MECHANISMS INVOLVED WITH
CONTROLLING GENE EXPRESSION

Author

Robert Holtz, President

BioInnovation Laboratories

7220 W. Jefferson Ave., Ste 112Lakewood, CO 80235 USA

ABSTRACT:

This chapter will describe the analytical methods used to assess the epigenetic mechanisms involved in controlling gene expression. Although the information in this chapter is presented as a general overview of the methods, it nonetheless contains a great deal of technical detail with the intent that knowledge of these details can assist in both experimental design by the research and development scientists and also help marketing groups to understand how the data were generated along with the benefits or limitations of using these techniques. For readers who are not analytical chemists, or biochemical experts, the chapter is designed to educate and enhance the understanding of the role that epigenetics plays in normal and abnormal skin function. While this field has made great strides, there is still much to learn. Being able to at least be conversant in the current technologies opens the door to ingredient and product developers to successfully interact with their analytical counterparts, either internally or externally, to move forward towards their goal of producing novel products.

The term “epigenetic” can be defined as heritable changes in gene expression or phenotype that do not involve changes in the underlying DNA sequence of the organism. Heritable changes are those capable of being passed from one generation to the next; i.e., hereditary. To illustrate the power and importance of epigenetics one need look no further than our own bodies. Human cells contain the same approximate 30,000 genes regardless of cell type—and yet when keratinocytes divide, the two daughter cells remain keratinocytes. The gene expression pattern is transmitted from the mother cell to the progeny while the DNA sequence in the two keratinocyte daughter cells is unchanged and still identical to fibroblasts or any other cell type in the human body.

The reason for this maintained pattern of gene expression is thought to involve an overlying epigenetic control mechanism. The process by which this epigenetic control of gene expression occurs involves at least two basic pathways: DNA methylation and histone modification. Both DNA methylation and histone modification can control gene expression by restricting the access of transcription factors to gene promoters. These two types of modification can induce changes in the structure of the chromatin, such as chromatin condensation, in the region where the gene is located. This essentially leaves the gene promoter blocked from required transcription factors resulting in a silenced gene. Both mechanisms play an essential role in the epigenetic control of gene expression. An understanding of this phenomenon is of critical importance to the development of optimal cosmetic and personal care products. Not only is it advantageous to understand the basic epigenetic control process in normal healthy skin, but it is equally important to realize that epigenetic control mechanisms can adversely change in response to cosmetically relevant stressors such as aging, inflammation, UV exposure, or exposure to other adverse environmental stresses. By understanding the mechanisms of the adverse epigenetic changes it then becomes possible to design and test products with the intent of returning the skin to its normal healthy state. This chapter will discuss useful in vitro methods that can be used to examine two of the better-understood epigenetic mechanisms and help understand the impact of adverse stressors on normal skin function. The topic of epigenetics and its role in skin aging is also discussed elsewhere in this book in the section Anti-Aging Pathways.

TABLE OF CONTENTS

11.7.1  Introduction

11.7.2  DNA Modifications: DNA Methylation

a.  DNA Methylation: Methyltransferases

b.  DNA Methylation: Pharmacological Agents

11.7.3  Histone Modifications

a.  Post-Translational Histone Modification Assays

b.  Histone-Modifying Enzyme Assays

c.  Pharmacological Agents That Impact Histone Modification

Conclusion

References

11.7.1 INTRODUCTION:

In 1951 Dr. G. Wyatt, at the time a young Ph.D. student, was working to improve current methods to separate DNA bases using paper chromatography. After finding a blend of isopropanol and HCl that gave a good separation, and well-defined “spots” for the four bases: adenine, guanine, cytosine, and thymine in test samples, Dr. Wyatt was surprised to find that upon running actual animal and plant DNA hydrolysate samples the assay produced an additional and unexpected fifth spot (Wyatt 1951). This fifth spot was quickly identified to be 5-methyl cytosine. While the presence of large amounts of 5-methylcytosine in human genomic DNA was interesting, its function remained largely unknown until 1975. In that year two papers were published predicting that programmed methylation and demethylation of cytosine bases in DNA would actually regulate gene expression (Holiday and Pugh 1975; Riggs 1975). The papers went on to suggest that the methylation of cytosine would not occur at random cytosine bases, but rather at specific DNA sequences containing key cytosine bases.

