Gene Regulation in Eukaryotes

• Some of these are expressed in all cells all the time. These so-called housekeeping genes are responsible for the routine metabolic functions (e.g. respiration) common to all cells.
• Some are expressed as a cell enters a particular pathway of differentiation.
• Some are expressed all the time in only those cells that have differentiated in a particular way. For example, a plasma cell expresses continuously the genes for the antibody it synthesizes.
• Some are expressed only as conditions around and in the cell change. For example, the arrival of a hormone may turn on (or off) certain genes in that cell.

How is gene expression regulated?

• Altering the rate of transcription of the gene. This is the most important and widely-used strategy and the one we shall examine here.
• However, eukaryotes supplement transcriptional regulation with several other methods:
  • Altering the rate at which RNA transcripts are processed while still within the nucleus. [Discussion of RNA processing]
  • Altering the stability of messenger RNA (mRNA) molecules; that is, the rate at which they are degraded. [Link to discussion of RNA interference]
  • Altering the efficiency with which ribosomes translate the mRNA into a polypeptide. [Examples]

• exons whose sequence encodes the polypeptide;
• introns that will be removed from the mRNA before it is translated [Discussion];
• a transcription start site
• promoters
  • a basal or core promoter located within about 40 base pairs (bp) of the start site
  • "upstream" promoters, which may extend over as many as 200 bp farther upstream
• enhancers
• silencers

Adjacent genes are often separated by an insulator which helps them avoid cross-talk between each other's promoters and enhancers (and/or silencers).

Transcription start site

The start site is where transcription of the gene into RNA begins.

The core promoter

• Transcription Factor IID (TFIID) which is a complex of
  • TATA-binding protein (TBP), which recognizes and binds to the TATA box
  • 13 other protein factors which bind to TBP, each other, and (some of them) to the DNA.
• Transcription Factor IIB (TFIIB) which binds both the DNA and pol II.

A core promoter, with little variation in its structure and binding factors, is found in all protein-coding genes. This is in sharp contrast to upstream promoters whose structure and associated binding factors differ from gene to gene.

Many different genes and many different types of cells share the same transcription factors — not only those that bind at the core promoter but even some of those that bind upstream. What turns on a particular gene in a particular cell is probably the unique combination of promoter sites and the transcription factors that are chosen.

An Analogy

The rows of lock boxes in a bank provide a useful analogy.

• your key, whose pattern of notches fits only the lock of the box assigned to you (= the upstream promoter), but which cannot unlock the box without
• a key carried by a bank employee that can activate the unlocking mechanism of any box (= the core promoter) but cannot by itself open any box.

Hormones exert many of their effects by forming transcription factors.

The complexes of hormones with their receptor represent one class of transcription factor. Hormone "response elements", to which the complex binds, are promoter sites. Link to a discussion of these.

Embryonic development requires the coordinated production and distribution of transcription factors.

Enhancers

Some transcription factors ("Enhancer-binding protein") bind to regions of DNA that are thousands of base pairs away from the gene they control. Binding increases the rate of transcription of the gene.

Enhancers can be located upstream, downstream, or even within the gene they control.

There are millions of enhancers in the human genome but which ones are active depends on the type of cell and the signals which it is receiving. Most genes, at least in Drosophila, are regulated by 2–3 enhancers, but some may be controlled by 8 or more. Multiple enhancers are particularly characteristic of "housekeeping" genes.

How does the binding of a protein to an enhancer regulate the transcription of a gene thousands of base pairs away?

One possibility is that enhancer-binding proteins — in addition to their DNA-binding site, have sites that bind to transcription factors ("TF") assembled at a promoter of the gene.

This would draw the DNA into a loop (as shown in the figure).

• a protein designated CTCF ("CCCTC binding factor"; named for the nucleotide sequence to which it binds). The CTCF at one site on the DNA forms a dimer with the CTCF at another site on the DNA binding the two regions together. CTCF has 11 zinc fingers. [View another example of a zinc-finger protein]
• cohesin — the same protein complex that holds sister chromatids together during mitosis and meiosis. [Link]

Visual evidence

• several (4) promoter sites for Sp1 about 300 bases from one end. Sp1 is a zinc-finger transcription factor that binds to the sequence 5' GGGCGG 3' found in the promoters of many genes, especially "housekeeping" genes.
• several (5) enhancer sites about 800 bases from the other end. These are bound by an enhancer-binding protein designated E2.
• 1860 base pairs of DNA between the two.

When these DNA molecules were added to a mixture of Sp1 and E2, the electron microscope showed that the DNA was drawn into loops with "tails" of approximately 300 and 800 base pairs.

At the neck of each loop were two distinguishable globs of material, one representing Sp1 (red), the other E2 (blue) molecules. (The two micrographs are identical; the lower one has been labeled to show the interpretation.)

Artificial DNA molecules lacking either the promoter sites or the enhancer sites, or with mutated versions of them, failed to form loops when mixed with the two proteins.

Significance of "Looping"

Silencers

Insulators

A problem:

As you can see above, enhancers can turn on promoters of genes located thousands of base pairs away. What is to prevent an enhancer from inappropriately binding to and activating the promoter of some other gene in the same region of the chromosome?

One answer: an insulator.

• stretches of DNA (as few as 42 base pairs may do the trick)
• located between the
  • enhancer(s) and promoter(s) or
  • silencer(s) and promoter(s)
  of adjacent genes or clusters of adjacent genes.

Their function is to prevent a gene from being influenced by the activation (or repression) of its neighbors.

The enhancer for the promoter of the gene for the delta chain of the gamma/delta T-cell receptor for antigen (TCR) is located close to the promoter for the alpha chain of the alpha/beta TCR (on chromosome 14 in humans). A T cell must choose between one or the other. There is an insulator between the alpha gene promoter and the delta gene promoter that ensures that activation of one does not spread over to the other.

All insulators discovered so far in vertebrates work only when bound by the CTCF protein.

The mechanism: the mother's allele has an insulator between the IGF2 promoter and enhancer. So does the father's allele, but in his case, the insulator has been methylated. CTCF can no longer bind to the insulator, and so the enhancer is now free to turn on the father's IGF2 promoter.

Many of the commercially-important varieties of pigs have been bred to contain a gene that increases the ratio of skeletal muscle to fat. This gene has been sequenced and turns out to be an allele of IGF2, which contains a single point mutation in one of its introns. Pigs with this mutation produce higher levels of IGF2 mRNA in their skeletal muscles (but not in their liver).

• Mutations need not be in the protein-coding portion of a gene in order to affect the phenotype.
• Mutations in non-coding portions of a gene can affect how that gene is regulated (here, a change in muscle but not in liver).

Gene regulation in bacteria