Genetics - A. V. Sivolob 2008

Gene Expression
Regulation of Gene Expression in Eukaryotes
Transcriptional Regulation

Several tens of thousands of eukaryotic genes require differential activation or repression at specific times, depending on Cell type, developmental stage, environmental conditions, and other factors.

METABOLISM/31.html">Transcription factors. As in prokaryotes, the key elements of the transcriptional regulation system are regulatory cis-acting sequence elements (proximal and distal promoter elements, see Fig. 2.11) and trans-regulators, namely protein transcription factors (TFs, which here refer to specific, non-basal transcription factors). However, the number of eukaryotic genes is significantly larger, and it is clear that each individual Gene cannot be controlled by its own unique transcription factor, because the factor itself is the product of a specific gene that must, in turn, be controlled by a certain factor.

The solution to The Challenge of regulating The activity of A large number of genes using a limited set of transcription factors lies in the modular architecture of eukaryotic promoters. This is illustrated in Fig. 2.15: three regulatory (e.g., proximal) sequence elements exhibit affinity for three transcription factors, and from these three pairs of interactions, six combinations can be formed. In reality, the number of such pairs is much greater, and the number of potential combinations is virtually endless. Each promoter can feature its own unique set of modules that distinguishes it from other promoters and, accordingly, its own specific repertoire of transcription factors required for gene activation.

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Fig. 2.15. The modular principle of transcription factor interaction with promoters

Closely related to the modular principle is the cooperativity of transcription factor interactions with adjacent cis-elements. Each individual TF typically has a relatively low affinity for its target sequence element. However, when two cis-elements are located close to each other and their respective TFs are capable of interacting mutually, the affinity of each factor increases, significantly enhancing complex stability.

Furthermore, the activation domains of TFs recruit protein Cofactors (coactivators), which drive the assembly of the enhanceosome and the preinitiation complex (see Fig. 2.12). The various Components of the enhanceosome act synergistically, increasing the overall Stability of the complex. Conversely, the absence of a few elements can trigger the destabilization and disassembly of the enhanceosome, given that the affinity of each individual element for DNA is relatively low. This ensures the dynamic nature of activation: the enhanceosome is not a fixed Structure, but rather assembles and disassembles at specific times. Notably, components of the multiprotein complexes assembled on promoters can also act in the opposite manner—blocking Transcription initiation—in which case they are termed repressors and corepressors.

The activity of a given gene depends on the presence of a specific set of transcriptional activators or repressors within The Cell. Consequently, the genes encoding the transcription factors themselves are controlled by complex regulatory networks operating during cell development and differentiation. As a result, a specific cell type synthesizes a distinct set of TFs, leading to the Activation of a corresponding specific set of genes.

At the same time, the expression of a particular gene can be rapidly modulated in response to external signals by altering the activity of already synthesized transcription factors. The two most critical mechanisms for this type of regulation are the Interaction of a specific TF—a hormone receptor—with Steroid Hormones, and cascades of post-translational modifications triggered by chemical signals (signal Transduction). In the absence of the hormone, the hormone receptor resides in the Cytoplasm in an inactive structural state. Upon entering the cytoplasm, the hormone binds to the receptor, inducing its activation. The hormone-receptor complex then translocates to The Nucleus, where it binds to a specific sequence element and initiates the cascade of enhanceosome assembly. An example of signal transduction is the binding of a protein hormone to a receptor on the outer surface of The cell membrane, which activates a membrane-proximal kinase on the inner side of the membrane. The kinase phosphorylates an inactive transcription factor, thereby converting it into its active form. Often, the membrane-proximal kinase triggers a phosphorylation cascade: it phosphorylates a protein that acquires kinase activity as a result, this new kinase phosphorylates another protein (or several different Proteins, thereby achieving signal Amplification and/or branching into multiple pathways directed toward several final targets), transforming it into a kinase, and so on, ultimately leading to the phosphorylation and activation of the transcription factor.

