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Though epigenetic modification is not well understood, scientists have a broad outline view. "Inside the nucleus of each cell there are proteins that bind to DNA and tell particular genes to be activated or repressed," explains Zon. The accessibility of binding targets on the DNA molecule depends in part on the chromosomes' shape and configuration at any given time. When the chromosomes are densely packed in the nucleus, they offer fewer binding targets. When they're in a more threadlike stretched-out arrangement, chromosomes are 50,000 times longer and that much more accessible to regulatory binding proteins.
Chromosomal DNA is packaged in a structure called chromatin, which contains small, spool-like proteins, or histones, around which the DNA is wrapped. These histone spools are like scaffolding that determines the configuration of the DNA and its availability for modification by regulatory proteins. Changes in chromatin structure caused by chemical modifications to the DNA or the histones are passed to successive generations of the same type of cell. Zon sums up the big picture like this: "Development from a single cell into a multicellular organism is regulated by environmental signals acting through changes in chromatin, affecting gene expression." The chromatin changes enable genes to be transcribed—that is, expressed—or to be repressed. Appropriate genes are thus turned on or silenced in different types of cells.
Conversely, Zon explains, "Reprogramming is the undoing of the chromatin changes back to the original conformation of the DNA. It's like a drug that erases the cell's memory of what it has been through."
Epigenetic regulation of gene expression is not a simple switch; many control factors have to operate in concert to achieve the appropriate level of expression. One of these factors is methylation—the addition of a "tag," recognizable by cellular proteins, onto one of the chemical bases, or nucleotides, that make up the DNA code. In methylation, a methyl group—a hydrocarbon unit notated as "CH3"—replaces a hydrogen atom on the base. Methylation generally represses gene expression, whereas removing the tag, or demethylation, allows the gene to be expressed.
Nuclear reprogramming occurs not only in cloning, but as a natural process just after a sperm and egg—both highly committed, differentiated cells—join in conception to create an embryo. For the embryo to take its first steps toward development, many genes in the sperm and egg that had been silenced must be reactivated.
"Very rapidly they demethylate, erasing the entire adult cell program," says David L. Garbers, an HHMI investigator at the University of Texas Southwest Medical Center at Dallas. "It's pretty cool, but none of this is well understood." Last November, Garbers reported on a method of keeping rat sperm precursor cells from differentiating into sperm proper. He maintained them—even after freezing and rethawing them—in a nearly stem cell-like state. "We're only one step removed from pushing these cells back a level to a state that is similar to embryonic stem cells," he says. If that could be done, it might be a model for creating embryonic stem cells without harvesting them from embryos.
Another question that weighs on the minds of cloning experts is whether some of the inefficiencies and abnormalities that plague the present technology result from incomplete reprogramming of the donor nucleus. Perhaps not all of the essential genes are activated or deactivated when the nucleus's adult program is erased. Jaenisch holds this view and points to an important embryonic gene called Oct-4 that was shown to be incompletely and randomly reactivated when adult cells were reprogrammed during cloning. This might be one of the reasons, he suggests, that most embryos created through nuclear-transfer techniques die.
Jaenisch and others in the cloning community are carrying out a range of experiments aimed at exploring this issue and, more broadly, trying to identify exactly what factors in the unfertilized egg are responsible for reprogramming.