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In the Part I, the basic approaches to understanding how a treatment or condition results in mRNA accumulation will be described. Although the focus and many of the examples will center on gene regulation by xenobiotics, the approaches are applicable to any treatment/condition that alters gene expression. The subsequent chapter will contain an overview of the molecular biology involved in each step of the process; however, details can be found elsewhere. For a good basic overview of transcriptional control of gene expression, the reader is directed to Molecular Biology of the Cell [1]. In addition, there have been several excellent review articles on eukaryotic transcriptional control [2–8]. Posttranscriptional gene regulation is, in general, more difficult to examine experimentally but is a very important determinant of cellular events. We will briefly discuss mRNA processing and stability, with emphasis on events altered by xenobiotics and the methods used to examine these events. Detailed laboratory procedures are available from several sources [9–13] and will not be emphasized here. Instead, we focus on the approaches to be used and the rationale behind these decisions. Translational and posttranslational regulation of gene control will not be examined in detail in this section, but they are discussed subsequently in Part II.

The detection of mRNA levels of a particular gene is one of the cornerstones of molecular biology. There are many ways that mRNA can be detected, each with its strengths and weaknesses. Surprisingly, few investigators give much thought as to whether their methodologies and approaches are appropriate. Yet, the quantification and interpretation of results depends on understanding some key points. Three key factors contribute to the complexity and difficulty in examining differentially expressed genes. First, genes are not present at the same abundance and can vary from less than one up to thousands of copies per cell. This has implications for methods that must be used to accurately detect and quantify the expression of an mRNA. Second, the intensity of response varies greatly from gene to gene; that is, when comparing two treatments or conditions, an mRNA can be twofold or several orders of magnitude different between samples. Certain methods have a robust linear range and can handle both levels of response, while others are biased toward either the low or high responder. Last, there are many ways to alter gene expression, and some or all of the particular mechanisms may be at play. Approaches must be used that are capable of isolating, or at least accounting for, the competing possibilities so that hypotheses can be tested confidently. We will briefly discuss these parameters as they pertain to examining altered gene expression, and how these factors impinge on developing an optimal model system.

An important concept about gene expression in the disease state, and the effects of xenobiotics or any external stimuli, is that often the events observed are cell-, species-, sex-, and development-specific. This is owing to the fact that combinatory gene expression is the norm in eukaryotes. In general, a combination of multiple gene-regulatory proteins, rather than a single protein, determines where and when a gene is transcribed. A single gene may be necessary but not sufficient to alter the phenotype of a cell. A good example of combinatorial gene control comes from muscle cell differentiation. Myogenic proteins (MyoD, myogenin; both are helix-loop-helix proteins) are key to causing certain fibroblasts to differentiate into muscle cells. It appears that MyoD regulates myogenin. If myogenin is removed, the cells will not differentiate, and if this gene is overexpressed in fibroblasts, they will convert to muscle. However, other cell types are not converted to muscle by myogenic proteins. This suggests that some cells have not accumulated the other regulatory proteins required.

The previous chapters have discussed how to determine whether a gene’s mRNA accumulates under a particular treatment or condition. In the present chapter, the exploration of the reason for this accumulation commences. The two major ways in which the steady-state levels of a transcript are altered are via transcriptional or posttranscription processes. For simplicity, effects at the chromatin level will be included in this chapter. A summary of the events leading to transcriptional regulation are given in and will be discussed in more detail. Further description of the molecular events associated with transcriptional regulation can be found elsewhere [1, 30].

Posttranscriptional processing is a very important process in eukaryotic mRNA accumulation, although it is often overlooked as a primary means of regulating gene expression [1,30]. Another key point is that in mammalian cells, transcription, processing, and stability are intertwined, and it is often difficult to dissociate an effect on one process from the others. When transcription of bacterial rRNAs and tRNAs is completed, they are immediately ready for use in translation with no additional processing required. Translation of bacterial mRNAs can begin even before transcription is completed owing to the lack of the nuclear-cytoplasmic separation that exists in eukaryotes. An additional feature of bacterial mRNAs is that most are polycistronic, which means that multiple polypeptides can be synthesized from a single primary transcript. This does not occur in eukaryotic mRNAs. In contrast to bacterial transcripts, eukaryotic RNAs (all three classes) undergo significant posttranscriptional processing and are transcribed from genes that contain introns.

