Antibody detecting

Related Terms

Acetyl, antibody, bisulfite sequencing, cancer, chemical modification, chromatin, chromatin immunoprecipitation, chromosome, cloning, defect, development, DNA, epigenetics, expression, gene, growth, H19 gene, histone, IGF2 gene, immunohistochemistry, immunoprecipitation, methyl, PCR, polymerase chain reaction, Western blotting.

Background

Human cells contain 46 chromosomes (22 pairs of autosomes, and one pair of sex chromosomes). Each individual has two copies of the 22 pairs of autosomal chromosomes, one pair inherited from the mother and one from the father. The pair of sex chromosomes includes an X chromosome from the mother and an X or a Y from the father. Females have two X chromosomes, whereas males have one X and one Y.
Individuals have two copies of most genes, which are sequences of deoxyribonucleic acid (DNA) that contain instructions for making proteins, one inherited from each parent. Researchers have found that the DNA of some genes (for example, IGF2) inherited from the mother has different modifications from the same genes inherited from the father, a phenomenon called imprinting. Imprinting is a normal process that occurs in all individuals, and it is needed for normal development. Modifications due to imprinting may cause the gene from one parent to be made at higher levels in a cell than the gene from the other parent.
DNA is located in a compartment of the cell called the nucleus and is contained in chromosomes. In addition to DNA, chromosomes also contain proteins, such as histones, which help to package the DNA and regulate gene expression. Unlike histones, most proteins are not involved in packaging DNA.
Chromosomes contain hundreds of genes, which provide the instructions for making proteins. Chromosomes also contain many other regulatory sequences that control how much of a gene will be made, when it will be made, and where in the body it will be made.
Epigenetics is a field of biological research that studies the effect of changes to DNA or chromosomes on the activity of genes. The field of genetics commonly focuses on how the changes in the order, or sequence, of chemical bases in DNA affect gene activity, whereas epigenetics focuses on changes in DNA other than sequence changes.
Typically, epigenetics focuses on changes to the DNA or the chromosomal proteins that affect the amount of a particular gene that is produced. The amount of a gene that is produced by a cell is referred to as the expression level.
The level of gene expression can be regulated by cells through chemically modifying DNA or the histone proteins. Through the addition or removal of specific chemical groups to or from the DNA or histone proteins, more or less of a gene can be produced. Several chemical groups, such as acetyl groups or methyl groups, that affect gene expression levels have been identified. Either the amount of a specific chemical modification or the location on the DNA or chromosome where the modification is made can influence the level of gene expression. Imprinted genes typically are differentially modified, meaning that one copy of the gene has a specific modification, but the other copy does not.
Imprinting is relevant to many biological processes, such as development. Also, defects in epigenetic modifications may lead to certain diseases. Because imprinting typically affects the level of gene expression, defects in imprinting are thought to lead to developmental defects or disease through deregulation of gene levels. For example, the IGF2 gene is thought to play a role in the progression of some human cancers, such as colorectal cancer. Some individuals with colorectal cancer have a loss of imprinting of the IGF2 gene, which causes the IGF2 genes from both the father and the mother to be made in an individual's cells. Researchers have found that individuals who have a loss of imprinting of the IGF2 gene have a greater risk of developing colorectal cancer.

