The region adjacent to the leucine repeats is highly basic in each of the zipper proteins, and could comprise a DNA-binding site. The 2 leucine zippers in effect form a Y-shaped structure, in which the zippers comprise the stem, and the 2 basic regions bifurcate simmetrically to form the arms that bind to DNA Figure 4.
This is known as the bZIP structural motif. It explains why the target sequences for such proteins are inverted repeats with no separation. Zippers may be used to sponsor the formation of homodimers or heterodimers. Its major component is Jun, the product of the gene c-jun , which was identified by its relationship with the oncogene v-jun carried by an avian sarcoma virus. The mouse genome contains a family of c-jun related genes, JunB and JunD.
All of them have leucine zippers that can interact to form homodimers or heterodimers for a review, see Wisdom, The other major component of AP1 is the product of another gene with an oncogenic counterpart : the c-fos gene, which is the cellular homologue of the oncogene v-fos carried by a murine sarcoma virus. Expression of c-fos activates genes whose promoters or enhancers possess an AP1 target site. The c-fos product is a nuclear phosphoprotein that is one of a group of proteins Fos-related antigens, FRA , which constitute a family of fos-like proteins.
Fos has also a leucine zipper. Fos cannot form homodimers, but can form a heterodimer with Jun. A leucine zipper in each protein is required for the interaction. The ability to form dimers is a crucial part of the interaction of these factors with DNA. Fos cannot by itself bind DNA, possibly because of its failure to form a dimer, but the Jun-Fos heterodimer can bind to DNA with same target specificity as the Jun-Jun dimer, and this heterodimer binds to the AP1 site with an affinity about 10X that of the Jun homodimer.
AP-1 proteins are transcription factors that contribute both to basal gene expression, as well as TPA-inducible gene expression. Many other stimuli, most notably serum, growth factors and oncoproteins, are also potent inducers of AP-1 activity ; it is also induced by tumor necrosis factor TNFa and interleukin I IL-1 , as well as by a variety of environmental stresses, such as UV radiation. AP-1 activity is important in growth control and play a key role in cell transformation. Furthermore, since two of AP-1 target genes are collagenase and IL-2, AP-1 is likely to be involved in inflammation and innate immune response.
AP-1 transcription factors have also been implicated in the control of cell death and proliferation ; indeed, they regulate the expression of target genes involved in the two processes. For example, AP-1 may promote cell proliferation by activating the cyclin D1 gene, whose regulatory sequences contain two AP-1 binding sites. The effect of c-Jun on p53 is likely to be direct, and exerted through a variant AP-1 site in the p53 promoter, but in this case c-Jun represses rather than activates transcription.
JunD also interacts with the p53 pathway, since expression of p19Arf is down-regulated by JunD. The INK4a locus is a transcription unit shared by the p19Arf and p16 genes, and recent results suggests that JunB regulates p16 expression , thus the growth inhibitory activity of JunB is likely to be in part dependent on p16 ; indeed, the p16 promoter contains 3 AP-1 binding sites. On the other hand, c-Jun down-regulates p16 transcription.
The pro-apoptotic capacity of c-Jun can be explained also by its ability to activate the FasL gene, which is induced by DNA damaging agents, and indeed contains one AP-1 binding site. It should be said that, despite the involvement of Jun and Fos in growth control and oncogenesis, positively regulated AP-1 target genes that mediate cell cycle progression were never identified.
The amphipathic helix-loop-helix HLH motif has been identified in some developmental regulators and in genes coding for eukaryotic DNA-binding proteins. The proteins that have this motif have both the ability to bind DNA and to dimerize. They share a common type of sequence motif: a stretch of aminoacids contains 2 amphipathic a -helices separated by a linker region the loop of varying length. The proteins in this group form both homodimers and heterodimers by means of interactions between the hydrophobic residues on the corresponding faces of the 2 helices.
The ability to form dimers resides with these amphipathic helices, and is common to all HLH proteins. Members of the group with such a region are called bHLH proteins. A dimer in which both subunits have the basic region can bind to DNA. The bHLH proteins fall into 2 general groups.
