Chapter 20: Genomics and Epigenetics

Joshua Reid; Lisa Limeri

20 Chapter 20: Genomics and Epigenetics

Joshua Reid and Lisa Limeri

Learning Objectives

By the end of this section, you will be able to do the following:

Define genomics
Describe genetic and physical maps
Describe genomic mapping methods
Describe three types of sequencing
Define whole-genome sequencing
Explain how individuals exposed to prenatal poverty, hunger, poor diet, smoking, stress, war or violence are prone to epigenetic influences
Describe how access to DNA is controlled by histone modification
Describe how DNA methylation is related to epigenetic gene changes

Genomics Introduction

Genomics is the study of entire genomes, including the complete set of genes, their nucleotide sequence and organization, and their interactions within a species and with other species. Genome mapping is the process of finding the locations of genes on each chromosome. The maps that genome mapping create are comparable to the maps that we use to navigate streets. A genetic map is an illustration that lists genes and their location on a chromosome. Genetic maps provide the big picture (similar to an interstate highway map) and use genetic markers (similar to landmarks). A genetic marker is a gene or sequence on a chromosome that co-segregates (shows genetic linkage) with a specific trait. Early geneticists called this linkage analysis. Physical maps present the intimate details of smaller chromosome regions (similar to a detailed road map). A physical map is a representation of the physical distance, in nucleotides, between genes or genetic markers. Both genetic linkage maps and physical maps are required to build a genome’s complete picture. Having a complete genome map of the genome makes it easier for researchers to study individual genes. Human genome maps help researchers in their efforts to identify human disease-causing genes related to illnesses like cancer, heart disease, and cystic fibrosis. We can use genome mapping in a variety of other applications, such as using live microbes to clean up pollutants or even prevent pollution. Research involving plant genome mapping may lead to producing higher crop yields or developing plants that better adapt to climate change.

Genetic Mapping

The study of genetic maps begins with linkage analysis, a procedure that analyzes the recombination frequency between genes to determine if they are linked or show independent assortment. Scientists used the term linkage before the discovery of DNA. Early geneticists relied on observing phenotypic changes to understand an organism’s genotype. Shortly after Gregor Mendel (the father of modern genetics) proposed that traits were determined by what we now call genes, other researchers observed that different traits were often inherited together, and thereby deduced that the genes were physically linked by their location on the same chromosome. Gene mapping relative to each other based on linkage analysis led to developing the first genetic maps.

Observations that certain traits were always linked and certain others were not linked came from studying the offspring of crosses between parents with different traits. For example, in garden pea experiments, researchers discovered, that the flower’s color and plant pollen’s shape were linked traits, and therefore the genes encoding these traits were in close proximity on the same chromosome. We call exchanging DNA between homologous chromosome pairs genetic recombination, which occurs by crossing over DNA between homologous DNA strands, such as nonsister chromatids. Linkage analysis involves studying the recombination frequency between any two genes. The greater the distance between two genes, the higher the chance that a recombination event will occur between them, and the higher the recombination frequency between them. Figure 20.1 shows two possibilities for recombination between two nonsister chromatids during meiosis. If the recombination frequency between two genes is less than 50%, they are linked.

Figure 20.1. Crossover may occur at different locations on the chromosome. Recombination between genes A and B is more frequent than recombination between genes B and C because genes A and B are farther apart. Therefore, a crossover is more likely to occur between them. (Credit)

Generating genetic maps requires markers, just as a road map requires landmarks (such as rivers and mountains). Scientists based early genetic maps on using known genes as markers. Scientists now use more sophisticated markers, including those based on non-coding DNA, to compare individuals’ genomes in a population. Although individuals of a given species are genetically similar, they are not identical. Every individual has a unique set of traits. These minor differences in the genome between individuals in a population are useful for genetic mapping purposes. In general, a good genetic marker is a region on the chromosome that shows variability or polymorphism (multiple forms) in the population.