Over the decades, since the initial discovery of methylated DNA bases and their involvement with controlling gene transcription, it became apparent that modified DNA was not alone in its ability to control gene transcription through nontraditional transcription methods. Histones, the proteins that help package DNA into well-defined chromatin structures, were also discovered to be able to be covalently modified in ways that controlled gene expression. The potential of DNA methylation and histone modification controlling gene expression was an exciting prospect since it indicated that genetic changes could be inherited without altering the basic DNA sequence. This discovery also added an additional level of control above and beyond the basic mechanisms of the previously known cellular machinery for determining which genes were turned on or off and had the potential to explain many biochemical phenomena affecting the human body.

The basic mechanism of gene expression involves the binding of transcription factors to the promoter region of the gene. Transcription factors are essentially control proteins that when activated can bind to the promoter region of the gene and help to recruit the necessary enzymes that are involved in transcribing an RNA copy of the gene, which is subsequently translated to the target protein that the gene is responsible for making. When an RNA copy of the gene is being transcribed the gene is essentially “on.” Conversely, when an RNA copy of the gene is not being made, the gene is referred to as “off” since without the transcribed RNA the final protein product of the gene cannot be made.

Transcription factors often work in concert with other regulatory proteins that can bind to repressor or enhancer regions also associated with the gene. Depending upon the combination of transcription factors and regulatory factors binding to the regulatory regions of the gene, gene expression control can go beyond simply being “on” or “off” and intermediate levels of control can be achieved, which can further enhance or repress the basal level of gene expression. However, as intricate as this basic control mechanism for gene transcription is, DNA methylation and histone modification can further increase the control of gene transcription. This is done by altering the structure of the DNA in such a way as to control the access of these transcription and regulatory proteins to the regions to which they need to bind. Methylated DNA can bind to special proteins with methylation binding domains and, in essence, coat the DNA in proteins that prohibit access to the DNA by transcription factors. In addition, both DNA methylation and histone modification can lead to changes in the chromatin structure, such as chromatin condensation. Such changes can again prevent or allow access of transcription factor to their DNA binding regions. Since DNA methylation and histone modification-based mechanisms control gene transcription at a level above normal transcription factors, they have been grouped into a class of new control mechanisms termed Epigenetic.

Epigenetic control mechanisms are now known to be heritable changes, that is, the changes in DNA methylation or histone modification can be passed from mother cell to daughter cell and play an important role in normal cell function such as maintaining cell differentiation. However, sometimes epigenetic changes can be detrimental to a cell’s performance. It is now thought that abnormal epigenetic control mechanisms may play a role in aging and cancer (Agrawal et al. 2010; Miller et al. 2011). Due to their ability to control global gene transcription, and their involvement in skin disease, the study of epigenetic control mechanisms has garnered a large amount of research interest by the skin care industry. Compounds that can influence epigenetic control mechanisms can be extremely beneficial in countering the effects of aging, preventing skin-based diseases, or even enhancing the normal function of the skin. This chapter describes some of the current methods used in skin care research when addressing epigenetic control mechanisms.

11.7.2 DNA MODIFICATION: DNA METHYLATION

It is now known that approximately 60% of the promoters within the human genome contain potential DNA methylation sites and that DNA methylation occurs primarily in cytosines that are followed immediately by a guanine residue (CpG) (Antequera and Bird 1993). These methylations are catalyzed by a family of DNA methyltransferases that consists of DNA methyltransferase (DNMT) 1, 3a, 3b, and 3l. This family of enzymes catalyzes the transfer of a methyl group from the methyl donor S-adenosylmethionine to cytosine resulting in the formation of 5-methylcytosine. Once donated to cytosine, this methyl group protrudes into the major groove of the DNA and can reduce the ability of transcription factors to both recognize and bind to their specific DNA binding site (DeAngelis et al. 2008). The attached methyl group can also be bound by proteins with methylcytosine binding sites (MCBs). This binding can impact the level of gene transcription by at least two mechanisms: (1) proteins with MCBs can block the access of transcription factors to gene regulatory regions by directly binding to the DNA regulatory region of interest, or (2) they can also recruit histone-modifying enzymes to their location. Histone-modifying enzymes can modify histones at a number of locations via changes in the methylation, acetylation, phosphorylation, or ubiquitinylation status of key amino acids (these changes will be discussed in greater detail later in the chapter). This type of modification can have the net result of altering the local chromatin structure in a manner that again impacts gene expression. For the most part, these changes tend to silence genes; however, there are a small handful of studies showing that the expression of some genes can also be enhanced by these epigenetic changes.