The basic approaches to understanding how a drug, chemical exposure, or disease results in altered protein level or activity will be described in this part, with particular emphasis on transcription factors. In addition, molecular approaches to understanding the role of a given protein in signaling pathways will also be examined. Exposure to a xenobiotic can alter protein activity or lev-els through a variety of mechanisms. We will explore the various approaches available to determine how a treatment leads to altered protein function by a mechanism other than by changes in m levels. This part examines the experimental approaches that can define the general mechanism(s) that lead to altered protein activity, as depicted in .

and in Part I have detailed the regulation of mRNA synthesis and stability. The next regulatory step in the process of protein production is the rate of protein synthesis or mRNA translation. Protein synthesis can be divided into three discreet steps; initiation, elongation, and termination. An important aspect of overall regulatory control of protein synthesis is regulation of peptide chain elongation by a group of eukaryotic elongation factors (eEFs). The activity of several eEFs is regulated by phosphorylation. For example, the kinase that stimulates eEF2 activity is regulated by calcium ions, calmodulin, and mitogen-activated protein (MAP) kinase. Also, low energy levels in the cell can lead to downregulation of the eEF2 kinase and thus peptide chain elongation activity. Other key contributors to translational control are the eukaryotic initiation factors (eIFs). One rate-limiting member of the eIF-4F translation initiation complex is the mRNA cap-binding protein eIF-4E. This protein binds the cap structure at the 5′terminus of mature mRNAs and recruits mRNAs to the eIF-4F complex, which then scans from the 5′cap through the untranslated region (UTR), unwinding secondary structure to reveal the translation initiation codon, which then leads to ribosome loading.

Development of global scale methods for protein profiling is important to complement existing mRNA analysis techniques, such as DNA microarray analysis. Especially considering that changes in protein levels can occur without any change in an individual protein’s mRNA levels. Changes in translational rates, protein stability, protein localization, or posttranslational modifications can all lead to altered protein levels or activity. Only through the global characterization of protein levels can a complete picture of possible changes evoked by a chemical treatment or altered cell phenotype be more fully established. After all, proteins usually are the end point of gene expression.

Determining whether or not a specific protein interacts with another can be accomplished in a number of ways, which can be divided into in vivo or in vitro approaches. The in vivo approaches include yeast two-hybrid, yeast threehybrid, mammalian two-hybrid, one-hybrid, and FRET analyses. While the most common in vitro approaches are glutathione-S-transferase (GST) pulldown assays, co-immunoprecipitation, immune depletion, gel-filtration or sucrose (or glycerol)-density gradient analysis, far-Western blot analysis, and chemical crosslinking. Each one of these assays has strengths and weaknesses and usually a combination of methods can lead a compelling case that a given interaction actually can occur within the cell. Many of these approaches utilize what can be considered transient overexpression of the proteins being studied and this can lead to interactions occurring that do not normally occur under physiologic conditions. Also, upon overexpression a protein may be found in subcellular compartments where they do not normally exist. For example, upon overexpression, a normally cytosolic protein may be found in significant concentrations in the nucleus. Nevertheless, the use of cellular overexpression systems usually yields information that is physiologically relevant. The following section outlines the various approaches that can be taken to document that one protein is capable of interacting with another.

Posttranslational modifications commonly occur on proteins within a cell and lead to changes in stability, subcellular localization, enzymatic activities, and other protein activities mediated through protein-protein interactions. Because of the large number of posttranslational modifications that occur, this chapter will focus on the modifications that are most frequently encountered when examining regulation of gene expression through transcription factor (TF) analysis. These modifications occur in the majority of proteins involved in transcriptional regulation, including transcription factors, coactivators, repressors, general transcription factors, histones, and RNA polymerases. Protein phosphorylation is probably the most studied posttranslational modification. Interestingly, phosphorylation has been demonstrated to influence a wide range of transcription factor activities, such as transactivation potential, DNA binding, half-life, subcellular localization, dimerization, or heterodimerization, and cofactor or ligand binding. Transcription factors can be phosphorylated on serine, threonine, and to a lesser extent on tyrosine residues. The mammalian cell contains a large number of protein kinases, the specific recognition motif for some being known, while others remain to be determined. For example, the recognition motif for casein kinase II is S/T-X-X-E. The overall level of protein phosphorylation is regulated by the balance between the level of protein-kinase versus protein-phosphatase activity in the cell.