D2.2 Gene expression

Gene expression is the use of the information in a gene to make a product, usually a protein, that affects the cell. Every cell in a human body carries the same genes, but each cell type uses a different selection of them, and that selection determines what the cell becomes and how it behaves.

Cells control expression mainly at the start of transcription, through proteins that bind to DNA, and through chemical tags on DNA and histones. Some of these tags can be passed to daughter cells or to offspring, and some are changed by the environment, without any change in the base sequence.

How genes affect the phenotype

A gene affects an organism only when it is used. Gene expression is the process by which the information in the base sequence of a gene is used to make a product that has an effect on the cell or organism. The observable characteristics that result, from the colour of a flower to the ability to digest milk, are the phenotype.

For most genes, expression happens in three stages. In transcription, RNA polymerase copies the base sequence of the gene into a molecule of mRNA. In translation, ribosomes use the sequence of codons in the mRNA to assemble a polypeptide. Finally, the polypeptide folds into a protein that carries out a function, for example as an enzyme, a structural protein, a hormone or a membrane transporter, and this function is what changes the phenotype.

The gene for the enzyme tyrosinase is a clear example. Tyrosinase catalyses the first step in the synthesis of melanin, the dark pigment of skin, hair and eyes. In pigment cells the gene is transcribed and translated, the enzyme is made, and melanin is produced. A mutation that stops tyrosinase working causes albinism, in which little or no melanin is made. The base sequence affects the phenotype only through the protein it codes for.

Cells do not express all of their genes all of the time. A cell controls which genes are expressed, how strongly and when, so that it makes only the proteins it needs. This control explains how cells with the same genes can become very different, how cells respond to their surroundings, and how some patterns of expression can be passed on to daughter cells and even to offspring.

Regulation of transcription

The main point of control for most genes is the start of transcription. Transcription is regulated by proteins that bind to specific base sequences in the DNA. Because each of these proteins recognises only one or a few sequences, the cell can switch individual genes on or off.

Promoters and transcription factors

Every gene has a promoter, a base sequence just before the start of the coding sequence where RNA polymerase binds. The promoter is not transcribed itself; it marks where and in which direction transcription begins. In eukaryotes, RNA polymerase cannot bind to a promoter on its own. A group of proteins called general transcription factors must first bind to the promoter and then recruit RNA polymerase.

General transcription factors are needed for transcription of almost every gene, so they do not determine which genes are expressed. Which genes are expressed depends on specific transcription factors that bind to other control sequences. Some are activators, which increase the rate of transcription; others are repressors, which decrease or block it.

Enhancers and silencers

An enhancer is a short base sequence that increases transcription of a gene when an activator protein binds to it. Enhancers can be thousands of base pairs away from the promoter, either before or after the gene. The DNA between them bends or loops, so that the activator bound to the enhancer can touch the proteins at the promoter and help RNA polymerase to start transcription.

A silencer is a base sequence that reduces transcription when a repressor protein binds to it. Repressors bound to silencers can block activators or prevent the transcription complex from assembling. Most genes have several enhancers and silencers, so their expression depends on the combination of activators and repressors present in a particular cell.

The pattern of specific transcription factors in a cell therefore determines which genes it expresses. The insulin gene is present in every cell of the body, but it is transcribed only in the β cells of the pancreas. These cells contain a set of transcription factors, including one called PDX1, that bind to enhancers near the insulin gene and activate it. Other cells lack this combination, so their insulin gene stays silent.

Controlling the breakdown of mRNA

Once an mRNA molecule has been made, it can be translated many times by successive ribosomes. The amount of protein made from a gene therefore depends not only on how fast the mRNA is produced, but also on how long each mRNA molecule lasts before it is broken down. Controlling the lifetime of mRNA is a way of regulating translation.

mRNA in the cytoplasm is broken down by nucleases, enzymes that hydrolyse the bonds between nucleotides in a nucleic acid. In eukaryotic cells, mRNA is protected at both ends: the 5′ cap at the start and the poly-A tail at the end. Over time, enzymes shorten the poly-A tail. When it becomes too short, the cap is removed, and nucleases rapidly break down the rest of the molecule.