Initial methods to examine DNA methylation used techniques based on either chromatography (Kuo et al. 1980), radiolabeling with tritiated S-adenosylmethionine (Duthie et al. 2000), or immunolabeling with anti-5-methylcytosine antibodies (Adouard et al. 1985). While these early methods were revealing, they were limited to simply determining the relative proportion of 5-methylcytosine within the genome and could not provide information on the methylation status of specific gene promoters or coding sequences. However, sometimes measuring the relative amount of global genomic methylation can be extremely informative. Such information is useful, for example when determining the impact of aging (Agrawal et al. 2010) or UV exposure (Mittal et al. 2003) on total genomic DNA methylation to determine if specific stressors can elicit general epigenetic changes before pursuing a more detailed study for specific changes. Such global measurements can be made with relative ease since the initially complicated and labor-intensive methods of chromatography and radiolabeling have now been replaced with rapid, high-throughput, sensitive ELISA-based methods (enzyme-linked immunosorbent assay). ELISA-based methods also have the advantages of low cost, relative to other methods for measuring DNA methylation. Thus, while ELISA-based methods may lack the ability to determine specific methylation sites, they have the benefit of being rapid and one of the least-expensive methods to measure changes in DNA methylation.

Since most DNA methylation ELISAs are competitive-based ELISAs designed to specifically measure 5’’-methyl-2’-deoxycytidine, the nucleoside version of the methylated DNA base, total genomic DNA must be isolated from the sample and broken down into individual nucleosides. DNA extraction from cells or tissues can be achieved using commercially available extraction kits. Once extracted, the DNA is re-suspended in water and denatured by incubating it for 5–10 minutes at 95°C. Denaturation will convert the double-stranded DNA molecule into two single strands. Denaturation is followed by immediate cooling on ice, after which the single stranded DNA is enzymatically digested to nucleoside monophosphates with nuclease P1, followed by the removal of the phosphate groups using alkaline phosphatase. The remaining nucleoside solution can then be assayed using any commercially available ELISA to quantitatively measure the amount of 5’-methyl-2’-deoxycytidine. The amount of remaining nucleoside solution can then be normalized to the amount of original starting DNA in order to determine the proportion of DNA that is methylated.

While measuring global changes in DNA methylation can be easily accomplished using ELISAs, sometimes it is essential to not only measure global changes in DNA methylation, but to also determine where in the genome those changes are occurring. For example, while it may be interesting to note that UV exposure of keratinocytes results in an increase in global DNA methylation, the next question would be which genes are specifically being impacted by epigenetic mechanisms, such as the genes involved in cell proliferation, the genes involved in DNA repair, or genes involved with other critical cellular functions.

In general, there are two alternative methods for determining specific changes in methylation status within defined regions of the genome. These two methods are based on either microarray formats or sequencing formats. Microarray formats are designed to assess changes in global DNA methylation status across the genome. However, while they can identify if changes in DNA methylation are occurring within specific regions of DNA, they cannot definitively determine which individual cytosines are methylated if more than one potential methylation site occurs within the region. In contrast, sequencing-based DNA methylation analysis can determine which individual cytosines have been methylated within a given DNA region. However, due to the expense and complexity of sequencing based methods, these analytical approaches are normally limited to a handful of DNA sequence targets and are not well suited for global genomic measurements. Thus, these latter two methods provide a tradeoff between either obtaining global measurements of genomic DNA methylation (with limited detail on specific regions), or obtaining very specific methylation data on individual cytosines within a set of very well-defined DNA regions without genome-wide information.

With microarray based DNA methylation analysis methods, the initial step is the isolation of genomic DNA from the sample. This can be accomplished using most commercially available DNA extraction kits or established lab methods (Sambrook et al. 1989). Once isolated, the genomic DNA must be randomly fragmented into DNA strands of approximately 200–1000 base pairs (bp) in size using either sonication or restriction enzyme digestion (Weng et al. 2009). Once fragmented, a portion of the DNA sample is then used to enrich for methylated DNA strands using affinity-based methods. With affinity-based methods, either an antibody that specifically recognizes 5-methylcytosine (Weber et al. 2005) or a protein containing an MCB (Rauch et al. 2006) is used to bind methylated DNA fragments. The antibody/protein DNA fragment complex is then precipitated, most commonly through the use of magnetic beads (Weng et al. 2009). Once separated from the antibody/protein used for precipitation, both the methylated DNA-enriched sample and the original DNA sample can be labeled with respectively colored fluorescent dyes and hybridized to a microarray containing complementary DNA probes specific for CpG-containing regions of the genome.