Methods

Research on imprinting typically focuses on measuring the amount of a specific chemical modification on DNA or on histone proteins. To measure chemical modifications, there are a variety of techniques that researchers may use.
Antibody detecting: Antibodies are commonly used in techniques that measure chemical modifications. Antibodies are a type of protein normally made in the body by immune system cells to fight off foreign invaders, such as viruses or bacteria. Because they have the ability to bind to specific proteins, however, antibodies may be used to detect specific chemical epigenetic modifications (for example, a modification on a histone protein, such as methylation).
Western blotting: In Western blotting, researchers extract proteins from cells or tissues and separate the proteins using a gel that is subjected to electrical charges. Researchers then transfer the proteins from the gel onto a membrane, usually a thin strip of nylon. Researchers may then look for a certain protein on the membrane using an antibody that is able to specifically detect that type of protein. For example, if a researcher is interested in whether a histone protein has been modified with an acetyl group, an antibody that detects acetylated histone could be used to probe the membrane and to detect the protein.
Immunohistochemistry: Immunohistochemistry is an antibody detection method in which researchers may use antibodies to study the level of a specific protein without extracting the proteins from cells. In immunohistochemistry, researchers apply an antibody directly to cells by pouring liquid containing the antibody onto the cells and observe whether the antibody is able to detect the protein of interest. Researchers commonly use a fluorescent detection system when performing immunohistochemistry, so that antibody binding can be detected by the emission of colored light. This detection system allows a researcher to determine whether an antibody has become bound to a target.
Chromatin immunoprecipitation: Chromatin refers to DNA and the proteins associated with DNA. Chromatin immunoprecipitation is a method that can be used to determine whether specific regions of DNA have undergone an epigenetic modification. To perform chromatin immunoprecipitation, researchers first extract DNA from cells and then cut it into small pieces using high-energy sound waves. Then an antibody that can detect a specific epigenetic modification is used to isolate all the pieces of DNA that contain that modification by binding to it specifically. Finally, a technique called polymerase chain reaction (PCR), which can amplify DNA containing a specific sequence, is used to check for a DNA region of interest.
For example, if researchers are interested in whether a specific gene is highly acetylated, which is a type of chemical modification, they would first use antibodies that can detect acetylated histones to isolate the DNA for all genes that are acetylated. Then they would perform PCR to specifically check whether the sequence of the gene they are interested in was isolated by the antibody.
Bisulfite sequencing: Bisulfite sequencing is commonly used in research labs to study imprinting. In some cases, epigenetic modifications may occur directly on the DNA and not on histone proteins. A method called bisulfite sequencing is commonly used to detect methylation, a type of chemical modification, directly on DNA. In bisulfite sequencing, DNA is treated with a chemical called bisulfite, which causes areas of DNA that have not undergone DNA methylation to mutate, or change in sequence. However, areas of DNA that have undergone DNA methylation are not affected by the bisulfite. Therefore, by comparing the sequences of bisulfite-treated DNA and DNA that has not been treated with bisulfite, researchers may be able to infer which regions are epigenetically modified.