Transcriptional regulation and Chromatin Structure. A fundamental feature of eukaryotes is that cellular DNA is organized into complex chromatin structures (Chapter 1). Nucleosomes and the chromatin fiber as a whole act as a general repressor of gene activity. Thus, they help ensure the global inactivation of most genes in a Introduction/5.html">Eukaryotic Cell, except for those whose activation is mediated by TFs. Transcriptional activation requires structural remodeling of chromatin, involving the decondensation of the chromatin fiber and the exposure of cis-elements from nucleosomes. Two primary tools operate in close coordination to achieve such remodeling: The system of post-translational histone modifications and ATP-dependent chromatin remodeling complexes that mediate nucleosome repositioning. Specific patterns of histone modifications also play the reverse role—ensuring the stable repression of specific chromatin regions.

Among other modifications, the Acetylation of Lys residues (at specific conserved positions) almost invariably correlates with transcriptional activation. Histones acetylated by histone acetyltransferases (HATs) accumulate at active promoters, whereas the action of histone deacetylases leads to inactivation. Acetyltransferases and deacetylases constantly and nonspecifically operate within chromatin, maintaining a baseline balance of histone acetylation and deacetylation. Upon activation of a specific promoter, acetyltransferases carry out targeted hyperacetylation, while the disappearance of the activation signal prompts deacetylases to restore the promoter to its basal inactive state. Deacetylases can also be specifically recruited to promoters by transcriptional repressors to maintain a stably deacetylated status.

HATs are integral components of multiprotein complexes that often serve as enhanceosome components. HATs frequently contain bromodomains—structural modules with a specific affinity for acetylated lysines. In other words, HATs recognize Lys residues already acetylated by other HATs and acetylate neighboring nucleosomes, thereby maintaining the acetylated status of a given chromatin region. Histone acetylation promotes chromatin fiber decondensation by reducing the positive charge of the primary Condensation factors, namely the histone tails. The unspooling of the fiber and the temporary dissociation of histone H1 create a "window of opportunity" for regulatory factors to bind to the internucleosomal linker DNA. Furthermore, acetylated Lysine residues on histones can be directly recognized by transcription factors and cofactors. For instance, the presence of a bromodomain within TFIID helps increase the local concentration of this basal transcription factor at acetylated chromatin regions.

Enhancing promoter accessibility during activation also requires other specialized mechanisms. Under physiological ionic strengths, the Electrostatic Interactions between DNA and histones are exceptionally strong, and nucleosomes maintain high stability, which virtually precludes even the sliding of a nucleosome along the DNA. Because nucleosome sliding is essential for exposing regulatory sites on DNA to transcription factors, Cells possess a specialized system: chromatin remodeling (CR) complexes, which frequently function as components of enhanceosomes. CR complexes are ATP-dependent multiprotein molecular machines that drive the movement of nucleosomes along the chromatin fiber (repositioning) and facilitate the temporary removal of nucleosomes from active promoters onto histone chaperone proteins. CR complexes engage in numerous interactions with nucleosomal DNA, histone tails, specific and general transcription factors, and histone acetyltransferases. Notably, the action of remodeling complexes does not necessarily result in transcriptional activation; it can also lead to repression, depending on the context of other functionally important influences with which the given complex cooperates.

One of the many possible scenarios for promoter activation is illustrated in Fig. 2.16: a nucleosome blocks the basal promoter; enhanceosome assembly is initiated within the linker region between nucleosomes, recruiting a HAT; the HAT acetylates histones; another HAT and a remodeling complex are recruited to the enhanceosome, which repositions the nucleosome; and RNA polymerase along with basal transcription factors bind to the basal promoter.

Fig. 2.16. One possible sequence of steps in eukaryotic promoter activation. HAT – histone acetyltransferase, Ac – acetate residues

During the transcription elongation stage, nucleosomes must also pose significant obstacles to the progression of RNA polymerase. At the same time, chromatin, even within active genes, generally preserves its nucleosomal structure. The elongation of RNA polymerase through chromatin is facilitated by a suite of elongation factors that, among other Functions, drive the disruption of nucleosomes ahead of the polymerase and their reassembly behind it.

Although the basic structure of interphase chromatin—the 30 nm fiber—acts as a barrier to transcriptional activation, stable repression requires an even higher degree of chromatin compaction. This additional compaction in heterochromatin and other repressed regions relies on specialized chemical markers. The most crucial among these are, once again, post-translational histone modifications (primarily the methylation of specific Lys residues) and DNA Methylation. The mechanisms underlying this type of repression are examined in Chapter 6.