In human cells, mRNA molecules last for anything from a few minutes to several days. The mRNA for some signalling proteins and transcription factors, such as the protein c-Fos, which is made when cells are stimulated to divide, is broken down within minutes. Its protein is therefore made only for a short time after the signal. At the other extreme, the mRNA for β-globin in developing red blood cells lasts for more than a day, so these cells can keep making haemoglobin in large amounts.

The two curves show the effect of mRNA lifetime. If transcription of a gene stops, an unstable mRNA has almost gone within three hours, so production of its protein stops quickly. A stable mRNA is still present after twelve hours, so its protein continues to be made long after the gene is switched off. Some mRNAs contain base sequences that are recognised by proteins which speed up or slow down their breakdown, so the lifetime can itself be regulated.

Epigenesis and the differentiation of cells

A multicellular organism develops from a single cell, the zygote. By mitosis the zygote gives rise to millions of cells with the same genes, yet these cells become very different: nerve cells, muscle cells, skin cells and many others. The process by which cells become specialised is differentiation.

Epigenesis describes how these patterns of differentiation develop as an embryo grows. Signals between cells and the position of each cell in the embryo switch particular sets of genes on and others off. Once a cell has started on a path of differentiation, its pattern of gene expression is maintained and passed on to its daughter cells, so that a liver cell divides to form more liver cells.

The changes that cause differentiation do not alter the base sequence of DNA. A skin cell and a nerve cell from the same person have the same genotype; they differ because different genes are expressed. Epigenetic changes therefore alter the phenotype of a cell without changing its genotype.

The term epigenetics is used for the study of these heritable changes and of the chemical tags that cause them. The word comes from "epi-", meaning above or on top of: epigenetic tags sit on top of the genetic code without changing it.

Genome, transcriptome and proteome

Three terms describe the genetic information of a cell at different stages of expression. They are easy to confuse, but each refers to a different set of molecules.

• The genome is all of the genetic information of a cell or organism: its entire DNA base sequence, including every gene and all the non-coding DNA.

• The transcriptome is all of the RNA molecules transcribed in a cell at a particular time.

• The proteome is all of the proteins produced by a cell, tissue or organism at a particular time.

Almost every cell in a multicellular organism has the same genome, because all its cells come from the zygote by mitosis. The transcriptome and proteome differ between cell types, and change within a single cell over time, because no cell expresses all of its genes. A pancreatic β cell transcribes the insulin gene but not the gene for β-globin; a developing red blood cell does the reverse. Genes needed by every cell, such as those for ribosomal proteins and ATP synthase, are expressed in all of them.

The pattern of gene expression in a cell determines how it differentiates. Each cell type expresses its own particular part of the genome, and the proteins made from those genes give the cell its structure and function.

Epigenetic tags: methylation of DNA and histones

Patterns of gene expression are held in place by chemical groups attached to DNA or to the histone proteins around which it is wound. These are called epigenetic tags. The two most studied are methyl groups (–CH₃) added to DNA and a range of chemical groups added to histones.

Methylation of DNA in promoters

In DNA methylation, an enzyme adds a methyl group to the base cytosine. In mammals this happens mainly where a cytosine is followed by a guanine (a CG pair in the sequence). Many promoters contain clusters of CG pairs. When the cytosines in a promoter are methylated, transcription factors and RNA polymerase cannot bind there, and proteins that recognise methylated DNA pack the region tightly. The gene downstream of a methylated promoter is therefore repressed.

Modification of histones

DNA in the nucleus is wrapped around histones to form nucleosomes. Each histone has a tail of amino acids that sticks out from the nucleosome, and enzymes can add or remove chemical groups on particular amino acids in these tails. This is histone modification.

• Acetylation (adding acetyl groups) neutralises the positive charge of lysine amino acids in the tails. The histones then bind DNA less tightly, the chromatin becomes loosely packed, and transcription factors can reach the genes. Acetylation activates transcription.

• Methylation of histones can either repress or activate transcription, depending on which amino acid in which histone is methylated. Some methylation tags attract proteins that pack the chromatin tightly and silence genes; others are found at actively transcribed genes and help to keep them switched on.