Class B consists of proteins that are expressed in a tissue-specific manner, including mammalian MyoD, Myf5, myogenin and MRF4 a group of transcription factors that are involved in myogenesis, called myogenic regulatory factors, MRFs. A common modus operandi for a tissue-specific bHLH protein may be to form a heterodimer with a ubiquitous partner. There is also a group of gene products that specify development of the nervous system in Drosophila melanogaster where Ac-S is the tissue-specific component, and da is the ubiquitous component.
The Myc proteins form a separate class of bHLH proteins. In addition, the MRFs have homology outside the bHLH domain, including a cysteine-histidine-rich stretch adjacent to the basic region and a serine-threonine-rich region at the C-terminus.
MyoD and Myf5 share an overlapping redundant function required for generating or maintaining muscle cell identity and activating myogenin ; myogenin is required at a later stage of muscle development, specifically during the terminal differentiation of myoblasts to myotubes. MRF4 may have a role in later development. MyoD is able to activate the expression of myogenin, p21, myosin heavy chain MHC , and desmin.
It binds cooperatively to 2 MyoD binding sites, and many MyoD target genes contain 2 or more sites. Indeed, MHC cis -regulatory region does not contain E boxes; it is possible that myogenic factors could bind sequences other than the CANNTG consensus, perhaps as components of complexes with other proteins. Proteins of this type have the same capacity to dimerize as bHLH proteins, but a dimer that contains one subunit of this type can no longer bind to DNA specifically.
The formation of muscle cells is triggered by a change in the transcriptional program that requires several bHLH proteins, including MyoD. Over-expression of Id can prevent myogenesis, so the removal of Id could be the trigger that release MyoD to initiate myogenesis. The members of the Myc family of oncogenes c-Myc , N-Myc and L-Myc code for nuclear phosphoproteins that appear to promote cell growth and transformation by regulating the transcription of target genes required for proliferation.
Mutations which disrupts the regulation or expression level of Myc are found in cancers. However, the mechanisms by which Myc activates transcription remain unclear.
Myc-Max transcriptional activity on either synthetic promoters or on putative cellular target genes is weak and variable on the order of fold. This suggests that maybe Myc is functioning facilitating the action of other transcription factors by opening up specific segments of chromatin. This may explain the relatively weak transactivation activity of Myc on its own. Myc also plays a role in the induction of apoptosis e. Other putative Myc target genes are odc ornithine decarboxylase , a -prothymosin involevd in cell cycle progression , nm23 inhibitor of metastatic progression , tert , and cad.
A potentially important function for Myc might be to regulate the rate of growth defined as an increase in cell mass and size that is thought to be required for cell cycle progression and cell division ; indeed, the Drosophila Myc ortholog dmyc is required to mantain the normal size of cells and organs. A majority of genes up-regulated following Myc induction in a variety of contexts, are involved in ribosome biogenesis, energy and nucleotide metabolism, and translational control. So, maybe the capacity of Myc to drive cell cycle progression is due, in part, to stimulation of growth.
The p53 gene was the first tumor-suppressor gene to be identified. The human p53 protein contains aminoacid and is structurally and functionally divided into 4 domains :. All these pathways inhibit p53 degradation, thus stabilizing p53 at high concentration. The amount of p53 protein in cells is determined mainly by the rate at which it is degraded.
The degradation proceeds through a process of ubiquitin-mediated proteolysis, involving Mdm2. This process is subjected to a feedback loop, since Mdm2 is a p53 target gene. The downstream events mediated by p53 take place by 2 major pathways : cell cycle arrest and apoptosis. E2F was originally discovered as a cellular activity that is required for adenovirus E1A transforming protein to mediate the transcriptional acitvation of the viral E2 promoter.
Rb is also an essential molecule in terminally differentiating cells, in which E2F target genes are irrevesibly repressed e. In this scenario, it is possible that Rb represses E2F transcription to varying degrees, even in a two step process. The tight association between HP1 and Suv39H1 would facilitate the modification of adjacent histone tails , and allow the silencing effect to be propagated throughout the locus.
