Enhancers

Enhancers, a type of cis-regulatory element, are DNA regions that are bound by Transcription Factors and which play a key role in eukaryotic gene regulation as a result of this interaction. Enhancers are made up of one or more Transcription Factor Binding Sites (TFBSs) which are short DNA sequence, typically about 6-12 nucleotides in length, that are recognized and bound in a sequence-dependent manner by a specific Transcription Factor.

On this page we will look at some of the general properties of enhancers and TFBSs and how they are involved in gene regulation. I should point out that in each of the figures here the regulatory elements are drawn upstream from the promoter. I do this just to keep the diagrams simple. In actual fact, regulatory elements are sometimes found downstream from the promoter, even within introns of the gene. Make sure that you have read the general overview before studying this material.

Defining Characteristics

Enhancers have two characteristics that differentiate them from the sequences located at the promoter, particularly the proximal promoter. These are:

• Their function does not depend on their specific location or distance from the promoter. They can be moved further from or closer to the promoter and still regulate the gene. This is not to say that they will function no matter where they are, it just means that they are not required to be in one specific location.


• Their function does not depend on their orientation relative to the promoter. They can be inverted and still regulate the gene.

These points apply to the entire set of TFBSs in an enhancer. The sites within an enhancer function as a unit and the relative locations and orientations of the TFBSs within the enhancer are important. The enhancer itself can be moved around or inverted and still function, but this is not generally the case for the individual TFBSs within the enhancer.

It should also be stressed that enhancers must be in cis-configuration with the gene (on the same DNA molecule) to function, and that, as stated briefly above, they cannot be moved just anywhere on the chromosome and still function; it is only that regulation is not strictly dependent on them being in a specific location. How far away can they be? That actually differs between organisms. In single-cell eukaryotes like yeast we find regulatory elements fairly close to the promoter, usually within a couple hundred nucleotides. In more complex eukaryotes like Drosophila we often find them a bit further away. In complex eukaryotes like vertebrates we often find them quite distal from the promoter, sometimes tens of thousands of nucleotides - or even more - away! Not only do they tend to be further away but we find many more of them per gene in vertebrates.

Binding by Transcription Factors

Let's make up a hypothetical TFBS and call it TFBS1. Since it is a TFBS it is recognized by a specific Transcription Factor: we will call this protein TF1. When TF1 is activated it binds to TFBS1 and then influences transcription initiation at the promoter. This is the basic idea of how TFBSs work.

Most Transcription Factors, when bound to their TFBS, increase, or activate, gene expression. These are generally called Activators. Other Transcription Factors decrease gene expression (when bound). These are generally referred to as repressive Transcription Factors or just Repressors. In the figure above, TF1 is an Activator.

If we now think about this function at the level of the enhancer, we can see that the binding of TF1 to TFBS1 is the same thing as the binding of TF1 to the enhancer that contains TFBS1. As a result we say that it is the enhancer that is regulating expression.

TFB Sites in Enhancers Function as a Unit

A couple of times I have said something about the TFBSs in an enhancer acting as a unit. What does this mean? What I have presented so far is a bit of a simplification of how the binding of TFs leads to gene activation. What geneticists actually observe is that activation by an enhancer requires that a particular set of the TFBSs in that enhancer be bound by TFs. What the enhancer seems to be doing is akin to reading (in a metaphorical sense!) the information in the set of TFs bound to it and activating gene expression only when specific sets are bound. How this is accomplished is not always understood but what it indicates is that the enhancer is really a unit, not a set of independent TFBSs.

There are multiple copies of each TFBS in a genome

The next important point is that it is not the case that there is just one copy of TFBS1 (or any TFBS) in the genome. There might be multiple copies within a single enhancer and there can also be copies in different enhancers, not just near one gene but near many different genes. When TF1 is activated it binds to all of these TFBS1 sites, meaning that it binds to all of the enhancers that contain a TFBS1. As a result, any promoter that is near an enhancer that has a TFBS1 site is regulated by TF1.

Activation of Transcription Factors

Transcription Factors bind to TFBSs but only when active. This is how changes in environmental or cellular conditions lead to changes in gene expression.

The upshot of this is that whatever conditions lead to the activation of TF1 will lead to a regulatory effect on any gene that has a TFBS1 in one (or more) of it's enhancers. As already noted, an enhancer functions as a unit and so whether or not activation of TF1 leads to activation of gene expression depends on other Transcription Factors being active and present.

General Features of Enhancer Function

To expand on the idea that there are multiple copies of any given TFBS in the genome we can examine a hypothetical set of enhancers near a set of genes as illustrated in the diagram below. This will allow us to study some important general features of enhancers. In the next diagram, E(X) represents an enhancer that has a TFBS for Transcription Factor X. (For example, any element labeled E2 has a TFBS2 in it.) This means that a gene with E(X) nearby is regulated by Transcription Factor X. It should be stressed that this terminology is NOT meant to indicate that this is the only TFBS in the enhancer, nor does it mean that all enhancers labelled E2 (for example) are the same; what it does mean is that all E2 enhancers have a copy of TFBS2, along with other TFBSs that we are not taking into consideration here. Also, rather than draw in all of the TFBSs for each enhancer we will now simplify it a bit and show an enhancer element as a unit, ignoring the individual TFBSs within, and Transcription Factors binding to the unit.

This figure illustrates some specific points observed in real regulatory elements:

• Each gene has numerous TFBSs arranged in several different enhancers. Basically, each gene has a unique set and arrangement of enhancers.


• As a result each gene is regulated by many different Transcription Factors. In general, the regulation of a gene involves a very complex interaction of Transcription Factor 'inputs'.


• The same TFBS can be found multiple times upstream from the same gene.


• The same TFBS is found upstream from different promoters. The result of this is that the same Transcription Factor will regulate multiple genes. Another way to think of this is that each Transcription Factor has multiple target genes.

For example, in the following diagram both TFBS1 and TFBS2 are present in enhancers that regulate Gene A. Thus, this gene will be regulated by both TF1 and TF2.

In the next example, since both Gene 1 and Gene 4 have E1 and E4 sequences upstream, each gene will be regulated by TF1 and TF4 as shown here.