Some genetic markers that scientists use in generating genetic maps are restriction fragment length polymorphisms (RFLP), variable number of tandem repeats (VNTRs), microsatellite polymorphisms, and the single nucleotide polymorphisms (SNPs). We can detect RFLPs (sometimes pronounced “rif-lips”) when the DNA of an individual is cut with a restriction endonuclease that recognizes specific sequences in the DNA to generate a series of DNA fragments, which we can then analyze using gel electrophoresis. Every individual’s DNA will give rise to a unique pattern of bands when cut with a particular set of restriction endonucleases. Scientists sometimes refer to this as an individual’s DNA “fingerprint.” Certain chromosome regions that are subject to polymorphism will lead to generating the unique banding pattern. VNTRs are repeated sets of nucleotides present in DNA’s non-coding regions. Non-coding, or “junk,” DNA has no known biological function; however, research shows that much of this DNA is actually transcribed. While its function is uncertain, it is certainly active, and it may be involved in regulating coding genes. The number of repeats may vary in a population’s individual organisms. Microsatellite polymorphisms are similar to VNTRs, but the repeat unit is very small. SNPs are variations in a single nucleotide.

Because genetic maps rely completely on the natural process of recombination, natural increases or decreases in the recombination level given genome area affects mapping. Some parts of the genome are recombination hotspots; whereas, others do not show a propensity for recombination. For this reason, it is important to look at mapping information developed by multiple methods.

Physical Maps

A physical map provides detail of the actual physical distance between genetic markers, as well as the number of nucleotides. There are three methods scientists use to create a physical map: cytogenetic mapping, radiation hybrid mapping, and sequence mapping. Cytogenetic mapping uses information from microscopic analysis of stained chromosome sections (Figure 20.2). It is possible to determine the approximate distance between genetic markers using cytogenetic mapping, but not the exact distance (number of base pairs). Radiation hybrid mapping uses radiation, such as x-rays, to break the DNA into fragments. We can adjust the radiation amount to create smaller or larger fragments. This technique overcomes the limitation of genetic mapping, and we can adjust the radiation so that increased or decreased recombination frequency does not affect it. Sequence mapping resulted from DNA sequencing technology that allowed for creating detailed physical maps with distances measured in terms of the number of base pairs. Creating genomic libraries and complementary DNA (cDNA) libraries (collections of cloned sequences or all DNA from a genome) has sped the physical mapping process. A genetic site that scientists use to generate a physical map with sequencing technology (a sequence-tagged site, or STS) is a unique sequence in the genome with a known exact chromosomal location. An expressed sequence tag (EST) and a single sequence length polymorphism (SSLP) are common STSs. An EST is a short STS that we can identify with cDNA libraries, while we obtain SSLPs from known genetic markers, which provide a link between genetic and physical maps.

Figure 20.2. A cytogenetic map shows the appearance of a chromosome after scientists stain and exam it under a microscope. (credit: National Human Genome Research Institute) — Figure 20.2. A cytogenetic map shows the appearance of a chromosome after scientists stain and exam it under a microscope. (credit)

Genetic and Physical Maps Integration

Genetic maps provide the outline and physical maps provide the details. It is easy to understand why both genome mapping technique types are important to show the big picture. Scientists use information from each technique in combination to study the genome. Scientists are using genomic mapping with different model organisms for research. Genome mapping is still an ongoing process, and as researchers develop more advanced techniques, they expect more breakthroughs. Genome mapping is similar to completing a complicated puzzle using every piece of available data. Mapping information generated in laboratories all over the world goes into central databases, such as GenBank at the National Center for Biotechnology Information (NCBI). Researchers are making efforts for the information to be more easily accessible to other researchers and the general public. Just as we use global positioning systems instead of paper maps to navigate through roadways, NCBI has created a genome viewer tool to simplify the data-mining process.

DNA sequencing

Although there have been significant advances in the medical sciences in recent years, doctors are still confounded by some diseases, and they are using whole-genome sequencing to discover the root of the problem. Whole-genome sequencing is a process that determines an entire genome’s DNA sequence. Whole-genome sequencing is a brute-force approach to problem solving when there is a genetic basis at the core of a disease. Several laboratories now provide services to sequence, analyze, and interpret entire genomes.

For example, whole-exome sequencing is a lower-cost alternative to whole genome sequencing. In exome sequencing, the doctor sequences only the DNA’s coding, exon-producing regions. In 2010, doctors used whole-exome sequencing to save a young boy whose intestines had multiple mysterious abscesses. The child had several colon operations with no relief. Finally, they performed whole-exome sequencing, which revealed a defect in a pathway that controls apoptosis (programmed cell death). The doctors used a bone-marrow transplant to overcome this genetic disorder, leading to a cure for the boy. He was the first person to receive successful treatment based on a whole-exome sequencing diagnosis. Today, human genome sequencing is more readily available and results are available within two days for about $1,000.

