12.5: Gene Regulation in Eukaryotes

Let’s recall an experiment described earlier and illustrated in Figure 12.12.

Figure 12.12: Replacement of an egg’s nucleus with that from an adult cell enabled the cloning of embryos and eventually, of complete organisms. This proved that adult cells contain all the genes required to program and to complete the development of the organism.

Results of this experiment provided the evidence that even very different cells of an organism contain the same genes. In fact, in any multicellular eukaryotic organism, every cell contains the same genes in their DNA. Therefore, the different cell types in an organism differ not in which genes they contain, but which sets of genes they express! Looked at another way, cells differentiate when they turn on new genes and turn off old ones. Thus, gene regulation produces different sets of gene products during differentiation, leading to cells that look and function differently in the organism.

220 An Experiment: All of an Organism's Cells Have the Same Genome

In eukaryotes many steps lie between the transcription of an mRNA and the accumulation of a polypeptide end-product—many more steps than occur in prokaryotes. Eleven of these eukaryotic steps are shown below in Figure 12.13, which lays out the pathway from gene to protein.

Figure 12.13: A potential to regulate cellular metabolism by controlling mRNA and/or protein levels or activity.

Theoretically, eukaryotic cells can turn or, turn off, speed up, or slow down any step(s) in this pathway, changing the steady-state concentration of a polypeptide in the cells. But the expression of a gene is usually controlled at only one or a few steps. As in bacteria, control of transcription initiation is a common mechanism for regulating gene expression (in principle if not in detail).

221-2 Many Options for Regulating Eukaryotic Genes

12.5.1. Complexities of Eukaryotic Gene Regulation

Eukaryotic gene regulation is more complex than in prokaryotes. This is in part because their genomes are larger and because they encode more genes. For example, the E. coli and human genomes house about five and twenty-five thousand genes respectively. Furthermore, unlike prokaryotes, eukaryotes produce more than twenty-five thousand proteins by alternative splicing to recombine mRNA coding regions and—in a few cases—by initiating transcription from alternative start sites in the same gene. And of course, the activity of many more genes must be coordinated without the benefit of multigene operons! Eukaryotic gene regulation is also more complicated because all nuclear DNA is wrapped in protein in chromatin and chromosomes. Last but not least, all organisms control gene activity with transcription factors that must penetrate chromatin to bind to specific DNA sequences (cis-regulatory elements). In eukaryotes, these elements can be proximal to (near) the promoter of a gene or distal to (quite far from) the gene they regulate.

Figure 12.14 maps a eukaryotic gene to show its typical components (e.g., its promoter, introns, and exons) and its associated cis-acting regulatory elements.

Figure 12.14: Eukaryotic regulatory DNA sequences (CIS elements) are proximal (near a gene promoter) or distal (far from the promoter). Here, a proximal element lies just left of the gene promoter, while distal elements (in this case, 'enhancers') lie upstream (to the left) of the promoter, downstream (to the right) of the last exon of the gene, and in the middle of an intron. Regulatory proteins (sometimes called 'TRANS' factors) bind to proximal and distal CIS elements to control transcription of regulated genes.

Enhancers are typical distal cis elements that recognize and bind transcription factors to increase the rate of transcription of a gene. Oddly enough, these short DNA elements can be in the 5′ or 3′ untranslated region of the gene or even within introns, often thousands of base pairs away from the promoter and transcription start-site of the genes they control. Upstream regulatory regions of eukaryotic genes (to the left of a gene promoter as shown in Figure 12.14) often have distal binding sites for more than a few transcription factors, some with positive (enhancing) and others with negative (silencing) effects. Of course, which of these DNA regions are active in controlling a gene depends on which transcription factor(s) are present in the nucleus. Sets of positive regulators can work together to coordinate and to maximize gene expression when needed and sets of negative regulators may bind negative regulatory elements to silence a gene.

