In addition to gross changes in chromatin affecting transcriptional activity, certain DNA elements facilitate or enhance initiation at the promoter and hence are termed enhancers. Enhancer elements, which typically contain multiple binding sites for transactivator proteins, differ from the promoter in notable ways. Enhancers can exert their positive influence on transcription even when separated by tens of thousands of base pairs from a promoter; enhancers work when oriented in either direction; and enhancers can work upstream (5′) or downstream (3′) from the promoter, or even when embedded within the transcription unit of a gene. Experimentally, enhancers can be shown to be promiscuous, in that they can stimulate transcription of any promoter in their vicinity, and may act on more than one promoter. The viral SV40 enhancer can exert an influence on, for example, the transcription of β-globin by increasing its transcription 200-fold in cells containing both the SV40 enhancer and the β-globin gene on the same plasmid (see following discussion and Figure 1); in this case the SV40 enhancer-β-globin reporter gene was constructed using recombinant DNA technology—see Chapter 39. The enhancer element does not produce a product that in turn acts on the promoter, since it is active only when it exists within the same DNA molecule as the promoter (ie, in cis, or physically linked to). Enhancer-binding proteins are responsible for this effect. The exact mechanism(s) by which these transcription activators work is subject to intensive investigation. Enhancer-binding trans-factors, some of which are cell-type specific, while others are ubiquitously expressed, have been shown to interact with a plethora of other transcription proteins. These interactions include chromatin-modifying coactivators, mediator, as well as the individual components of the basal RNA polymerase II transcription machinery. Ultimately, transfactor-enhancer DNA binding events result in an increase in the binding and/or activity of the basal transcription machinery on the linked promoter. Enhancer elements and associated binding proteins often con vey nuclease hypersensitivity to those regions where they reside (see Chapter 35). Recently, while analyzing regulatory sequences that control cellular identity (and other genes essential for cell function) in mammalian genomes investigators have identified large tandem clusters of various enhancer elements in tandem arrays. These sequence elements have been termed super enhancers. Not surprisingly, the cis-linked genes modulated by super-enhancers are highly expressed. It is highly likely that such super-enhancers contribute importantly to the formation of the biomolecular condensates described earlier. A summary of the properties of enhancers is presented in Table 1.

Fig1. A schematic illustrating the methods used to study the organization and action of enhancers and other cis acting regulatory elements. These model chimeric genes, all con structed by recombinant DNA techniques in vitro (see Chapter 39), consist of a reporter gene that encodes a protein that can be readily assayed, and that is not normally produced in the cells to be studied, a promoter that ensures accurate initiation of transcription, and the indicated enhancer (regulatory response) elements. In all cases, high level transcription from the indicated chimeras depends on the presence of enhancers, which stimulate transcription ≥100-fold over basal transcriptional levels (ie, transcription of the same chimeric genes containing just promoters fused to the indicated reporter genes). Examples (A) and (B) illustrate the fact that enhancers (eg, here SV40) work in either orientation and upon a heterologous promoter. Example (C) illustrates that the metallothionein (mt) regulatory element (which under the influence of cadmium or zinc induces transcription of the endogenous mt gene and hence the metal-binding mt protein) will work through the herpes simplex virus (HSV) thymidine kinase (tk) gene promoter to enhance transcription of the human growth hormone (hGH) reporter gene. In a separate experiment, this engineered genetic construct was introduced into the male pronuclei of single-cell mouse embryos and the embryos placed into the uterus of a surrogate mother to develop as transgenic animals. Offspring have been generated under these conditions, and in some the addition of zinc ions to their drinking water effects an increase in growth hormone expression in liver. In this case, these transgenic animals have responded to the high levels of growth hormone by becoming twice as large as their normal litter mates. Example (D) illustrates that a glucocorticoid response element (GRE) enhancer will work through homologous (PEPCK gene) or heterologous gene promoters (not shown; ie, HSV tk promoter, SV40 promoter, β-globin promoter, etc) to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene.

Table1. Summary of Novel Histone PTMs (2011 to 2020)

