sites where we can further study how these switches interact within the cell,” co-senior author Chia-Lin Wei, a senior group leader at the Genome Institute of Singapore’s Genome Technology and Biology Group, said in a statement. “Hopefully, this will eventually allow us to unlock the secrets of stem cells.”