Enhancers, Insulators, and Chromatin

Some DNA regions affect the activity of genes that are amazingly far away in the linear sequence of the molecule.

The best known way that genes turn on and off--and thus determine a cell's fate--is when special proteins bind to target DNA sequences right next to different genes--within a few tens of bases. Together with other DNA-binding proteins, these sequence-specific transcription factors promote or discourage transcription of the sequence into RNA. This mechanism is an example of what's called cisregulatory action, because the gene and the target sequence are on the same molecule.

There's another type of sequence that affects genes on the same DNA molecule, but these can be so far away--tens of thousands of bases--that it seems odd to call them cis-regulatory elements. These "enhancers" can be upstream or downstream of the gene they regulate, or even inside of it, in an intron that doesn't code for amino acids. In fact, some of them affect genes on entirely different chromosomes.

Enhancers have important roles in regulating the activity of genes during development, "waking up" in specific tissues at specific times.

The flexible location makes it hard to find enhancers in the genome, and researchers have also struggled to find clear sequence signatures for them. In ongoing work that I described last year for the New York Academy of Sciences, Eddy Rubin and his team at Lawrence Berkeley Labs instead looked for sequences that were extremely conserved during evolution. Although evolutionary conservation is not a perfect indicator, when they attached a dye near these sequences in mice, they often found telltale coloration appearing in particular tissues as the mouse embryos developed.

Years of effort have uncovered many important clues about how enhancers exert their long-distance effects, but still no complete picture. Most researchers envision that the DNA folds into a loop, bringing the enhancer physically close to the promoter region at the start of a gene. Proteins bound to the enhancer region of the DNA, including sequence-specific proteins that can also be called transcription factors, can then directly interact with the proteins bound near the gene, and enhance transcription of the DNA.

But the enhancement effect can be turned off, for example, when researchers insert certain sequences in the DNA sequence between the gene and the enhancer. These "insulator" sequences seem to restrict the influence of the enhancer to specific territories of the genome. Naturally occurring insulators serve the same restrictive function, but they can also be turned off, for example by chemical modification, providing yet another way to regulate gene activity.

If enhancers work by looping, it seems surprising that an intermediate sequence could have such a profound effect. Researchers have proposed various other explanations as well.

In addition to stopping the influence of enhancers, many insulators restrict the influence of chromatin organization. Biologists have long recognized that the histone "spools" that carry DNA can pack in different ways that affect their genetic activity. In a simplistic view, tight packing makes it hard for the transcription machinery to get at the DNA. This chromatin packing can be modified in the cell, and is one important mechanism of epigenetic effects that persistently affect gene expression even through cell division.

As a further confirmation of the close relationship, Bing Ren of the Ludwig Institute and the University of California at San Diego has successfully used known chromatin-modifying proteins to guide him to enhancers, in work that I summarized from the same meeting last year.

One model that combines some of these ingredients says that loops of active DNA are tethered to some fixed component of the nucleus, and that enhancers can only affect genes on the same loop. If insulators act as tethers, this naturally explains how it limits interactions to particular regions (which are then lops). There is still much to be learned, but enhancers and the chromatin packing appear to be tightly coupled.

I suspect that enhancers have been somewhat neglected both because their action mechanism is so confusing and because definitive experiments have been difficult. But recent experiments done in a collaboration between Rubin's and Ren's teams have used a protein called p300, which binds to the enhancer complex, to identify new enhancers with very high accuracy. Moreover, the binding changes with tissue and development just as the enhancer activity does. These and other experiments are opening new windows into these important regulatory elements.