Strategies Used in Sequencing Projects

The basic sequencing technique used in all modern day sequencing projects is the chain termination method (also known as the dideoxy method), which Fred Sanger developed in the 1970s. The chain termination method involves DNA replication of a single-stranded template by using a primer and a regular deoxynucleotide (dNTP), which is a monomer. The primer and dNTP mix with a small proportion of fluorescently labeled dideoxynucleotides (ddNTPs). The ddNTPs are monomers that are missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain (Fig. 20.3).

Figure 20.3 A dideoxynucleotide is similar in structure to a deoxynucleotide, but is missing the 3' hydroxyl group (indicated by the box). When a dideoxynucleotide is incorporated into a DNA strand, DNA synthesis stops. — Figure 20.3 A dideoxynucleotide is similar in structure to a deoxynucleotide, but is missing the 3′ hydroxyl group (indicated by the box). When a dideoxynucleotide is incorporated into a DNA strand, DNA synthesis stops. (Credit)

Scientists label each ddNTP with a different color of fluorophore. Every time a ddNTP incorporates in the growing complementary strand, it terminates the DNA replication process, which results in multiple short strands of replicated DNA that each terminate at a different point during replication. When gel electrophoresis processes the reaction mixture after separating into single strands, the multiple newly replicated DNA strands form a ladder because of the differing sizes. Because the ddNTPs are fluorescently labeled, each band on the gel reflects the DNA strand’s size and the ddNTP that terminated the reaction. The different colors of the fluorophore-labeled ddNTPs help identify the ddNTP incorporated at that position. Reading the gel on the basis of each band’s color on the ladder produces the template strand’s sequence (Fig 20.4).

Figure 20.4 This figure illustrates Frederick Sanger's dideoxy chain termination method. Using dideoxynucleotides, the DNA fragment can terminate at different points. The DNA separates on the basis of size, and we can read these bands based on the fragments’ size. — Figure 20.4 This figure illustrates Frederick Sanger’s dideoxy chain termination method. Using dideoxynucleotides, the DNA fragment can terminate at different points. The DNA separates on the basis of size, and we can read these bands based on the fragments’ size. (Credit)

Early Strategies: Shotgun Sequencing and Pair-Wise End Sequencing

In shotgun sequencing method, several DNA fragment copies cut randomly into many smaller pieces (somewhat like what happens to a round shot cartridge when fired from a shotgun). All of the segments sequence using the chain-sequencing method. Then, with sequence computer assistance, scientists can analyze the fragments to see where their sequences overlap. By matching overlapping sequences at each fragment’s end, scientists can reform the entire DNA sequence. A larger sequence that is assembled from overlapping shorter sequences is called a contig. As an analogy, consider that someone has four copies of a landscape photograph that you have never seen before and know nothing about how it should appear. The person then rips up each photograph with their hands, so that different size pieces are present from each copy. The person then mixes all of the pieces together and asks you to reconstruct the photograph. In one of the smaller pieces you see a mountain. In a larger piece, you see that the same mountain is behind a lake. A third fragment shows only the lake, but it reveals that there is a cabin on the shore of the lake. Therefore, from looking at the overlapping information in these three fragments, you know that the picture contains a mountain behind a lake that has a cabin on its shore. This is the principle behind reconstructing entire DNA sequences using shotgun sequencing.

Originally, shotgun sequencing only analyzed one end of each fragment for overlaps. This was sufficient for sequencing small genomes. However, the desire to sequence larger genomes, such as that of a human, led to developing double-barrel shotgun sequencing, or pairwise-end sequencing. In pairwise-end sequencing, scientists analyze each fragment’s end for overlap. Pairwise-end sequencing is, therefore, more cumbersome than shotgun sequencing, but it is easier to reconstruct the sequence because there is more available information.

Next-generation Sequencing

Since 2005, automated sequencing techniques used by laboratories are under the umbrella of next-generation sequencing (deep sequencing or massively parallel sequencing), which is a group of automated techniques used for rapid DNA sequencing. These automated low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to 500 base pairs) in the span of one day. These sequencers use sophisticated software to get through the cumbersome process of putting all the fragments in order.

Evolution Connection: Comparing Sequences

A sequence alignment is an arrangement of proteins, DNA, or RNA. Scientists use it to identify similar regions between cell types or species, which may indicate function or structure conservation. We can use sequence alignments to construct phylogenetic trees. The following website uses a software program called BLAST (basic local alignment search tool).