One of the best-understood mammalian enhancer systems is that of the β-interferon gene. This gene is induced upon viral infection of mammalian cells. One goal of the cell, once virally infected, is to attempt to mount an antiviral response—if not to save the infected cell, then to help to save the entire organ ism from viral infection. Interferon production is one mechanism by which this is accomplished. This family of proteins is secreted by virally infected cells. Secreted interferon interacts with neighboring cells to cause an inhibition of viral replication by a variety of mechanisms, thereby limiting the extent of viral infection. The enhancer element controlling induction of the β-interferon gene, which is located between nucleotides −110 and −45 relative to the transcription start site (+1), is well characterized. This enhancer consists of four distinct clustered cis-elements, each of which is bound by unique trans-factors. One cis-element is bound by the transacting factor NF-κB , one by a member of the interferon regulatory factor (IRF) family of transactivator factors, and a third by the heterodimeric leucine zipper factor ATF-2/c-Jun (see following discussion). The fourth factor is the ubiquitous, abundant architectural transcription factor known as HMG I(Y). Upon binding to its A + T-rich binding sites, HMG I(Y) induces a significant bend in the DNA. There are four such HMG I(Y) binding sites interspersed throughout the enhancer. It is believed that these sites play a key role in facilitating the formation of a unique 3D structure in concert with the afore mentioned three trans-factors, by inducing a series of critically spaced DNA bends. Consequently, HMG I(Y) likely induces the cooperative formation of a unique, stereospecific structure within which all four factors are active when viral infection signals are sensed by the cell. The putative structure formed by the cooperative assembly of these four factors has been termed the β-interferon enhanceosome (Figure 2), so named because of its proposed structural similarity to the nucleosome, which is also a unique three-dimensional protein-DNA structure that wraps DNA about a core assembly of proteins. The enhanceosome, once formed, induces a large increase in β-interferon gene transcription upon virus infection. Thus, it is thought that it is not simply the protein occupancy of the linearly apposed cis element sites that induces β-interferon gene transcription— rather, it is the formation of the enhanceosome proper that provides appropriate surfaces and 3-dimensional organization for the efficient recruitment of coactivators that results in the enhanced formation of the PIC on the cis-linked promoter and thus transcription activation.

Fig2. Formation and putative structure of the enhanceosome formed on the human β-interferon gene enhancer. Diagrammatically represented at the top is the distribution of the multiplecis-elements (HMG, PRDIV, PRDI-III, PRDII, NRDI) composing the β-interferon gene enhancer. The intact enhancer mediates transcriptional induction of the β-interferon gene (IFNB1) over 100-fold upon virus infection of human cells. The cis-elements of this modular enhancer represent the binding sites for the trans-factors HMG I(Y), cJun-ATF-2, IRF3-IRF7, and NF-κB, respectively. The factors interact with these DNA elements in an obligatory, ordered, and highly cooperative fashion as indicated by the arrow. Initial binding of four HMG I(Y) proteins induces sharp DNA bends in the enhancer, causing the entire 70- to 80-bp region to assume a high level of curvature. This curvature is integral to the subsequent highly cooperative binding of the other trans-factors since bending enables the DNA-bound factors to make critical direct protein–protein interactions that both contribute to the formation and stability of the enhanceosome and generate a unique 3D surface that serves to recruit chromatin-modifying coregulators that carry enzymatic activities (eg, Swi/Snf: ATPase, chromatin remodeler and P/CAF: histone acetyltransferase) as well as the general transcription machinery (RNA polymerase II and GTFs). Although four of the five cis-elements (PRDIV, PRDI-III, PRDII, NRDI) independently can modestly stimulate (~10-fold) transcription of a reporter gene in transfected cells, all five cis-elements, in appropriate order, are required to form an enhancer that can appropriately stimulate transcription of IFNB1 (ie, ≥100-fold) in response to viral infection of a human cell. This distinction indicates a strict requirement for appropriate enhanceosome architecture for efficienttrans-activation. Similar enhanceosomes, involving distinct cis- andtrans-factors and coregulators, are proposed to form on many other mammalian genes.

cis-Acting DNA elements that decrease the expression of specific genes are termed silencers. Silencers have also been identified in a number of eukaryotic genes. However, because fewer of these elements have been intensively studied, it is not possible to formulate accurate generalizations about their mechanism of action. That said, it is clear that as for gene activation, chromatin level covalent modifications of histones, and other proteins, by silencer-recruited repressors and co recruited multisubunit corepressors likely play central roles in these regulatory events.

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مواضيع ذات صلة

Several Structural Motifs Compose the DNA-Binding Domains of Regulatory Transcription Factor Proteins

Reporter Genes Are Used to Define Enhancers & Other Regulatory Elements That Modulate Gene Expression

Epigenetic Mechanisms Contribute Importantly to the Control of Gene Transcription

The Chromatin Template Contributes Importantly to Eukaryotic Gene Transcription Control

The Genetic Switch of Bacteriophage Lambda (λ) Provides Another Paradigm for Understanding the Role of Conditional Regulatory Protein-DNA Interactions in Transcriptional Control in Eukaryotic Cells

Analysis of Lactose Metabolism in E. coli Led to the Discovery of the Basic Principles of Gene Transcription

Biologic Systems Exhibit Three Types of Temporal Responses to a Regulatory Signal

Regulated Expression of Genes is Required for Development, Differentiation, & Adaptation

Posttranslational Processing Affects the Activity of many Proteins

Like Transcription, Protein synthesis can be described in three phases: Initiation, Elongation, & Termination

Mutations result when changes occur in the Nucleotide Sequence

The Genetic Code is Degenerate, Unambiguous, Nonoverlapping, Without Punctuation, & Universal