Epigenetic tags can be added and removed by enzymes, so they are reversible. They are also copied when cells divide, which allows a pattern of gene expression to be inherited by daughter cells. Because tags are stable through cell division but can also be removed, they can maintain differentiation for a lifetime and still change in response to the environment.

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Epigenetic inheritance

Epigenetic tags can be passed on when cells divide. When DNA is replicated, each new double helix has one old strand, which carries the original methyl groups, and one new strand, which has none. An enzyme called a maintenance methyltransferase recognises cytosines that are methylated on the old strand and adds methyl groups to the matching cytosines on the new strand. The pattern of DNA methylation is therefore copied into both daughter cells after mitosis. Histone modifications are also partly re-established on the new nucleosomes.

Epigenetic inheritance through mitosis is what keeps differentiated cells stable. A liver cell that divides produces two liver cells, because the tags that silence non-liver genes are copied with the DNA. In this way a phenotypic change can be inherited by daughter cells without any change in the base sequence.

Some tags can also pass through meiosis into gametes and so to the next generation. Most tags are removed during gamete formation and after fertilisation, but a few persist. Where they do, offspring can inherit a pattern of gene expression that was set in a parent, sometimes in response to the environment the parent experienced.

Agouti mice: diet, methylation and coat colour

Mice carrying the Avy allele of the agouti gene show the effect of methylation on phenotype. When the Avy allele is unmethylated it is expressed in every cell, and the mice are yellow, obese and prone to diabetes. When it is methylated it is silenced, and the mice are brown and of normal weight. Genetically identical mice can therefore look completely different. When pregnant mothers were fed a diet rich in methyl donors (folic acid, vitamin B₁₂, choline and betaine), more of their offspring had a methylated Avy allele and a brown coat. A change in the mother's diet changed the phenotype of her offspring through an epigenetic tag.

Honeybees: one genome, two castes

Female honeybees (Apis mellifera) develop into either a fertile queen or a sterile worker, although they have the same genome. The difference depends on diet: larvae fed royal jelly throughout development become queens. When the gene for DNA methyltransferase 3, an enzyme that adds methyl groups to DNA, was silenced in young larvae, many of them developed into queens with fully developed ovaries without being fed royal jelly. Differences in DNA methylation are part of the mechanism by which diet sets the caste.

Environmental effects on gene expression

The environment of a cell or organism can change its pattern of gene expression. Some effects are short-term responses, such as a bacterium switching on genes when a food becomes available. Others alter epigenetic tags and can last for years. In each case the base sequence is unchanged, but the phenotype changes.

Air pollution and DNA methylation

Air pollution is one environmental factor that changes methylation tags on DNA. In a study in California, children living in Fresno, a city with high levels of ambient air pollution, were compared with children living in Palo Alto, where air pollution is much lower. In the Fresno children, the promoter of a gene called FOXP3 was more heavily methylated in their regulatory T cells. These are cells of the immune system that damp down inflammation, and FOXP3 is the gene that allows them to function.

The extra methylation repressed FOXP3, so the regulatory T cells worked less well. Higher methylation was linked with greater exposure to pollution and with more severe asthma. The results suggest that pollutants such as polycyclic aromatic hydrocarbons from vehicle exhaust can alter epigenetic tags in immune cells, and that this change in gene expression contributes to the harmful effects of pollution on health. This is an example of interaction between an organism and its environment, acting at the level of molecules.

Case study: the Dutch Hunger Winter

From November 1944 to May 1945, a German blockade of food supplies caused a famine in the western Netherlands, at the end of the Second World War. Official food rations fell to an average of about 667 kcal per day, a fraction of normal needs, and lower still at times. The famine had a clear start and end, and the Netherlands kept detailed birth records, so it has become a natural experiment on the long-term effects of poor nutrition before birth.

About six decades later, researchers measured DNA methylation in the blood of people who had been conceived during the famine and compared it with their same-sex brothers or sisters conceived before or after it. Comparing siblings controls for many genetic and family factors, because siblings share half their alleles and were raised in the same home. They studied a region of the gene IGF2, which codes for insulin-like growth factor 2, a hormone important for growth before birth. IGF2 is an imprinted gene, and the region studied carries methylation tags that control its expression.

Methylation is given as the mean fraction of methylated CG sites in the IGF2 region. Source: Heijmans et al., PNAS 105:17046, 2008.

