VARIEGATION VISUALIZED

There is more to silencing than silencing. We have emphasized that a gene, expressed weakly at its ordinary chromosomal location, can be effectively turned off when positioned near the telomere. But about once every 10–20 cell divisions, the silenced gene spontaneously switches on (to a low level). Then, about 10–20 generations later, it switches off again. We say, there- fore, that expression of the gene is “variegated.” Variegation in yeast is eas- ily observed with the ADE2 gene, as discussed in the panel.

We do not have a detailed understanding of the molecular events under- lying variegation. A reasonable scenario might go as follows. The gene is ini- tially silenced by the barrier presented by the silencing heterochromatin, as we have indicated. But that barrier is weak and it might be disrupted (occa- sionally) by the binding of the transcriptional machinery; that binding might occur spontaneously, or it might be helped by a weak activator.

So why, once it is switched on, does the gene tend to stay on for sever- al generations? A reasonable explanation would be that once disrupted, reformation of the silencing heterochromatin, which presumably requires the binding of multiple proteins, is a relatively rare event. Moreover, the active state might be self-perpetuating: HATs associated with the tran- scriptional machinery could modify nucleosomes, which in turn would have an increased affinity for that machinery. Similarly, once the silenced

VARIEGATION VISUALIZED

A yeast colony arises from a single cell; many generations produce the million or so cells found in a typical large colony growing on an agar plate. Should the cells contain a gene whose product confers a charac- teristic color, and if the color is restricted to the cell in which that pro- tein is produced, then variegation is easily observed. Thus, if such a gene, lacking a strong activator, is placed near a telomere, the colony will be mixed: some cells will be colored, some uncolored, as gene expression variegates.

Consider an example in which the cells bear an ADE2gene near the telomere. When the gene is on (even to a very low level) the cells are white, whereas when it is totally off, the cells are bright red. The red color arises from accumulation of the substrate of the enzyme encoded by ADE2. The typical colony bearing ADE2at the telomere is sectored: mostly red with white streaks. When cells are picked from the white sec- tors and replated, they give rise to new colonies, which are mostly white with red sectors.

EPIGENETICS

The classical definition of epigenetics is “a change in the state of expression of a gene that does not involve a mutation, but that is nevertheless inherited in the absence of the signal (or event) that initiated that change.” Many of the regulatory events we have discussed are epigenetic. For example, referring back to the λ case in bacteria, transient expression of one protein (CII) acti- vates transcription of a gene (cI), the product of which then keeps that gene on, a state that is inherited.

Another example is found with the lac genes. A low level of lactose in the medium does not activate the lac genes in ordinary cells. Cells that are preinduced with a pulse of lactose at higher concentration will maintain that induction if the lactose level is then reduced (to the low level in question). The effect is caused by induction of a permease (by the initial, high, levels of lactose) that concentrates lactose in the cell. Once permease is produced, it can maintain a high level of lactose in the cell even when the concentration of that sugar in the medium is low.

Many of the gene regulatory events underlying development of a higher organism involve these kinds of epigenetic changes. In contrast, there is an array of epigenetic phenomena in eukaryotes—particularly in higher eukary- otes and in the yeast Schizosaccharomyces pombe—that evidently cannot be explained solely by the kinds of mechanisms alluded to above. The key dif- ference is that in these cases, there is a different effect on one of two homol- ogous genes or chromosomes in the same cell—a so-called cis effect on gene expression. The classic example is X-chromsome inactivation in mammals, in which the genes on one of the two X chromosomes in each cell is, at an early stage of development, largely inactivated, and this state is stably maintained throughout development.

Our understanding of mechanisms underlying such cis-epigenetic effects is rudimentary. It has been suggested that in at least some such cases, histone modifications that can help or hinder gene expression can, once established, be inherited.

We have mentioned that acetylated histones are recognized by so-called bromo domains found in several components of the transcriptional machinery, including HATs. A similar picture describes histone methylation: enzymes (found in higher eukaryotes and in the yeast S. pombe) methylate specific lysines in histone tails; those modified nucleosomes are recognized by pro- teins bearing chromo domains, including proteins that are themselves histone methylases. Phosphorylation of histones provides another means of creating such a “histone code.”

The difficulty with such models is in understanding how the effect would be transmitted upon replication; i.e., the modified histones would have to be inherited (and perpetuated) only on the chromosomes that are the descen- dants of the one originally modified. We do not know whether such a sce- nario holds in any specific case. See also Footnote 19.

state were formed, it would tend to be self-perpetuating because the Sir proteins, some of which are HDACs, themselves have higher affinity for unacetylated histones.

Silencing at the HM loci is stronger than that at the telomere, but there too, a strong activator overcomes the effect. Variegation is not ordinarily observed with a gene placed at these loci, but it is observed if one of the relevant Sir proteins is mutated.42

Variegation is often cited as an example of “epigenetic” change.