Eight human genes have been identified as components of the E2F transcriptional activity ; these genes have been divided into 2 distinct groups : the E2Fs , and the DPs 1 and 2. The protein products of these two groups heterodimerize through a leucine zipper domain to give rise to functional E2F activity, and all possible combination of E2F-DP complexes exist in vivo. E2F1,2 and 3 are potent transcriptional activators " activating E2Fs " ; by contrast, E2F4 and E2F5 seem to be primarily involved in the " active repression " of E2F responsive genes by recruiting the pocket proteins and their associated histone modifying enzymes.
Finally, E2F6 also acts as a transcriptional repressor, but through a distinct, pocket-protein-independent manner. E2F1 was cloned by virtue of its ability to bind Rb. E2F1, 2 and 3 could contribute to the repression of E2F-responsive genes by recruiting Rb.
However, the key role of these E2Fs is the activation of genes that are essential for cellular proliferation and the induction of apoptosis. E2F1, 2 and 3 are potent transcriptional activators of E2F responsive genes. Overexpression of any of these proteins is sufficient to induce quiescent cells to re-enter the cell cycle ; beside their role in the control of cell proliferation, de-regulation of E2F activity can trigger apoptosis.
Apoptosis can be either pdependent, and in this case it is likely to be mediated by the transcriptional activation of p19Arf , a known E2F-responsive gene, or pindependent. In normal cells, the " activating " E2Fs are specifically regulated by their association with Rb, but not with the related pocket proteins p and p Their release is triggered by the phosphorylation of Rb in late G1 and correlates closely with the activation of E2F target genes.
The analysis of mutant mice for E2F1 have evidenced its tumor-suppressor properties E2F1 deficient mice develop a broad spectrum of tumors , maybe through its ability to induce apoptosis. These E2Fs are important in the induction of cell cycle exit and terminal differentiation. They were originally identified and cloned by virtue of their association with p and p The sequence of these proteins diverge considerably from those of the activating E2Fs.
Significant levels of E2F4 and 5 are detected in quiescent G0 cells, whereas E2F1, 2 and 3 are primarely restricted to actively dividing cells. In addition, whereas the activating E2Fs are specifically regulated by Rb, E2F5 is mainly regulated by p, and E2F4 associates with each of the pocket proteins at different points in the cell cycle.
As E2F4 is expressed at higher levels than the other E2F-family members, it accounts for at least half of the Rb-, p and passociated E2F activity in vivo. They also have a different subnuclear localization : E2F1, 2 and 3 are constitutively nuclear, whereas E2F4 and 5 are predominantly cytoplasmatic, but it was shown that association with Rb or p is enough to induce their nuclear localization in vivo.
As these complexes associate with HDACs in vivo, E2F4 and 5 are thought to be crucial in mediating the transcriptional repression of E2F-responsive genes. This member of the family lacks the carboxy-terminal sequences that are responsible for both pocket protein binding and transactivation. Overexpression studies showed that E2F6 can repress E2F responsive genes. This suggests that the transcriptionally repressive properties of E2F6 are mediated through its ability to recruit the PcG complex.
Bmi-1 is involved in the regulation of senescence and tumorigenity, and its transforming activity seems to depend on its ability to repress the p16INK4A and p19ARF tumor-suppressor genes, which are expressed from the INK4A locus ; it is widely inferred that Bmi-1 mediates the direct transcriptional repression of INK4A through its participation in the PcG complex. The most important structural difference between eukaryotic and prokaryotic DNA is the formation of chromatin in eukaryotes.
Chromatin results in the different transcriptional "ground states" of prokaryotes and eukaryotes Table 1. Sequence-specific transcription factors are considered the most important and diverse mechanisms of gene regulation in both prokaryotic and eukaryotic cells Pulverer, 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. See Figure 1. 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 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. 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, As you have seen, the state of chromatin structure at a specific region in eukaryotic DNA, along with the presence of specific transcription factors, works to regulate gene expression in eukaryotes.