People exposed to famine around the time of conception had lower methylation of IGF2 at a mean age of 58, while those exposed only late in pregnancy showed no clear difference. The results suggest that the very earliest stage of development is a sensitive period, when epigenetic tags are being set up after fertilisation. A shortage of nutrients at this time altered the tags, and the altered pattern was then copied through the many cell divisions that followed and persisted into late adulthood.

The study shows correlation rather than proof of cause, and the methylation difference was measured in blood cells, not in the tissues where IGF2 acts. Even so, it was among the first evidence in humans that conditions early in life can leave lasting epigenetic marks, and it has been supported by later studies of the same cohort.

Removal of epigenetic tags from gametes

Most epigenetic tags are removed in two waves of reprogramming. The first happens as the cells that will become eggs and sperm develop in the embryo; the second happens in the early embryo just after fertilisation. Removing the tags resets the genome, so that the cells of the early embryo can express any gene and become any type of cell. Without this reset, tags from the specialised cells of the parents could restrict the development of the offspring.

Removal is not complete, however. A small number of genes keep tags that were added in either the father or the mother, so that only one of the two alleles is expressed, depending on which parent it came from. This is genomic imprinting. For an imprinted gene, the allele from one parent is silenced by methylation and only the allele from the other parent is used. In mammals, IGF2 is normally expressed only from the allele inherited from the father.

Ligers and tigons

Lions (Panthera leo) and tigers (Panthera tigris) are separate species but can produce hybrid offspring in captivity. The cross can be made in two directions, and the two kinds of hybrid differ in size even though both have one set of chromosomes from each species.

The most widely accepted explanation involves imprinted genes that control growth. Some growth-promoting genes are expressed only from the allele inherited from the father, and some growth-limiting genes are expressed only from the allele inherited from the mother. In lions, where a male mates with several females, the paternal growth-promoting genes are thought to be strongly expressed, and lionesses carry maternal growth-limiting genes that balance them. In tigers, both sides of this balance are thought to be weaker.

• A liger receives strong growth-promoting genes from its lion father and only weak growth-limiting genes from its tiger mother, so growth is not held back and it becomes very large.

• A tigon receives weaker growth-promoting genes from its tiger father and strong growth-limiting genes from its lion mother, so it is no larger, and often smaller, than its parents.

The genotypes of ligers and tigons are the same in kind (half lion, half tiger), but their phenotypes differ because of epigenetic tags inherited from each parent. This shows that the removal of tags from the gametes is incomplete, and that the parent of origin of an allele can matter.

Monozygotic twin studies

Monozygotic twins (identical twins) develop from a single zygote that splits into two embryos early in development. They therefore have the same genome. Any differences between them in phenotype must be caused by the environment, by random events in development, or by differences in epigenetic tags, not by differences in their DNA base sequence.

This makes monozygotic twins a natural controlled experiment: the genotype is fixed, so the effect of the environment on gene expression can be studied. Twin researchers compare the twins of each pair with each other. If a trait differs more between monozygotic twins as they grow older, the difference is likely to come from the environment acting over time.

In one study, the patterns of DNA methylation and histone acetylation were compared in 40 pairs of monozygotic twins aged from 3 to 74. Young twin pairs had almost identical epigenetic tags. Older pairs differed much more, and the differences were greatest in twins who had lived apart for longer or had different lifestyles. The epigenetic tags of the twins drifted apart over their lives, probably in response to differences in diet, smoking, physical activity and other factors, and also by chance errors in copying tags during cell division.

The difference in epigenetic tags helps to explain why one monozygotic twin can develop a condition, such as type 1 diabetes or schizophrenia, while the other does not. Twin studies are limited, however. Twins share a womb and usually a home, so their environments are similar, and there are relatively few twin pairs available for any particular condition. Differences found in one tissue, such as blood, may not apply to others.

External factors that change the pattern of gene expression

Cells respond to chemical signals from outside by changing which genes they express. Two examples show how: a hormone acting on human cells, and a nutrient acting on a bacterium.

