Regulation of Transcription and Gene Expression in Eukaryotes

Transcription factors (TFs) are regulatory proteins whose function is to activate (or more rarely, to inhibit) transcription of DNA by binding to specific DNA sequences. TFs have defined DNA-binding domains with up to 10⁶-fold higher affinity for their target sequences than for the remainder of the DNA strand. These highly conserved sequences have been used to categorize the known TFs into various "families," such as the MADS box-containing proteins, SOX proteins, and POU factors (Remenyi et al., 2004). Transcription factors can also be classified by their three-dimensional protein structure, including basic helix-turn-helix, helix-loop-helix, and zinc finger proteins. These different structural motifs result in transcription factor specificity for the consensus sequences to which they bind.

Sequence-specific transcription factors are considered the most important and diverse mechanisms of gene regulation in both prokaryotic and eukaryotic cells (Pulverer, 2005). In eukaryotes, regulation of gene expression by transcription factors is said to be combinatorial, in that it requires the coordinated interactions of multiple proteins (in contrast to prokaryotes, in which a single protein is usually all that is required).

Many genes, known as housekeeping genes, are needed by almost every type of cell and appear to be unregulated or constitutive. But at the core of cellular differentiation, manifested in the variety of cell types observed in different organisms, is the regulation of gene expression in a tissue-specific manner. The same genome is responsible for making the entire cadre of cell types, each of which has its own function—for example, red blood cells exchange oxygen, muscle cells expand and contract, and cells in the immune system recognize pathogens. Genes that regulate cell identity are turned on under very specific temporal, spatial, and environmental conditions to ensure that a cell is able to perform its designated function.

Take the example of the gene for beta globin, a protein used in red blood cells for oxygen exchange. Every cell in the human body contains the beta globin gene and the corresponding upstream regulatory sequences that regulate expression, but no cell type other than red blood cells expresses beta globin. Scientists can use a technique called DNA footprinting to map where transcription factors bind to specific regulatory sequences. When Reddy et al. examined the beta globin promoter in different cell types, they found that the transcription factors that could bind to the promoter sequences required for beta globin expression were expressed only in erythroblasts (immature adult red blood cells). (See Figure 1). The two consensus sequences in the beta globin promoter known for binding transcription factors, CCAAT and CACC, were protected in the erythroid cells (E), but not the other cell types (Reddy et al., 1994).

RNA polymerase in prokaryotes can access almost any promoter in a DNA strand without the presence of activators or repressors. Thus, the "ground state" of DNA expression in prokaryotes is said to be nonrestrictive, or "on." In eukaryotes, however, the ground state of expression is restrictive in that, although strong promoters might be present, they are inactive in the absence of some sort of recruitment to the promoter by transcription factors. For instance, RNA polymerase II, which transcribes mRNA, cannot bind to promoters in eukaryotic DNA without the help of transcription factors (Struhl, 1999). In many eukaryotic organisms, the promoter contains a conserved gene sequence called the TATA box. Various other consensus sequences also exist and are recognized by the different TF families. Transcription is initiated when one TF binds to one of these promoter sequences, initiating a series of interactions between multiple proteins (activators, regulators, and repressors) at the same site, or other promoter, regulator, and enhancer sequences. Ultimately, a transcription complex is formed at the promoter that facilitates binding and transcription by RNA polymerase.

As in prokaryotes, eukaryotic repressor molecules can sometimes bind to silencer elements in the vicinity of a gene and inhibit the binding, assembly, or activity of the transcription complex, thus turning off expression of a gene. Positive regulation by TFs that are activators is common in eukaryotes. Considering the restrictive transcriptional ground state, it is logical that positive regulation is the predominant form of control in all systems characterized to date. Many activating TFs are generally bound to DNA until removed by a signal molecule, while others might only bind to DNA once influenced by a signal molecule. The binding of one type of TF can influence the binding of others, as well. Thus, gene expression in eukaryotes is highly variable, depending on the type of activators involved and what signals are present to control binding.

Even when transcription factors are present in a cell, transcription does not always occur, because often the TFs cannot reach their target sequences. The association of the DNA molecule with proteins is the first step in its silencing. The associated DNA and histone proteins are collectively called chromatin; the complex is tightly bonded by attraction of the negatively charged DNA to the positively charged histones (Table 1). The state of chromatin can limit access of transcription factors and RNA polymerase to DNA promoters, contributing to the restrictive ground state of gene expression. In order for gene transcription to occur, the chromatin structure must be unwound.

Chromatin structure contributes to the varying levels of complexity in gene regulation. It allows simultaneous regulation of functionally or structurally related genes that tend to be present in widely spaced clusters or domains on eukaryotic DNA (Sproul et al., 2005). Interactions of chromatin with activators and repressors can result in domains of chromatin that are open, closed, or poised for activation. Chromatin domains have various sizes and different extents of stability. These variations allow for phenomena found solely in eukaryotes, such as transcription at various stages of development and epigenetic memory throughout cell division cycles. They also allow for the maintenance of differentiated cellular states, which is crucial to the survival of multicellular organisms (Struhl, 1999).