Under “Basic Blast,” click “Nucleotide Blast.” Input the following sequence into the large “query sequence” box: ATTGCTTCGATTGCA. Below the box, locate the “Species” field and type “human” or “Homo sapiens”. Then click “BLAST” to compare the inputted sequence against the human genome’s known sequences. The result is that this sequence occurs in over a hundred places in the human genome. Scroll down below the graphic with the horizontal bars and you will see a short description of each of the matching hits. Pick one of the hits near the top of the list and click on “Graphics”. This will bring you to a page that shows the sequence’s location within the entire human genome. You can move the slider that looks like a green flag back and forth to view the sequences immediately around the selected gene. You can then return to your selected sequence by clicking the “ATG” button.

Use of Whole-Genome Sequences of Model Organisms

British biochemist and Nobel Prize winner Fred Sanger used a bacterial virus, the bacteriophage fx174 (5368 base pairs), to completely sequence the first genome. Other scientists later sequenced several other organelle and viral genomes. American biotechnologist, biochemist, geneticist, and businessman Craig Venter sequenced the bacterium Haemophilus influenzae in the 1980s. Approximately 74 different laboratories collaborated on sequencing the genome of the yeast Saccharomyces cerevisiae, which began in 1989 and was completed in 1996, because it was 60 times bigger than any other genome sequencing. By 1997, the genome sequences of two important model organisms were available: the bacterium Escherichia coli K12 and the yeast Saccharomyces cerevisiae. We now know the genomes of other model organisms, such as the mouse Mus musculus, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis. elegans, and humans Homo sapiens. Researchers perform extensive basic research in model organisms because they can apply the information to genetically similar organisms. A model organism is a species that researchers use as a model to understand the biological processes in other species that the model organism represents. Having entire genomes sequenced helps with the research efforts in these model organisms. The process of attaching biological information to gene sequences is genome annotation. Annotating gene sequences helps with basic experiments in molecular biology, such as designing PCR primers and RNA targets.

Link to Learning

Click through each genome sequencing step at this site.

Genome Sequence Uses

DNA microarrays are methods that scientists use to detect gene expression by analyzing different DNA fragments that are fixed to a glass slide or a silicon chip to identify active genes and sequences. We can discover almost one million genotypic abnormalities using microarrays; whereas, whole-genome sequencing can provide information about all six billion base pairs in the human genome. Although studying genome sequencing medical applications is interesting, this discipline dwells on abnormal gene function. Knowing about the entire genome will allow researchers to discover future onset diseases and other genetic disorders early. This will allow for more informed decisions about lifestyle, medication, and having children. Genomics is still in its infancy, although someday it may become routine to use whole-genome sequencing to screen every newborn to detect genetic abnormalities.

In addition to disease and medicine, genomics can contribute to developing novel enzymes that convert biomass to biofuel, which results in higher crop and fuel production, and lower consumer cost. This knowledge should allow better methods of control over the microbes that industry uses to produce biofuels. Genomics could also improve monitoring methods that measure the impact of pollutants on ecosystems and help clean up environmental contaminants. Genomics has aided in developing agrochemicals and pharmaceuticals that could benefit medical science and agriculture.

It sounds great to have all the knowledge we can get from whole-genome sequencing; however, humans have a responsibility to use this knowledge wisely. Otherwise, it could be easy to misuse the power of such knowledge, leading to discrimination based on a person’s genetics, human genetic engineering, and other ethical concerns. This information could also lead to legal issues regarding health and privacy.

Epigenetics Introduction

Eukaryotic gene expression is more complex than prokaryotic gene expression because the processes of transcription and translation are physically separated. Unlike prokaryotic cells, eukaryotic cells can regulate gene expression at many different levels. Epigenetic changes are heritable changes in gene expression that do not result from changes in the DNA sequence. Eukaryotic gene expression begins with control of access to the DNA. Transcriptional access to the DNA can be controlled in two general ways: chromatin remodeling and DNA methylation. Chromatin remodeling changes the way that DNA is associated with chromosomal histones. DNA methylation is associated with developmental changes and gene silencing.

Epigenetic Control: Regulating Access to Genes within the Chromosome

The human genome encodes over 20,000 genes, with hundreds to thousands of genes on each of the 23 human chromosomes. The DNA in the nucleus is precisely wound, folded, and compacted into chromosomes so that it will fit into the nucleus. It is also organized so that specific segments can be accessed as needed by a specific cell type.

The first level of organization, or packing, is the winding of DNA strands around histone proteins. Histones package and order DNA into structural units called nucleosome complexes, which can control the access of proteins to the DNA regions (Fig 20.5). Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string (Fig 20.5).

