TRANSCRIPTION FACTORS - GENETIC SWITCHES
At the molecular level, transcription factors are the biochemical flavors or scents of the environment. They act as the gene's "on-off switches" and "volume controls." Like tiny chemical keys fitting into chemical locks, each such factor fits into a specific location on a strand of DNA (called a binding site). Gene control is the result of the binding of transcription factors with their specific sites on the gene's regulatory section, that section of the gene which contains the controls for its activities. These sites are found in clusters (modules) located within DNA.
At the moment of conception a spermatazoan, carrying a half set (called "haploid") of chromosomes combines with a oocyte (egg) also equipped with a haploid set of chromosomes. The result is a single cell embryo, equipped with a complete double (diploid) set of chromosomes. This single cell reproduces through the process of mitosis to generate two identical daughter cells, each of which in turn similarly produces two daughter cells, bringing the total number to four identical cells forming a small growing ball. This simplified description of sexual reproduction and embryo growth predominates in nature, being found over an enormous range of organisms, from invertebrates such as jellyfish through mammals such as man.
Each one of the cells at this early stage of embryo development contains a full set of genetic instructions (genes), with all the "instructions" necessary for continued growth and cellular specialization ("differentiation") until the mature form of the organism is reached. If you were to take a sea urchin, or even a human, embryo at the two-cell stage and separate those cells (a form of cloning), you would get two complete, identical organisms. A great mystery of this process is how the identical cells of this small ball begin to differentiate, forming different tissue types, skeletal structures and organ systems. After all, how can one embryonic cell "know" that it is destined to become the ancestor of a brain, while its immediate neighbor will be the ancestor of an intestinal tract? Although, it had been assumed that embryonic cells are somehow sensitive to biochemical clues in their environment, there has been no real evidence that genes in these cells are "smart" enough to pick up such signals.
That evidence may have been found as the result of research on the sea urchin Strongylocentrotus purpuratus. The cells of this humble invertebrate contain a sort of "genetic ear" which is attuned to subtle chemical signals in the environment. This gene, Endo16, produces its protein products (is expressed) in the cells that eventually become the gut of the urchin embryo. Gene activity is controlled by chemicals known as transcription factors (also called regulatory factors"). At the molecular level, transcription factors are the biochemical flavors or scents of the environment. They act as the gene's "on-off switches" and "volume controls." Like tiny chemical keys fitting into chemical locks, each such factor fits into a specific location on a strand of DNA (called a binding site). Gene control is the result of the binding of transcription factors with their specific sites on the gene's regulatory section, that section of the gene which contains the controls for its activities. These sites are found in clusters, known as modules, located within DNA. Each module contains a specific and definite number of transcription factors (much like words in a sentence are constructed by a definite sequence and quantity of letters1). These modules, each with its unique collection and arrangement of transcription factors, when clustered together, affect the time and place of a gene's activation.
It was previously thought that genes could only bind to a handful of transcription factors. Data from sea urchin research indicates that genes can pick up and use many environmental cues and that the module may be the fundamental unit directing the pattern of growth in the early embryo.
The Endo16 gene is found in every cell of the sea urchin. Yet, its protein product accumulates only in the portion of the animal that gives rise to endodermal tissues such as the gut. The search for the answer focused upon the gene's cis-regulatory region, a section of DNA, located just before the section that contains the protein-encoding sequences, which was already known to be involved in regulating the volume of transcription. Endo16 has one such region. Detailed analysis of the gene turned up nearly three dozen transcription binding sites, grouped into seven clusters/modules. As an experiment, researchers separated the seven modules and attached them, in varying combinations, to a bacterial gene that produces an easily detectable "reporter" enzyme2 when it is active. When these gene structures ("constructs") were injected into embryonic sea urchins, varying levels of the reporter enzyme indicated that the concentration of transcription factors varied throughout the animal so that the gene structures were activated in some areas, but not in others.