Upon being scanned and normalized, the two-color microarray provides an indication of the specific areas of the genome that contain methylated DNA. Current methylation arrays are considered tiling arrays. By this term we mean that the potentially thousands of bases that form the DNA sequence for any given gene (including both the promoter and coding portion of the gene) are divided into smaller portions and thereafter split into sequential features on the microarray chip. Thus, although each specific probe region on the array chip may only contain a DNA sequence corresponding to a 50–100 base portion of the gene of interest, the next probe set will cover the next adjacent DNA region. This process is then continued until the entire gene sequence is covered in the same manner that multiple individual tiles cover a single floor. With each probe for the region containing a portion of the whole DNA sequence for that region, this collection of multiple probes can provide complete coverage of methylated areas. Since most microarray chips now contain at least 250,000 probe sets, a good portion of the human DNA methylome can be covered with a single array experiment in a matter of a few days.

As indicated above, while DNA microarray-based methods for measuring DNA methylation can determine if methylated cytosines are present within a given region of DNA, they cannot definitively determine which cytosine within the region is methylated or if more than one cytosine is methylated. If a single probe region contains more than one cytosine methylation site then, depending upon the DNA fragmentation pattern, the array can potentially give the same signal if just one site within the probe region is methylated or if all of the sites within the probe are methylated, since all it takes is just one methylated cytosine to immunoprecipitate a DNA fragment. Generating smaller DNA fragments prior to enriching for methylated DNA may somewhat help to increase the ability of the microarrays to determine the level of methylation in regions with multiple methylation sites. However, to truly observe which specific cytosines are methylated within the genome requires true sequencing-based methods. Yet, some evidence suggests that within a given region of DNA, all of the potential cytosine methylation sites share the same methylation status (Eckhardt et al. 2006). Therefore it may be enough to know if a region contains some methylated cytosines, without having to determine the details on all potential methylation sites within the region, since it can be inferred that they all share the same methylation status.

As you can see, microarray-based DNA methylation studies can provide a lot of data. With at least 250,000 data points the prospect of analysis can be quite daunting; however, the wealth of data obtained in these types of experiments is an extremely valuable asset. This is especially true since the field of epigenetic research is still relatively young and there are only a limited number of studies that have begun to address the epigenetic control mechanisms pertaining to skin cells. Since the main cell types of the skin (keratinocytes, fibroblasts, and melanocytes) have not been well characterized with respect to normal patterns of DNA methylation, let alone how stress may alter this pattern of DNA methylation, then experiments that examine global changes are the best option when specific targets are not known.

With respect to skin care research, DNA methylation microarray data can describe which regions of the genome are methylated and therefore potentially silenced. This type of data is especially useful when comparing different types of cellular stress since it will tell you how the cell is responding to the stress at an epigenetic level, allowing for the determination of whether the response is ultimately beneficial or harmful to the cell. It is these epigenetic responses to stress that represent an example of interesting targets in the product development and marketing aspect of cosmetic research. These epigenetic responses provide targets against which active ingredients can be screened to determine if the actives can either augment a beneficial epigenetic change or reduce a harmful response.

In contrast to microarray-based methods to measure genomic DNA methylation, sequencing-based methods can provide a very detailed analysis of precisely which cytosine bases have been methylated. The key to this precision is the reaction of both cytosine and 5-methylcytosine with the acid salt sodium bisulfite. When exposed to sodium bisulfite, cytosine is deaminated to form uracil (Hayatsu et al. 1970). Although 5-methylcytosine will also react with sodium bisulfite, the reaction rate is significantly slower than the reaction with cytosine. This reduction in reaction rate has allowed for the development of a method that will convert nonmethylated cytosines into uracil residues while methylated cytosines would remain as essentially cytosine residues (Frommer et al. 1992). Since sequencing reactions involve amplification of the target DNA via PCR, the uracil (which is normally found in RNA and base pairs with adenine) in the bisulfite-treated DNA sample is converted to a thymine residue (which is the DNA base that binds to adenine). This reaction results in the 5-methylcytosine remaining a cytosine. By comparing the DNA sequences of non-sodium bisulfite–treated regions of the genome to the same region after sodium bisulfite treatment, any remaining cytosine bases in the sodium bisulfite–treated DNA represent methylated cytosines, while any cytosines in the original sequence that have been converted to thymines were nonmethylated cytosines (Zhang and Jeltsch 2010).