222-2 Transcription Factors Bind DNA Near and Far

In eukaryotes we saw that the initiation of transcription involves many transcription factors (TFs) and RNA polymerase II acting at a gene promoter to form a transcription-preinitiation complex. TFIID (or the TATA-binding protein) is one of the first factors to bind, causing the DNA in the promoter region to bend, much like the CAP-protein in bacteria. Once bound, TFIID recruits other transcription factors to the promoter. As in bacteria, bending the DNA loosens H-bonds between bases, facilitating the unwinding of the double helix near the gene. As the eukaryotic DNA bends, regulatory proteins on distal enhancer sequences are brought close to proteins on regulatory elements that are more proximal to the promoter, as shown in Figure 12.15 (below).

Figure 12.15: Assembly of a eukaryotic transcription complex on a regulated gene: distal regulatory protein/DNA interactions can cause DNA bending, recruiting transcription-initiation factors and proximal regulatory factors to the transcription complex to control gene expression.

Nucleotide methylation sites may facilitate regulatory protein-enhancer binding. When such regulatory proteins, here called activators of transcription, bind to their enhancers, they acquire an affinity for protein cofactors, which allow recognition and binding to other proteins in a transcription-initiation complex. This results in bending the DNA, which then makes it easier for RNA polymerase II to initiate transcription from the appropriate strand of DNA.

232-2 Assembling a Eukaryotic Transcription-Initiation Complex

It is worth reminding ourselves that allosteric (shape) changes in proteins allow DNA protein interactions (in fact, any interaction between macromolecules). The lac repressor we saw earlier is a transcription factor with helix-turn-helix DNA-binding motifs. This motif and two others (zinc finger and leucine zipper) that characterize DNA-binding proteins are illustrated in Figure 12.16.

Figure 12.16: Three DNA-binding motifs commonly found in trans-acting (i.e., regulatory) protein factors.

The DNA-binding motifs shown (zipper, helices, zinc fingers) bind regulatory DNA sequences that are “visible” to the transcription factor in the major groove of the double helix.

224-2 Transcription Factor Domains: Motifs Bind Specific DNA Sequences

We will look next at some common ways in which eukaryotic cells are signaled to turn genes on or off or to increase or decrease their rates of transcription. As we describe these gene regulatory strategies, remember that eukaryotic cells regulate gene expression in response to changes in extracellular as well as intracellular environments. These can be unscheduled, unpredictable changes in blood or extracellular fluid composition (e.g., ions and small metabolites), or they can be dictated by changes in long-term genetic programs of differentiation and development. Some changes in gene expression even obey circadian (daily) rhythms, like the ticking of a clock.

In eukaryotes, changes in gene expression, expected or not, are usually mediated by the timely release of chemical signals from specialized cells (e.g., hormones, cytokines, and growth factors). We will focus on some better-understood models of the gene regulation that is caused by these chemical signals.

12.5.2. Regulation of Gene Expression by Hormones That Enter Cells and Those That Don’t

Gene-regulatory (cis) elements in DNA and the transcription factors that bind to them have coevolved—but not only that! Eukaryotic organisms have evolved complete pathways that respond to environmental or programmed developmental cues and that lead to an appropriate cellular response. In eukaryotes, chemicals released by some cells signal other cells to respond, effectively coordinating the activity of the whole organism. Hormones released by cells in endocrine glands are well-understood signal molecules. Hormones affect target cells elsewhere in the body.

12.5.2.a How Steroid Hormones Regulate Transcription

Steroid hormones cross the cell membranes to have their effects. Common steroid hormones include testosterone, estrogens, progesterone, glucocorticoids, and mineral corticoids. Once inside the target cell, such hormones bind to a steroid-hormone receptor protein to form a steroid-hormone-receptor complex.

CHALLENGE

Hydrophobic steroid hormones must get past a hydrophilic cell surface before, and one after on their way through a lipoidal membrane interior on their way into a cell? How might they do this? Also, discuss how steroid hormones get to receptors already in the nucleus.

The receptor may be in the cytoplasm or in the nucleus, but in the end the hormone receptor complex must bind to DNA-regulatory elements of a gene, either to enhance or to silence transcription. Therefore, a steroid hormone must cross the plasma membrane and may also need to cross the nuclear envelope. You can follow the binding of a steroid hormone to a cytoplasmic receptor in Figure 12.17. Here the hormone (depicted as a red triangle in the illustration) enters the cell. An allosteric change in the receptor releases a protein subunit called Hsp90 (the black rectangle). The remaining hormone-bound receptor enters the nucleus.