However, this complex interplay between proteins that serve as transcriptional activators or repressors and accessibility to the regulatory sequence is still just part of the story. Epigenetic mechanisms, including DNA methylation and imprinting , noncoding RNA , post-translational modifications, and other mechanisms, further enrich the cellular portfolio of gene expression control activities. Pulverer, B. Sequence-specific DNA-binding transcription factors. Nature Milestones doi: Reddy, P.
Genomic footprinting and sequencing of human beta-globin locus: Tissue specificity and cell line artifact. Journal of Biological Chemistry , — Remenyi, A. Combinatorial control of gene expression. Nature Structural and Molecular Biology 11 , — doi Struhl, K. Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell 98 , 1—4 Gene expression is defined as a process through which the information encoded in a gene is used to direct the synthesis of a functional gene product 4.
As a process, it may explain why organisms containing mostly the same DNA exhibit different cell types and functions 5. Gene expression is an intricate process and involves the coordination of multiple dynamic events, which are subject to multi-level regulation 6.
Those regulatory levels include the transcriptional level, the post-transcriptional level, the translational level and the post-translational level. Regulating gene expression is crucial in living organisms 7. Gene regulation is essential in cellular differentiation in multicellular organisms, since it can contribute to the function and the structure of a specific cell, and is an integral part of organism development 4. All of the above prove that apart from inherited genetic information, cell function and structure are influenced by information that is not encoded in the DNA sequence.
This information has also been termed epigenetic information 5. Epigenetics is defined as both heritable alterations in gene activity and expression and also stable, long-term alterations in the transcriptional potential of a cell that may not be heritable 8.
Epigenetics comprises of a number of mechanisms, which include DNA methylation, histone modification, post-translation modifications, chromatin remodeling and various forms of regulatory RNA molecules. These mechanisms seem to influence gene expression 5. Gene transcriptional regulation is a fundamental part of both tissue-specific gene expression and gene activity in response to stimuli 9.
The main regulators of gene transcription are transcription factors TFs. TFs are defined as proteins that can bind specific DNA sequences to control transcription Each cellular life form follows different strategies for the initiation and regulation of transcription.
Bacteria have two distinct mechanisms for the initiation of transcription, the promoter-centric mechanism, in which specific TFs interact with the promoter in order to alter its ability to bind RNA polymerase or RNA-centric mechanism, in which TFs interact with RNA in order to alter its promoter preference In eukaryotes, a number of TFs interact with their cognate DNA motifs and recruit transcriptional cofactors to alter the chromatin environment. Lastly, the archaea transcriptional mechanism can be summarized as a simplified version of the eukaryotic transcriptional mechanism Archaea feature a transcriptional apparatus that includes additional RNA polymerase subunits and basal TFs that direct transcription initiation and elongation.
The above underline the importance of TFs in both the initiation and regulation of gene transcription. The activation of TFs is quite complex and may involve multiple intracellular transduction pathways or direct activation through specific molecules that bind, known as ligands TFs mostly regulate gene activity by binding to specific short DNA base pair patterns termed motifs or cis -regulatory elements CREs in upstream, intron, or downstream regions of target genes.
They can also act by interacting with other genomic locations that may be distant to the primary DNA sequence These are defined as gene regulatory regions. The interaction between DNA and TFs goes beyond the structural and sequence level since several other factors participate in the process, such as the influence of cofactors, epigenetic modifications and the cooperative binding of other TFs Thus, gene regulation involves a large number of molecular mechanisms.
Therefore, an in-depth examination of the evolution of TFs, which takes into account the interaction with all the molecular factors mentioned above, and the manner through which TFs influence the evolution of other molecular mediators, is essential to the understanding of organism evolution.
TF function involves two basic features: i The ability to recognize and bind short, specific sequences of DNA within regulatory regions; and ii the ability to recruit or bind proteins that participate in transcriptional regulation Consequently, the evolution of TFs mainly depends on alterations in binding sites, binding partners and expression patterns Moreover, as an integral part of gene expression, they are closely related to the evolution of epigenetic mechanisms 5.