These modules interact to determine when and where the gene is activated. Although each cell in the organism has an identical set of genes, the genes for the gut, for example, only activate in the place where gut cells should develop (differentiate). For example, constructs containing modules A or B expressed the reporter enzyme not just in the basal portions, but in areas destined to become skeletal member and ectodermal cells. When modules containing constructs E and F were added, enzyme expression was prevented in the future ectodermal cells. When constructs C and D were added, enzyme expression was prevented in future skeletal cells. Therefore, it appears that modules A and B cause the Endo16 gene to be active in all three areas as a response to transcription factors found in those areas. Modules C, D, E, and F detect and respond to chemicals particular to certain areas of the developing embryo (destined to become the ectoderm or skeleton) and block the activity of modules A and B. In other sea urchins studied, an even more complex regulatory mechanism was detected, involving three cis-regulatory regions, more binding sites, transcription factors and greater module complexity. Researchers mutated sections of the gene, deleting various binding sites. Using the same reporter enzyme system, researchers showed that each and every one of the transcription factors performs a specific regulatory role. Some act like switches, turning modules "on" or "off", while others amplify the module's activity or cause it to be confined to specific areas of the developing embryo. Overall, the arrangement of transcription factors bound to binding sites and clustered into modules is much like a computer program, with transcription factors acting as "bits" of data, just like "zeros" and "ones" in electronic computing.
We have seen, above, that for an organism to develop normally, the concentration of transcription factors cannot be identical everywhere in its cellular environment. Concentrations must vary and form gradients. Using Drosophila (the common fruit fly) as an experimental subject, scientists explored the mechanisms by which transcription factors form such gradients. From a molecule's point of view, concentration gradients ARE its neighborhood. One such fruit fly transcription factor3 is Bicoid. The concentration of Bicoid is highest at the front end (or what is destined to become the front end) of the fly larva which "tells" a growing fruit fly embryo what the structure of the front end of the fly should look like. Bicoid must be present above a certain minimum concentration in order to bind to the regulatory site of the target gene (the Hunchback gene). Below that threshold, it will not bind to a gene. Genes are so sensitive to concentration gradients of transcription factors (morphogens) that a set of two or three genes may respond (be turned "on" or "off", for example) by the same morphogen, although each might require a different concentration to be activated.
How does a gradient get established in the first place? This is the same as asking, why, for example, does a morphogen exist in higher concentrations in one area? In fruit flies a gene called Dorsal determines back from front (dorsal from ventral). Like Bicoid, it is activated as the result of concentration gradients of morphogens. At the earliest stage of fruit fly development, a signal of some sort acts to trigger development of a Dorsal gradient. This, in turn, triggers a cascade of other gradients affecting other genes. The initial "signal" which causes the Dorsal gradient probably is the result of a gradient of some chemical that develops in the space surrounding the fertilized egg. Once the dorsal side of the egg is determined (as the result of a higher concentration of Dorsal in one area), and the embryo begins to grow in size, diffusion may take over as a factor that causes gradients to form. As the embryo grows in size, the physical distance (and number of cells) separating the new dorsal region from what will become the ventral region increases. Protein products produced by genes in dorsal cells will have further to travel as they diffuse into their environment. They will be found in steadily lesser concentrations the further they are away from the dorsal region. Eventually, their concentrations drops to the point where they no longer activate genes. In fact, this lack of activation of genes that are normally sensitive to Dorsal may permit other genes, such as genes that are required for the ventral region (but were suppressed so long as dorsal genes were active), to now become active.
From the above discussion you may obtain an appreciation for the wonderful sophistication and complexity of the process of gene control. Genes that produce transcription factors that control other genes are themselves controlled by transcription factors that may be environmentally produced, or produced by yet other genes. A single transcription factor may, depending upon its concentration, affect gene activity of several genes. Concentration gradients that develop through ordinary physical processes (such as diffusion) determine fundamental outcomes such as top from bottom, front from back.
Footnotes:
1 Consider the words "begin" and "cease" as representing a sequence of binding sites, forming a chemical message. The sentence (representing a sequnce of modules) "the process must 'begin" carries a message to start a process. The same sentence, using a different word, might read "the process must 'cease'", a message to stop a process. The "process" might be the production of a protein. In genetic terms, one command might turn a gene on, the other off. Each "letter" represents a transcription factor bound to a binding site. Each "word" represents a module, or cluster of such sites. The sentence itself, made up of four "words" (modules) carries a message, i.e. "begin" or "cease", representing "on" or "off".
2 See "Firefly Genes Light the Way" in the issue of the Double Helix, Vol. 4:2, December 1995.
3 Also called morphogens, for their ability to cause changes in the morphology or gross structure of an organism.