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Jun 1, 2012 (Vol. 32, No. 11)

Reprogramming Alters Cells’ Fate

  • Pioneer Transcription Factors

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    Pioneer factors are distinguished by being able to bind silent genes in chromatin. By binding silent chromatin, pioneer factors can enable the binding of other regulatory factors to the DNA when such factors are induced by different stimuli. Relevant stimuli include those that occur during embryonic development, hormonal induction, and during pathogenic processes that activate genes inappropriately, leading to disease. [University of Pennsylvania School of Medicine]

    Pioneer transcription factors sound as if they should be riding in a covered wagon blazing a trail across the Wild West. While that’s not their role, they do, however, take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression.

    Transcription factors help this process as they seek out and bind specific regulatory sequences in DNA. That may be no easy feat to accomplish as DNA is coated with and condensed into a thick fiber of chromatin. In each eukaryotic cell, ~2 meters of DNA is packaged into a nucleus of only several microns in diameter.

    “We discovered pioneer transcription factors in 1996 and were surprised to find that when they first attached to chromatin, they did not immediately activate the corresponding genes at that site,” said Kenneth S. Zaret, Ph.D., professor, department of cell and developmental biology, University of Pennsylvania School of Medicine. “By contrast, the pioneer factor endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.”

    Dr. Zaret notes that at any one time, the vast majority of potential DNA-binding sites are not occupied, suggesting that most nuclear DNA isn’t easily accessible. The question prompted is: how do pioneer factors perform their role? An example is the pioneer transcription factor FoxA, a member of the Forkhead box family of transcription factors.

    “FoxA factors engage and subsequently help activate silent genes. They are expressed in the foregut endoderm of the mouse and are necessary for induction of the liver program. We looked at the genomic location of FoxA in the adult mouse liver. We found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes.”

    These studies help explain the progression of cells to cancer. “We found that in sites near where FoxA bound to silent genes, there were motifs for the transcriptional repressors Rfx1 and type II nuclear hormone receptors (HHR-II). We then confirmed protein binding and subsequent repression of adjacent FoxA sites at a novel and otherwise silent enhancer element for Cdx2. Cdx2 mediates differentiation of intestinal epithelial cells and is not normally expressed in esophageal cells. Thus, Rfx1 restricts FoxA1 activity at silent genes.

    “We next examined Rfx1 in human esophageal epithelium and in adenocarcinoma and determined that Rfx1 levels decline during esophageal cancer progression, which could allow FoxA to activate Cdx2 in esophageal cells. Overall this suggests that when such networks are perturbed, cancer progression may result. It also suggests that Rfx 1 expression may serve as a biomarker for esophageal cancer progression.

    “These studies illustrate how evaluating the binding of pioneer factors to silent genes may reveal the basis for how cell perturbations deregulate gene expression and progress to cancer,” Dr. Zaret concluded. “Thus, understanding how silent genes can be activated provides insights not only for development but also for cellular programming and pathogenesis.”

  • Germ Cells to Neurons

    While transcription factors are critical elements for inducing the identity of specific cell types in multicellular organisms, they also are cell-type specific and can be limited when ectopically expressed in other cells, notes Oliver Hobert, Ph.D., professor, department of biochemistry and molecular biophysics, Columbia University Medical Center. According to Dr. Hobert, the generally accepted paradigm in the field is that transcription factors can exert their activities only within a specific cellular context.

    Dr. Hobert indicates that one of the goals in the field is to develop ways to overcome such cellular context dependency. That is, one could envision generating specific cell types using a cocktail of transcription factors to reprogram cells for the creation of in vitro models for specific diseases or to provide a source of material for cellular replacement therapies.

    To better elucidate the mechanism for context dependency, Dr. Hobert and colleagues conducted cellular reprogramming studies in the nematode, Caenorhabditis elegans. They initially monitored the effect of expressing the zinc finger transcription factor CHE-1 that programs gustatory neurons called ASE neurons. Next they established from an RNA interference library that the chromatin factor lin-53 controls context dependency of CHE-1.

    Dr. Hobert and colleagues determined that to convert germ cells into specific neuron types, mitotic germ cells required not only the addition of the specific transcription factor, but also the removal of inhibitory factors—in this case the histone chaperone lin-53, a component of several histone modifying and remodeling complexes. They also found they could chemically inhibit histone deacetylases to achieve germ cell to neuron conversion.

    Dr. Hobert suggests these studies have important ramifications. That is, removing even a single chromatin factor, while also inducing individual transcription factors, dramatically impacts neuronal development. He feels this data is a testament to the simplicity but elegance of programs that control neuronal differentiation.

    The emerging picture is that distinct sets of factors orchestrate and coordinate a terminal differentiation regulatory routine. Thus, cellular reprogramming depends not only on expressing the correct transcription factors for inducing a specific neuronal fate, but also removing the inhibitory mechanisms that govern transcription factor activity.

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