Nerd Alert: How embryonic cell differentiation works?

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Studies Find Elusive Key to Cell Fate in Embryo

By NICHOLAS WADE
Published: April 25, 2006

For three billion years, life on earth consisted of single-celled organisms like bacteria or algae. Only 600 million years ago did evolution hit on a system for making multicellular organisms like animals and plants.

The key to the system is to give the cells that make up an organism a variety of different identities so that they can perform many different roles.

So even though all the cells carry the same genome, each type of cell must be granted access to only a few of the genes in the genome, with all the others permanently denied to it.

People, for instance, have at least 260 different types of cells, each specialized for a different tissue or organ, but presumably each type can activate only some of the 22,500 genes in the human genome.

The nature of the system that assigns cells their various identities is a central mystery of animal existence, one that takes place at the earliest moments of life when the all-purpose cells of the early embryo are directed to follow different fates. Biologists at the Broad and Whitehead Institutes in Cambridge, Mass., have now delved deep into this process and uncovered what seems to be a crucial feature of how a cell's fate is determined, even though much remains to be understood.

They have discovered a striking new feature of the chromatin, the specialized protein molecules that protect and control the giant molecules of DNA that lie at the center of every chromosome.

The feature explains how embryonic cells are kept in a poised state so that all of the genome's many developmental programs are blocked, yet each is ready to be executed if the cell is assigned to that developmental path.

The developmental programs, directing a cell to become a neuron, say, or a liver cell, are initiated by master regulator genes. These genes have the power to reshape a cell's entire form and function because they control many lower genes.

They do so by producing proteins known as transcription factors that bind to special sites on the DNA and control the activity of the lower-level target genes.

A question of interest for biologists studying cell identity is what regulates the master regulator genes. The answer has long been assumed to lie in the chromatin, which determines which genes are accessible to the cell and which are excluded. The chromatin consists essentially of millions of miniature protein spools around each of which the DNA strand is looped some one and half times.

The spools, however, are not mere packaging. They can lock up the DNA they are carrying so that it is inaccessible.

Or they can unwind a little, so that the strand becomes accessible to the transcription factors seeking to copy a gene on the DNA and generate the protein it specifies.

Working backward from that knowledge, biologists have spent much effort trying to learn how the state of the spools is determined.

They have learned there are protein complexes — essentially sophisticated cellular machines — that travel along the chromosome and mark the spools with chemical tags placed at various sites on the spool.

A complex known as polycomb — the name comes from the anatomy of fruit flies, in which it was first discovered — tags spools at a site called K27.

This is a signal for another set of proteins to make the spools wrap DNA tight and keep it inaccessible.

Another complex tags spools at their K4 site, which has the opposite effect of making them loosen their hold on the DNA.

The chromosomes of the body's mature cells are known to have long stretches of K27-tagged spools, where genes are off limits, and other regions where the spools are tagged on K4, allowing the cell to activate the local genes.

The Broad Institute scientists have made use of new techniques that let them visualize which spools along a chromosome carry the K27 or K4 tags.

They decided to map the tags in embryonic cells because of the interest of seeing how the process of determining cell fate is initiated.

In the current issue of Cell, a team led by Bradley E. Bernstein and Eric S. Lander reports that they looked at the chromatin covering the regions where the master regulator genes are sited.

They found to their surprise that these stretches of chromatin carried both kinds of tags, as if the underlying genes were being simultaneously silenced and readied for action.

These bivalent domains, as the biologists called the ambiguously tagged stretches of chromatin, were puzzling at first but make sense in terms of what embryonic cells are meant to do.

Each cell must avoid being committed to any particular fate for the time being, so all its master regulator genes must be repressed by tight winding of the spools that hold their DNA. But the cell must be ready at any moment to activate one specific master regulator as soon as its fate is determined.

The Broad team then looked at the chromatin state of the master regulator genes in several kinds of mature cell.

As was now predictable, they found that the bivalent domains had resolved into carrying just one type of mark, mostly the K27 tag, indicating the master genes there were permanently repressed.

But in each kind of mature cell one or more of the domains had switched over to carrying just the K4 tags, within which genes would be active.

"We think the bivalent state is keeping the embryonic cells poised," Dr. Bernstein said. "It's very special; we didn't see it in any other kind of cell."

Dr. Bernstein's team worked with mouse cells, but its findings have been confirmed in human embryonic stem cells by Tong Ihn Lee and Richard A. Young of the Whitehead Institute.

They and their colleagues started not with the bivalent domains but with the polycomb complex that gives the spools their K27 tag.

Working with human embryonic stem cells, the Lee-Young team mapped where a component of the polycomb complex was attached to the chromatin.

They found it had sought out some 200 sites where many of the master regulator genes of human cells are located. The Whitehead team's article, also published in the current Cell, indicates that in mice and people, just as in fruit flies, the same ancient mechanism is used to make the crucial decisions that determine cell fate.

"This is a very nice piece of work and will be widely interesting because it is fundamental," said Allan Spradling, an expert on embryonic development at the Carnegie Institution of Washington, referring to both teams' findings.

The new findings raise the question of how the embryonic cell knows where on its chromosomes the bivalent domains should be established. Dr. Bernstein and Dr. Lander believe that the answer lies in the structure of the DNA itself.

The bivalent domains occur at regions on the chromosome where some of the DNA sequence is highly conserved, meaning the same sequence is found in widely differing species.

Because DNA is always subject to mutation, a highly conserved sequence is a sure sign of DNA that plays some vital role. These particular sequences, however, do not contain genes, so must be conserved for some other reason.

The highly conserved non-gene sequences were first detected in the dog genome, which was decoded last year. It was in trying to figure out what these regions did that the Broad team stumbled across the bivalent domains.

Although only half of the highly conserved regions contain master regulator genes, something in their DNA structure may be the signal that tells the cell where to create the bivalent domains. This is the crucial step before cells differentiate and take on their various specialized roles.

"We don't know the trigger for differentiation — that is our next step — but I think we now have the key set of genes to look at," Dr. Young said.

Dr. Young's team has studied another aspect of embryonic stem cells which ties into the new finding about bivalent domains. Three genes, known as oct4, sox2 and nanog, are known to be particularly active in the cells and are regarded as a hallmark of the embryonic state.

Dr. Young showed last year that the genes make transcription factors that act on each other's control sites in ways that in effect form a circuitry for controlling the master regulator genes.

He has now found that these transcription factors bind at many of the bivalent domains created by the polycomb complex.

Though it's not yet clear how the whole system works, it seems that the settings on the chromatin spools determine in general what genes are accessible while at a lower level of control the transcription factors control which of the accessible genes are in fact activated.

Because human and other cells can assume so many roles and identities, biologists have long wondered how the status of a cell should be defined, but the new findings may begin to offer a definition.

"This is about as fundamental as you can get," Dr. Spradling said. "We don't really understand what we mean when we say cell state — it hasn't been converted to an understanding in terms of molecular biology."

But a working definition of cell status may be almost at hand, in Dr. Lander's view, in terms of a cell's chromatin state and the transcription factors that can bind to its available genes.

This, after all, is what determines the identities of the various cell types central to an animal's existence. "We are just beginning to get a glimpse of how that central mystery plays out," he said.