A hormone: oestradiol and the endometrium

Oestradiol is the main oestrogen, a steroid hormone secreted by the ovaries. Because it is lipid-soluble, it diffuses through the plasma membrane of its target cells. Inside the cell it binds to an oestrogen receptor, an intracellular receptor protein. The hormone–receptor complex acts as a transcription factor: it binds to specific base sequences called oestrogen response elements near target genes and activates their transcription.

In the endometrium, the lining of the uterus, oestradiol switches on genes that cause cells to divide, so the lining thickens during the first half of the menstrual cycle. Oestradiol also activates the gene for the progesterone receptor. This prepares the endometrium to respond to progesterone after ovulation, when progesterone maintains the thickened lining ready for implantation. Cells without oestrogen receptors are not affected by the hormone, which is why oestradiol acts only on certain tissues.

A biochemical: lactose and the lac operon

The bacterium Escherichia coli can use lactose, the sugar in milk, as an energy source, but it makes the enzymes to do so only when lactose is present. The genes for these enzymes form an operon: a group of genes transcribed together from one promoter and controlled by a single operator sequence. The lac operon contains three genes: lacZ codes for β-galactosidase, which hydrolyses lactose into glucose and galactose; lacY codes for a permease that transports lactose into the cell; and lacA codes for a transacetylase.

A separate gene, lacI, codes for a repressor protein. The repressor is made all the time and controls the operon.

1. Lactose absent. The repressor binds to the operator, a base sequence between the promoter and the genes. RNA polymerase can bind to the promoter but cannot move past the repressor, so the genes are not transcribed. The cell does not waste energy making enzymes it cannot use.

2. Lactose present. Some lactose is converted to allolactose, an isomer of lactose. Allolactose binds to the repressor at a site away from its DNA-binding site, changing its shape. The repressor can no longer bind to the operator and is released.

3. Transcription. RNA polymerase transcribes lacZ, lacY and lacA into a single mRNA, which is translated to make the three enzymes. The bacterium takes up and breaks down lactose.

4. Lactose used up. As lactose is broken down, allolactose levels fall. The repressor returns to its original shape, binds to the operator again and switches the operon off.

The lac operon was described by François Jacob and Jacques Monod in 1961, from experiments on mutant bacteria that made the enzymes all the time or never. It was the first model of how genes are switched on and off, and it showed that some DNA sequences code for no protein but act as control switches.

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The two examples differ in detail, but in each case a chemical from outside the cell binds to a regulatory protein and changes whether that protein binds to DNA, so the pattern of transcription changes without any change in the base sequence.

Exam practice

Gene expression is examined at HL only. Paper 1A questions often test definitions (genome, transcriptome, proteome) and the effects of methylation and acetylation. Paper 1B and Paper 2 Section A use data from twin studies, methylation measurements or bacterial enzyme activity. Paper 2 extended responses ask for explanations of how transcription is regulated, how epigenetic tags are inherited, or how the lac operon responds to lactose. The command terms most often used are distinguish, outline, explain, suggest and evaluate.

Gene expression connects several other subtopics: B2.3 Cell specialisation (differentiation of stem cells), C2.1 Chemical signalling (steroid hormones and intracellular receptors) and D3.1 Reproduction (oestradiol in the menstrual cycle).

Self-check

• To do:

  I can outline gene expression as transcription, translation and protein function, with an example.

• To do:

  I can explain how promoters, enhancers, silencers and transcription factors regulate transcription.

• To do:

  I can explain how the lifetime of mRNA and its breakdown by nucleases regulate translation.

• To do:

  I can define epigenesis and explain that epigenetic changes alter the phenotype but not the genotype.

• To do:

  I can distinguish the genome, transcriptome and proteome of a cell.

• To do:

  I can describe the effects of methylation of promoters and of histones, and of histone acetylation.

• To do:

  I can explain how epigenetic tags are inherited by daughter cells and sometimes by offspring.

• To do:

  I can describe the effect of air pollution on DNA methylation and interpret data such as the Dutch Hunger Winter study.

• To do:

  I can explain genomic imprinting and the difference between ligers and tigons.

• To do:

  I can explain how monozygotic twin studies show the effect of the environment on gene expression.

• To do:

  I can explain how a hormone (oestradiol) and a biochemical (lactose) change the pattern of gene expression.