Figure 12.17: Once in a target cell, some steroid hormones bind a receptor protein in the cytoplasm, changing its shape. The resulting hormone-receptor complex translocates to the nucleus where it acts as a transcription factor, binding to regulatory DNA sequences to activate, repress or modulate gene transcription. Some steroid hormones find their receptors already in the nucleus!

The fascinating thing about Hsp90 is that it was first discovered in cells subjected to heat stress. When the temperature gets high enough, cells shut down most of transcription and instead transcribe Hsp90 and/or other special heat-shock genes. The resulting heat-shock proteins seem to protect the cells against metabolic damage until temperatures return to normal. Of course, most cells never experience such high temperatures, so the evolutionary significance of this protective mechanism is unclear. But as we now know, heat-shock proteins have critical cellular functions, in this case blocking the DNA-binding site of a hormone receptor until a specific steroid hormone binds to it.

Back to hormone action! No longer associated with the Hsp90 protein, the receptor bound to its hormone cofactor now binds to a cis-acting transcription-control element in the DNA, turning the transcription of a gene on or off. The hormone receptors for some steroid hormones are already in the nucleus of the cell, so the hormone must cross not only the plasma membrane but also the nuclear envelope in order to access the receptor. As for steroid hormone functions, we’ve seen their roles in activating genes for gluconeogenesis. Steroid hormones also control sexual development and reproductive cycling in females, sexual development and sperm maturation in males, salt and mineral homeostasis in the blood, metamorphosis in arthropods, and more, all by regulating gene expression.

226 Steroid Hormones Regulate Gene Transcription

12.5.2.b How Protein Hormones Regulate Transcription

Compared to steroids, proteins are large and soluble and have highly charged surfaces. Even protein-derived hormones like adrenalin, though small, are charged and hydrophilic. Large and/or hydrophilic signal molecules cannot get across the phospholipid barrier of the plasma membrane. To have any effect at all, they must bind to receptors on the surface of cells. Typically, these receptors are membrane glycoproteins. The information carried by protein hormones must be conveyed into the cell indirectly by a process called signal transduction. One of two well-known pathways of signal transduction is shown in the left panel in Figure 12.18.

Figure 12.18: Some signal molecules can't enter cells. They get their message into the cell by signal transduction. Here, a large or polar signal molecule (e.g., a hormone) binds a membrane receptor (1). The binding changes the shape of the receptor (2), which can then bind to a cell membrane protein (3), which becomes an activated enzyme. This enzyme catalyzes the formation of cAMP (the structure seen at the right). cAMP binds and activates the first in a series of protein kinases (4), starting a cascade of protein phosphorylations, a phosphorylation cascade (5) that leads to a cellular response, e.g., the transcription regulation shown here, or changes in cytoplasmic activities.

In this pathway, the binding of the hormone to a cell-surface receptor results in formation of cAMP, a second messenger (Figure 12.18, right panel). cAMP forms when the hormone-receptor in the membrane binds to and activates a membrane-bound adenylate cyclase enzyme. Once formed, cAMP binds to and activates a protein kinase to initiate a protein phosphorylation cascade in the cytoplasm. The phosphorylation cascade is a series of consecutive protein phosphorylations, the last of which is often an activated transcription factor which enters the nucleus and bind to a cis-regulatory DNA sequence to turn a gene on or off or modulate its expression. As suggested in the illustration, that last phosphorylation can also lead to one of many different cytoplasmic responses. Whether by regulating genes or their enzyme products, signal transduction regulate biochemical pathways to control cellular metabolism. cAMP was the first second-messenger metabolite to be discovered. When hormones bind to a membrane receptor that is itself a protein kinase, hormone-receptor binding causes an allosteric change, activating the linked receptor kinase. This starts a phosphorylation cascade without a second messenger. The cascade ends with the activation of a transcription factor, as illustrated in Figure 12.19.

Figure 12.19: Enzyme-linked receptors transduce hormonal signals directly, activating receptor kinases on the cytoplasmic cell surface, initiating a phosphorylation cascade, which lead to any of several responses.

We’ll take a closer look at signal transduction in another chapter.

227 Signal Transduction Can Lead to Gene Regulation