Research

Individuals have two copies of most genes, one inherited from the father and one from the mother. Researchers have found that some genes an individual inherits from the mother have different epigenetic modifications from the same genes inherited from the father, a phenomenon called imprinting. Commonly, the DNA of imprinted genes has higher levels of methylation, a type of chemical modification that causes them to be expressed at lower levels. Because imprinting typically affects the level of gene expression, defects in imprinting are thought to lead to developmental defects or disease through deregulation of gene levels. Imprinting is thought to affect the level of gene expression by interfering with the ability of a cell to produce RNA for a specific gene.
Gene expression: Researchers who study imprinting attempt to better understand how different chemical modifications on DNA or histone proteins can influence the expression of genes. Some types of chemical modifications (such as acetylation) appear to cause genes to be expressed at higher levels, whereas other types of chemical modifications (such as methylation) cause genes to be expressed at lower levels.
For example, DNA that is methylated at a specific position appears to cause nearby genes to be expressed at lower levels. Commonly, imprinted genes are methylated, causing them to be expressed at lower levels. For example, researchers have found that the H19 gene, which is thought to be involved in growth and development, is imprinted. The H19 gene that an individual inherits from the father has higher levels of methylation and is not expressed, whereas the H19 gene that an individual inherits from the mother is not methylated and is expressed. Individuals who inherit an imprinted H19 gene from the father and one from the mother have normal growth and development, even though the gene from the mother is expressed.
Researchers have also identified specific enzymes responsible for attaching chemical modifications to DNA or to histones. For example, researchers have found that proteins called DNA methyltransferases are responsible for adding methyl groups to DNA. Methyl groups are chemical modifications that may affect the level of gene expression.
DNA contains four different chemical compounds called bases: cytosine, thymine, guanine, and adenine. In any given person, these bases are found in a particular order along the chromosomes, and it is the order of these bases that stores information for making genes. Researchers have found that the base cytosine becomes methylated near genes that undergo imprinting.
Development and disease: Using model organisms, such as mice or flies, researchers have found that the imprinting of genes is important for normal development. For example, researchers have found that if a gene called IGF2, which is normally expressed only from the copy inherited from the father, is not properly imprinted in mice, higher levels of IGF2 will be expressed. The increased amount of IGF2 causes problems with development, and the mice develop a condition similar to Beckwith-Wiedemann syndrome, in which developing organs in the fetus become abnormally large.
Additionally, the gene IGF2 is thought to play a role in the progression of some human cancers, such as colorectal cancer. Some individuals with colorectal cancer have a loss of imprinting of the IGF2 gene such that the gene does not become chemically modified, which causes the IGF2 genes from both the father and the mother to be made in the individual's cells. Researchers have found that individuals who have a loss of imprinting of the IGF2 gene have a greater risk of developing colorectal cancer.
Cloning: Imprinting has important consequences for the field of cloning. Cloning is a technique in which a researcher removes the DNA from the egg of an organism and replaces it with new DNA. The egg with the new DNA can then be used to produce an organism that contains the new DNA in all the cells of its body or to produce embryonic stem cells. An embryonic stem cell is a type of cell that has the potential to develop into any type of tissue or organ, such as blood, skin, or liver. Embryonic stem cells could potentially be used therapeutically to help replace damaged tissues.
Attempts at reproductive cloning often fail, and the success rate of generating a healthy, living organism through cloning is very low. The low success rate of cloning may occur because some genes are not imprinted in cloned organisms. The aberrant expression of these genes may lead to developmental defects and death.

Implications

Fighting diseases: Imprinting may be used to better understand some diseases. This is because defects in epigenetic modifications may cause specific genes to become expressed at higher or lower levels, and this aberrant expression may play a role in causing the disease. By identifying genes that are not properly regulated, scientists can better understand how a disease is caused. This information could potentially be used develop drugs to fight diseases caused by defects in imprinting, such as cancer.

Limitations

Not applicable.

Future research

Researchers are working to extend their knowledge of where different epigenetic modifications are located in the human genome, which is the entire DNA sequence of every chromosome contained in an organism. Current large-scale efforts are underway in which researchers are studying the entire human genome to better understand which regions have epigenetic modifications, such as methylation. This future research should give researchers a better global understanding of how many genes in humans are imprinted and what those genes are.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography

Cheng X, Blumenthal RM. Mammalian DNA methyltransferases: a structural perspective. Structure. 2008 Mar;16(3):341-50.
Cui H. Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer. Dis Markers. 2007;23(1-2):105-12.
Jinno Y, Ikeda Y, Yun K, et al. Establishment of functional imprinting of the H19 gene in human developing placentae. Nat Genet. 1995 Jul;10(3):318-24.
Johns Hopkins University School of Medicine. .
Lees-Murdock DJ, Walsh CP. DNA methylation reprogramming in the germ line. Epigenetics. 2008 Jan-Feb;3(1):5-13.
National Center for Biotechnology Information. .
Natural Standard: The Authority on Integrative Medicine. .
Rideout WM 3rd, Eggan K, Jaenisch R, et al. Nuclear cloning and epigenetic reprogramming of the genome. Science. 2001 Aug 10;293(5532):1093-8.
Sun FL, Dean WL, Kelsey G, et al. Transactivation of Igf2 in a mouse model of Beckwith-Wiedemann syndrome. Nature. 1997 Oct 23;389(6653):809-15.
The Epigenome Network of Excellence. .