Gene networks in human embryonic stem cells (hESCs) have been found to serve two purposes at once. They maintain pluripotency, and they keep apoptosis, or programmed cell death, on a hair trigger. This discovery, from a study led by researchers from Brigham and Women’s Hospital and Harvard Medical School (HMS), suggests that faulty embryonic stem cells have a built-in mechanism to ensure that they are destroyed before they can compromise the functioning of future cells and tissues.
The researchers used genome-wide genetic screening to both overexpress and inactivate (“knock out”) tens of thousands of genes that govern embryonic stem cell proliferation and differentiation into the three germ layers. In the course of this work, the researchers introduced genetic alterations that caused pluripotency dissolution and simultaneously increased apoptosis resistance.
Details of the work appeared October 28 in the journal Genes and Development, in an article titled, “Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.” The article offers new insights into cancer genetics and suggests novel approaches for regenerative medicine research.
“We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification,” the article’s authors wrote. “Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers.”
As pluripotent cells—progenitors of every type of cell type in the body—hESCs are of central interest to developmental and regenerative biologists. Many genes driving hESC functioning have previously been identified, but powerful tools that shed light on the interrelated activities of these genes have only emerged more recently.
In the current study, the researchers in the Brigham laboratory of Stephen Elledge, PhD, used an integrated genome-scale loss- and gain-of-function screening approach. “Our methods allowed us to create an ‘atlas’ of nearly every gene in the human genome and determine what its overexpression or loss does to the most fundamental first steps of human development,” said Kamila Naxerova, PhD, a former postdoctoral fellow in the Elledge laboratory and the lead author of the Genes and Development article. “Instead of looking at genes one by one, we looked at thousands of genetic alterations at the same time to determine how they affect the proliferation of embryonic stem cells, and, subsequently, the development of the three germ layers that serve as the raw material for human tissues.”
The screening experiment involved knocking out roughly 18,000 genes and overexpressing 12,000 genes. When genes known to control pluripotency were deleted—genes such as OCT4 and SOX2—the hESCs surprisingly increased their resistance to death, indicating that under normal circumstances pluripotency regulators also contribute to apoptosis pathways.
These interrelated behaviors were especially evident in a pluripotency regulator known as the SAGA complex. The researchers demonstrated for the first time that hESCs died less readily in the absence of the SAGA complex. In addition, its absence inhibited the development of all three germ layers (the endoderm, mesoderm, and ectoderm), testifying to the SAGA complex’s central role in a range of hESC activities. Finally, the researchers observed that many of the genes that regulate the formation of the three germ layers also are known contributors to the growth of cancers when they are over- or underexpressed in somatic cells.
Beyond offering a new perspective on the genetic basis of cancers, the study’s high-throughput genetic screening approach may inform future work in regenerative biology.
“Elucidating how hESC function is controlled by genetics is essential for our understanding of developmental biology and regenerative medicine,” said co-corresponding author Stephen Elledge, PhD, the Gregor Mendel professor of genetics and of medicine at the Brigham and HMS. “Our study provides the most extensive examination of gene functionality in hESCs to date.”
“Genetic screens present a wonderful opportunity to probe how genetic networks contribute to interrelated cellular behaviors like growth, differentiation, and survival,” added Naxerova, who is now an assistant professor in the Center for Systems Biology at Massachusetts General Hospital. “This approach can help regenerative and developmental biologists systematically map out genetic networks that are involved in the formation of particular tissues and manipulate those genes to more efficiently grow different kinds of human tissues from stem cells.”