The current literature on TF evolution provides a broad range of information. Firstly, gene duplication and gene loss as crucial drivers of evolution 21 , 22 are subsequently important drivers of TF evolution.
Regardless of organism complexity, they are present in all domains of life. Duplication and deletion can influence transcriptional regulatory networks by increasing or reducing the number of TFs with specific binding preferences 23 , Following the duplication of a TF gene, the two resulting gene copies are likely the same.
Since they share the same sequence, including the DBD sequence, they bind to the same target genes. Ensuing mutations in the DNA binding domain sequence can lead to one of the TF copies to switch to regulating different target genes.
On a more lineage-specific level, TFs display several differences. Although the basal transcription machinery has long been considered universally conserved, it is currently accepted that it too diversifies during evolution. The size and subunit composition of the basal transcription machinery increase highly during evolution, consisting of roughly 6 subunits in bacteria, up to 15 in the archaea, and a large number in eukaryotes, which have at least 3 different RNA polymerases Significant differences are apparent between prokaryotes and eukaryotes.
Firstly, some DBDs are specific to evolutionary lineages; e. Moreover, eukaryotic TFs are relatively longer than other eukaryotic proteins with a different function, while this association is reversed in prokaryotes. This phenomenon may be due to the fact that eukaryotic TFs have a number of long intrinsic disordered segments that are needed to leverage the formation of a multi-protein transcription protein complex Another characteristic specific to eukaryotes are the repeats of the same DBD family in one polypeptide chain.
This characteristic may be the result of a mechanism eukaryotes use that increases the length and diversity of DNA binding recognition sequences using a limited number of DNA binding domain families Several factors seem to affect the evolution, emergence, disappearance and function of CREs.
These factors include insertion and deletion mutational mechanisms, slippage processes, tje large rearrangement of promoter regions, co-operation amongst TFs and the existence of initial sequence distributions that are biased towards the mutational neighborhood of strongly binding sequences 30 , Insertion and deletion mutational mechanisms can lead to the slow emergence of binding sites out of a random sequence, while factors that accelerate these processes may include the already sufficient genomic sequence from which sites can evolve and the possible co-operativity between adjacent TFs Furthermore, since the interaction of TFs' with TF binding sites is integral in gene regulation, a mutation in either TF or binding site hinders that interaction and may lead to dysfunctional gene expression.
Therefore, in order to maintain proper gene expression levels, TF evolution and CREs evolution are closely intertwined They specifically bear a co-evolutionary association, where in order to sustain proper interaction, a mutation in one interacting partner could be compensated by a corresponding mutation in its' interacting partner during the course of evolution Although prokaryote individual TFs can recognize long DNA motifs that are alone capable of defining the genes they may regulate, organisms with larger genomes are characterized by TFs that recognize sequences too short to be able to define unique genomic positions.
Moreover, the development of multicellular organisms requires molecular systems that are complex and able to execute combinational processes. In an effort to overcome these obstacles, organisms have evolutionary developed co-operative recognition of DNA by multiple TFs TFs can collaborate through a variety of mechanisms, with each co-operative mechanism determining the specifics of the regulatory interaction.
Some of the mechanisms through which TFs cooperate include protein-protein interaction and indirect co-operation A prime example of protein-protein interaction among TFs is the formation of functional dimers. A number of eukaryotic TFs proteins are not able to bind DNA sequences as monomeric proteins and require physical interaction with an identical molecule or one within the same family to form functional dimers that are able to bind targeted DNA sequences.
It has been suggested that, at first, TFs function as monomers, something supported by the fact that TFs in less complex organisms can sufficiently bind target sequences as monomers Several promoters that include symmetrical palindromic repeats of the DNA-recognition motif could have potentially brought two or more copies of the same TF protein into proximity.
If, by chance, an interaction domain with only one interaction sequence appeared, then this would help establish the formation of a TF complex on DNA because this specific complex would recognize a larger DNA motif These events would lead to more relaxed evolutionary constraints on the TF DBD within a redundant duplicate gene and would allow the emergence of a DNA-binding domain that binds with less affinity, but is still functional.
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