Part A depicts a nucleosome composed of spherical histone proteins that are fused together. A double-stranded D N A helix wraps around the nucleosome twice. Free D N A extends from either end of the nucleosome. Part B is an electron micrograph of D N A that is associated with nucleosomes. Each nucleosome looks like a bead. The beads are connected together by free D N A. Nine beads strung together is approximately 150 n m across. — Figure 20.5. DNA is folded around histone proteins to create (a) nucleosome complexes. These nucleosomes control the access of proteins to the underlying DNA. When viewed through an electron microscope (b), the nucleosomes look like beads on a string. (Credit)

These beads (histone proteins) can move along the string (DNA) to expose different sections of the molecule. If DNA encoding a specific gene is to be transcribed into RNA, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow for the transcriptional machinery (RNA polymerase) to initiate transcription (Fig 20.6).

Nucleosomes are depicted as wheel like structures. The nucleosomes are made up of histones, and have D N A wrapped around the outside. Each histone has a tail that juts out from the wheel. When D N A and the histone tails are methylated, the nucleosomes pack tightly together so there is no free D N A. Transcription factors cannot bind, and genes are not expressed. Acetylation of histone tails results in a looser packing of the nucleosomes. Free D N A is exposed between the nucleosomes, and transcription factors are able to bind genes on this exposed D N A. — Figure 20.6. Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing. (Credit)

In females, one of the two X chromosomes is inactivated during embryonic development because of epigenetic changes to the chromatin. What impact do you think these changes would have on nucleosome packing?

How closely the histone proteins associate with the DNA is regulated by signals found on both the histone proteins and on the DNA. These signals are functional groups added to histone proteins or to DNA and determine whether a chromosomal region should be open or closed (Fig 20.7 depicts modifications to histone proteins and DNA). These tags are not permanent, but may be added or removed as needed. Some chemical groups (phosphate, methyl, or acetyl groups) are attached to specific amino acids in histone “tails” at the N-terminus of the protein. These groups do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins. DNA is a negatively charged molecule and unmodified histones are positively charged; therefore, changes in the charge of the histone will change how tightly wound the DNA molecule will be. By adding chemical modifications like acetyl groups, the charge becomes less positive, and the binding of DNA to the histones is relaxed. Altering the location of nucleosomes and the tightness of histone binding opens some regions of chromatin to transcription and closes others.

The DNA molecule itself can also be modified by methylation. DNA methylation occurs within very specific regions called CpG islands. These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) found in the promoter regions of genes. The cytosine member of the CG pair can be methylated (a methyl group is added). Methylated genes are usually silenced, although methylation may have other regulatory effects. In some cases, genes that are silenced during the development of the gametes of one parent are transmitted in their silenced condition to the offspring. Such genes are said to be imprinted. Parental diet or other environmental conditions may also affect the methylation patterns of genes, which in turn modifies gene expression. Changes in chromatin organization interact with DNA methylation. DNA methyltransferases appear to be attracted to chromatin regions with specific histone modifications. Highly methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive.

Illustration shows a chromosome that is partially unraveled and magnified, revealing histone proteins wound around the D N A double helix. Histones are proteins around which DNA winds for compaction and gene regulation. Methylation of D N A and chemical modification of histone tails are known as epigenetic changes. Epigenetic changes alter the spacing of nucleosomes and change gene expression. Epigenetic changes may result from development, either in utero or in childhood, environmental chemicals, drugs, aging, or diet. Epigenetic changes may result in cancer, autoimmune disease, mental disorders, and diabetes. — Figure 20.7. Histone proteins and DNA nucleotides can be modified chemically. Modifications affect nucleosome spacing and gene expression. (Credit)

Epigenetic changes are not permanent, although they often persist through multiple rounds of cell division and may even cross generational lines. Chromatin remodeling alters the chromosomal structure (open or closed) as needed. If a gene is to be transcribed, the histone proteins and DNA in the chromosomal region encoding that gene are modified in a way that opens the promoter region to allow RNA polymerase and other proteins, called transcription factors, to bind and initiate transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration. In this closed configuration, the RNA polymerase and transcription factors do not have access to the DNA and transcription cannot occur (Fig 20.7).

Acknowledgements

Adapted from Clark, M.A., Douglas, M., and Choi, J. (2018). Biology 2e. OpenStax. Retrieved from https://openstax.org/books/biology-2e/pages/1-introduction

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