Sequencing-based methods can share some common approaches to DNA methylation analysis with microarray-based methods. In a manner similar to array-based methods, sequencing analysis can start with DNA samples that have been enriched for methylated DNA using the affinity/precipitation-based methods described above (Maunakea et al. 2010; Salpea et al. 2012). The enrichment for DNA methylation reduces the complexity and significantly reduces the cost of the subsequent sequencing data analysis. Rather than subject the entire genome to sodium bisulfite treatment, and then search for pockets of methylation, using the methylation- enriched DNA samples only those regions that contain DNA methylation are sequenced. Yet, despite using the methylated DNA enriched samples for analysis, there is still a considerable amount of sequencing that needs to be accomplished in order to obtain a detailed image of changes in the entire human DNA methylome.

As an alternative to the sequencing method described above, which uses DNA samples enriched for methylation to determine widespread DNA methylation, it is possible to use PCR to amplify small regions of interest to see if these discrete regions are methylated. All that is required in this method is to isolate genomic DNA from the sample, take a portion of that sample, and expose it to sodium bisulfite to differentiate between methylated and nonmethylated cytosines. Thereafter, PCR primers are designed that will amplify the region of interest in both the native DNA and bisulfite-converted DNA. The PCR products from both approaches would then be sequenced and compared to determine sites of DNA methylation within a discrete region of the genome.

a. DNA Methylation: Methyltransferases

DNA methylation is catalyzed by a family of DNA methyl transferases (DNMTs). DNMT1 appears to be the main isoform of the enzyme responsible for maintaining the methylation status of existing methylation patterns, while DNMTs 3a, 3b, and 3L are responsible for de novo patterns of DNA methylation (Yang et al. 2010). These enzymes catalyze the transfer of a methyl group from the methyl donor S-adenosyl methionine to cytosine, resulting in the formation of 5-methylcytosine.

Currently several companies produce commercial assays that are available to measure changes in DNMT activity in either purified enzyme preparations or in nuclear extracts from samples of treated cells or tissues. For DNMTs, there are two basic assay forms that are commercially available. Both forms of the assay require a source of the enzyme (either purified or nuclear extract), along with a DNA substrate containing CpG repeats. The first type of DNMT assay uses an ELISA-based format and ultimately measures the level of DNA methylation. The DNA substrate is immobilized on a 96 well plate and incubated with the DNMT enzyme source in the presence of S-adenosyl methionine. After the incubation period the plate is washed and then incubated with either an anti-5-methylcytosine antibody or a tagged MCB protein. After washing the plate again, the antibody or MCB protein is detected using an appropriate detection molecule coupled to a signal-generating system that is generally of the colorimetric, fluorescent or luminescent type. The intensity of the signal generated will be proportional to the amount of 5-methylcytosine, which in turn will be proportional to the amount of DNMT activity in the sample.

The second type of DNMT activity assay is based on the accumulation of S-adenosylhomocysteine, which is formed from S-adenosyl methionine after it donates its methyl group (Dorgan et al. 2006). In this type of assay a DNMT enzyme source is combined with the DNA substrate and S-adenosyl methionine, but additional enzymes are added to the reaction mix to convert any S-adenosylhomocysteine to S-ribosylhomocysteine. The S-ribosylhomocysteine is used as a reactant—leading to the ultimate formation of a compound that can be measured spectrophotometrically or fluorometrically. This second type of assay has the advantage of not requiring wash steps and, since the entire reaction mix is combined at the same time, it allows for real-time monitoring of DNMT activity.

b. DNA Methylation: Pharmacological Agents

Commercially available assay kits offer the ability to both assess the level of DNMT activity in cells or tissues undergoing various treatments. They also provide the ability to screen potential materials for their ability to either inhibit, or stimulate, the activity of DNMTs by using purified forms of the enzyme family. Compounds that can impact DNMT function not only have a significant impact in potentially treating disease states that have a basis in altered DNA methylation patterns (i.e., cancer), but can also function as molecular tools for helping to understand the mechanisms behind the epigenetic controls involved with gene expression. As tools for skin care research, compounds that impact DNMT activity can play an important role as controls for DNA methylation-based studies. Table 1 provides a list of some well-established DNMT regulators.

Table 1. DNA Methylation-Related Agents

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