Researchers headed by a team at the University of Illinois (U of I) Urbana-Champaign now report on the discovery of a protein that plays a key role in prompting stem cells in the body to switch off their pluripotency and adopt a final functional state. Their studies indicated that the molecule, known as BEND3, acts to shut down the expression of hundreds of genes associated with differentiation, so maintaining the cell’s stem cell-like status. Their findings indicated that only when BEND3 is downregulated can cells differentiate into their designated form and function. And once cells differentiate, they usually stop actively proliferating.
The findings are relevant to understanding normal development, and may be useful in cancer research, suggested U of I Urbana-Champaign cell and developmental biology professor and department head, Supriya Prasanth, PhD, who led the research. “In most cancers, cells are going through this rampant proliferation because cell-cycle regulators are not functioning properly. The prognosis of how cancer cells will respond to treatment often relates to its status of differentiation. The more differentiated a tumor is, the better the prognosis.”
Prasanth and colleagues reported on their findings in PNAS, in a paper titled, “BEND3 safeguards pluripotency by repressing differentiation-associated genes,” in which they concluded, “Our results support a model in which transcription repression mediated by BEND3 is essential for normal development and to prevent differentiation.”
Embryonic stem cells and other pluripotent cells divide rapidly and have the capacity to become nearly any cell type in the body. Scientists have long sought to understand the signals that prompt stem cells to switch off pluripotency and adopt their final type and function. Stem cells have the capacity to repopulate a cancer tumor after it has shrunk during treatment, Prasanth said. Finding a molecular switch that will shift cancer cells away from proliferation and toward differentiation could aid in cancer treatment.
BEN domain–containing proteins have been identified as an important class of factors that are involved in modulating gene expression, but the molecular basis of how they regulate chromatin function and transcription remains to be established, the authors noted. “The mammalian genome encodes several BEN domain proteins, but we know little about the functionality of these proteins.” The Prasanth lab focuses on cell cycle regulators, and the team’s early studies identified BEND3 as a potentially important player in the system. The researchers found that when BEND3 bound to strategic locales along the chromosome, it reduced or blocked the expression of dozens of genes. And when BEND3 is removed, gene expression rebounds.
“We previously identified BEN domain–containing protein BEND3, a highly conserved protein among vertebrates that associates with heterochromatic structures and functions as a transcriptional repressor,” the authors noted. “More recently, a proteomic screen identified BEND3 as a factor bound exclusively to gene promoters, indicative of its role in transcriptional regulation.” Prasanth continued, “When you do these gene-expression studies, you can see hundreds of genes go up, hundreds down. But what does it really mean?”
Through their new work, the investigators confirmed that BEND3 is highly expressed in pluripotent cells, and binds to promoters of genes involved in differentiation. “It is noteworthy that BEND3 expression was high in embryonic stem cells and in iPSCs [induced pluripotent stem cells],” they wrote. “We found that BEND3 binds to 800 gene promoters, and a subset of these genes showed altered expression upon loss of BEND3, consistent with the notion that BEND3 impacts the expression of these genes directly.” The study results indicated that many of the genes repressed by BEND3 promote cell differentiation. Conversely, they wrote, “The removal of BEND3 from pluripotent cells results in cells exhibiting a differentiated phenotype concomitant with up-regulation of the differentiation-inducing gene expression signature.”
A detailed analysis of pathways that were specifically affected by alterations in the expression of genes involved in neuronal differentiation following BEND3 loss highlighted changes in MAPK pathway genes and the p53 pathway which the scientists commented, is known to play an active role in promoting differentiation in human embryonic stem cells.
BEND3 is not the only regulator of the cell-differentiation pathway; it binds to and interacts with many other molecular regulators of this process, Prasanth said. But its presence or absence appears critical to determining a cell’s fate, making it an attractive target for potential medical interventions when the process goes awry.
Illinois graduate students Fredy Kurniawan and Neha Chetlangia spearheaded the reported work, with postdoctoral researcher Mohammad Kamran, PhD, in collaboration with the laboratory of U of I cell and developmental biology professor Kannanganattu Prasanth, PhD, and Mirit Aladjem, PhD, a senior investigator at the National Institutes of Health’s National Cancer Institute.
In an accompanying paper published in the journal Genes and Development, the Prasanth lab and collaborators at the Memorial Sloan Kettering Cancer Center provided structural insights into BEND3-mediated gene regulation.